75 Years of Chromatography (Journal of chromatography library)

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 17

75 years of chromatographya historical dialogue

JOURNAL OF CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G.H. Wagman and M.J. Weinstein Volume 2

Extraction Chromatography edited by T. Braun and G. Ghersini

Volume 3

Liquid Column Chromatography. A Survey of Modem Techniques and Applications edited by Z. Deyl, K. Macek and J. Jan&

Volume 4

DetecJors in Gas Chromatography by J. SevElk

Volume 5

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen

Volume 7 Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei Volume 8

Chromatography of Steroids by E. Heftmann

Volume 9

HPTLC - High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser

Volume 0

Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya

Volume 1 Liquid Chromatography Detectors by R.P.W. Scott Volume 12

Affmity Chromatography by J. Turkovg

Volume 13

Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber

Volume 14

Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman

Volume 16

Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger

Volume 17

75 Years of Chromatography - A Historical Dialogue edited by L.S. Ettre and A. Zlatkis

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 17

75 years of chromatographya historical dialogue edited by L. S. Ettre Instrument Division, The Perkin-Elmer Corporation, Norwalk, Connecticut 06856

A. Zlatkis Chemistry Department, University of Houston, Houston, Texas 77004

ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam - Oxford - New York 1979

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 211, 1000 AE Amsterdam, The Netherlands

Distributors f o r the United States and Canada: ELSEVIER/NORTH-HOLLAND INC. 52, Vanderbilt Avenue New York. N.Y. 10017

PUBLISHER'S NOTE Within the limits of examination by the Editors, the authors in this volume have had a completely free hand in giving their views on the varioua topics covered. The publisher can therefore accept no responsibility as to the accuracy of any statement, account or view expressed in it.

Library of Congress Cataloging in Publication D a t a

Main entry under t i t l e :

75 years of chromatography. (Journal of chromatography library ; V. 17) 1. Chromatographic analysis-Addresses, essays, lectures. I. E t t r e , Leslie S. 11. Zlatkis, Albert. 111. Series.

QV79.C4%8

ISEN c-44t-417544

544'.92

ISBN 0-444-41754-0 (Vol. 17) ISBN 0-444-41616-1 (Series) Q

78-23917

'

Elsevier Scientific Publishing Company, 1979

AU rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, P.O. Box 330, 1000 AH Amsterdam, The Netherlands Printed in The Netherlands

V

CONTENTS

Introduction Contributors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

............................................... ...............................................

............................................ 1 ................................................ 11 .............................................. 21 ............................................. 31 ............................................ 43 ............................................. 53 .............................................. 67 ............................................ 75 .......................................... 87 ........................................... 99 ........................................... 109 ............................................. 115 ............................................ 125 .............................................1 3 1 .......................... 141 ............................................. 151 ........................................... 159 ............................................. 167 ............................................... 173 ............................................ 187 .............................................. 193 .......................................... 201 ........................................... 209 ........................................... 219 . .......................................... 231 ............................................. 237 ............................................. 247 ............................................. 255 ............................................ 265 .......................................... 277 .......................................... 285 ................................ 297 ............................................ 309 ........................................ 315 ............................................ 323 ........................................... 333 ......................................... 339 ............................................... 345 ....................................... 351

E . R . Adlard H Boer E Cremer D.H. D e s t y G Dijkstra L.S. E t t r e P Flodin C.W. Gehrke J . C . Giddings E Glueckauf M . J . E . Golay D.W. Grant E Heftmann G . E . Hesse E . C . Horning and M.G. Horning C Horvhth J . F . K . Huber A.T. James J . Janhk R . E . Kaiser A . Karmen J . G . Kirchner J.J. Kirkland A.V. K i s e l e v E . s z Kovhts E . Lederer M . Lederer A Liberti S.R. Lipsky J . E . Lovelock A.J.P. Martin S Moore and W.H. S t e i n H.W. P a t t o n C.S.G. P h i l l i p s J.O. Porath V. Pretorius G.R. P r i m a v e s i N.H. Ray L Rohrschneider

. . .

. .

. .

.

.

.

VII XI

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

....................................... ........................................... G . - l . Schwab ........................................... R.D. Schwartz .......................................... C.D. Scott ............................................. R.P.W. Scott ........................................... G.T. Seaborg and G . H . Higgins .......................... M.S. Shraiber .......................................... L . R . Snyder ............................................. E . Stahl ............................................... H.H. Strain ............................................ F.H. Stross ............................................ R . L . M . Synge ........................................... R . Teranishi ........................................... J.J. van Deemter ....................................... A . A . Zhukhovitskii ..................................... A . Zlatkis ............................................. Those who are no longer with us (by L . S . Ettre) ........ K . I . Sakodynskii

G . Schomburg

361 367 375 381 391 397 405 413 419 425 437 443 447 453 461 467 473 483

VII

INTRODUCTION

~ A A A , f160 T O T a w e h a 1J-Epv;)aeal ~ I ~ V W V (Sweet is the memory of past labor) Euripides , Andromeda

The 75th anniversary of the invention of chromatography is being celebrated this year. It was on March 21, 1903, that a young Russian scientist presented a paper On a New Category of Adsorption Phenomena and Their Application t o Biochemical Analysis at the regular meeting of the Biological Section of the Warsaw Society of Natural Sciences. In this paper he reported his first experiments on the isolation of plant pigments in which he utilized the new technique of adsorption separation. He did not as yet use the name "chromatography" - this was done only in the two detailed papers published three years later - despite this, however, all the fundamentals of chromatography were outlined in this paper. In the seventy-five years since its inception - and particularly in the last 47 years since its 'rebirth' - chromatography underwent a development unparalleled in any other analytical technique. This development is characterized by the close cooperation of scientists from practically every part of the world. Most of these were not "analytical chemists" in the usual sense: Tswett himself was a botanist while among the pioneers of the 1 9 3 0 ' ~Lederer, ~ Zechmeister, Strain and Hesse were organic chemists, Schwab was a physical chemist and Martin and Synge biochemists. In the generation of the 1950's one finds, in addition to these disciplines, chemical engineers, petroleum chemists, physicists and even physicians. Indeed, the whole development of modern liquid and gas chromatography is a fascinating story as a human endeavour involving a relatively small number of individuals. A . J . P . Martin, in 1951, began his Nobel lecture with these words:

"If enough h i s t o r i e s , w r i t t e n while the ideas are s t i l l f r e s h i n t h e minds of the people concerned, are available f o r a v a r i e t y of discoveries of inventions, i t may eventually be possible t o lay down some of the p r i n c i p l e s required t o f a c i l i t a t e the obtaining of f r u i t f u l r e s u l t s i n s c i e n t i f i c research i n general. Clearly also t h e background of knowledge a t t h e time the advance was made w i l l be b e s t understood if the h i s t o r y i s as recent as possible. ''

VIII This statement gave us the impetus for this book.

It is well known that Tswett's work was not appreciated immediately; however, after its 'reinvention' by E. Lederer in 1931, liquid chromatography became a fully developed technique in less than 15 years including its major variants such as thin-layer, partition and paper chromatography. After a number of fundamental studies in gas (adsorption) chromatography in the 1940's and early 1950's, the paper by James and Martin triggered the exponential development of gas-liquid partition chromatography (GLPC) which, in less than 10 years, became one of the most widely used analytical techniques. The development of ion-exchange chromatography and size-exclusion chromatography was also accomplished in a relatively short time. Finally, the adaptation of the theory and practice of GLPC in liquid chromatography resulted in what is currently referred to as highperformance liquid chromatography developed in the last decade. While this book is being edited, we can observe a similar transition from classical thin-layer chromatography to modern high-performance TLC. All of these developments took place in a little more than a generation's time and most of the leading pioneers are still among us. Encouraged by Martin's quoted statement we have decided to commemorate the seventy-fifth anniversary of the invention of chromatography by compiling the personal recollections of the pioneers involved in this hectic period. In this way, we present "the background of knowledge at the time the advance was made" for future generations of chromatographers; also, by recording the many different - and sometimes contradictory - viewpoints of the individual researchers we have a unique occasion to demonstrate the manner in which science works. We have attempted to include as many of those pioneers as possible who began their involvement well before 1960, contributed to the development of the technique and were active in chromatography for an extended period of time. In a few cases younger scientists are also included who joined the field in the early 1960's but had a significant role in the evolution of chromatography. We fully realize that the list of contributors is far from complete. A number of pioneers were unable to participate in this venture because of other committments; furthermore, some simply could not be included because of the limited space. However, we believe that our contributors represent an even cross-section of the whole field of chromatography and thus, this book presents a fair report of its evolution. A few remarks regarding the organization of this compilation is a l s o in order. The contributions are given alphabetically. Each begins with a biographical note about the author; these notes were written by the editors and represent a summary of the individual's activities. The recollections of the individuals which follow were written by them and we tried to do as little editing as possible so that each contribution would reflect the pioneer's experiences in his own words, even if we might disagree with some of the state-

IX ments. The figures accompanying the chapters represent either illustrations from original publications or personal photographs. We are very grateful to all of the authors who understood the importance of this volume for future generations and agreed to participate in it. Indeed, our real gratitude should be for their contribution to the advancement of our common field: chromatography. December 15. 1978

This Page Intentionally Left Blank

XI

CONTRIBUTORS

The affiliation and address of each contributor is given in the language of the country. Transliteration of Russian text from Cyrillic is done according to the rules used by Chemical Abstracts. The same rule is followed in the text of the contributions. The only exemption is the spelling of the name of Tswett where we follow the spelling used by him in his own publications. The correct transliteration would be Tsvet. E.R. ADLARD, Shell Research Ltd., Thornton Research Centre, P.O. Box 1, Chester CH1 3SH, United Kingdom. H. BOER, Koninklijke/Shell Laboratorium, Badhuisweg 3, 1031 CM Amsterdam-Noord, The Netherlands. E. CREMER, Institut fur Physikalische Chemie, Universitat Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria. D.H. DESTY, The British Petroleum Co., Ltd., BP Research Centre, Sunbury-on-Thames, Middlesex TW16 7LN, United Kingdom. G. DIJKSTRA, Analytisch Chemisch Laboratorium, Rijksuniversiteit Utrecht, 3522 AD Utrecht, The Netherlands.

L.S. ETTRE, Instrument Division, The Perkin-Elmer Corporation, Norwalk, Connecticut 06856, U.S.A. P. FLODIN, Institutionen for Polymerteknologi, Chalmers Tekniska Hogskola, S-40220 Goteborg, Sweden. C.W. GEHRXE, Department of Biochemistry, Experiment Station Chemical Laboratories, University of Missouri, Columbia, Missouri 65211, U.S.A. J.C. GIDDINGS, Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, U.S.A. E. GLUECKAUF, Chemistry Division, Building 10.30, Atomic Energy Research Establishment, Harwell, Oxfordshire OX11 ORA, United Kingdom. M.J.E. GOLAY, Clair-Azur, CH-1095 Lutry, Vaud, Switzerland D.W. GRANT, The British Carbonization Research Association, Wingerworth, Chesterfield, Derbyshire S42 6JS, United Kingdom.

E. HEFTMANN, Western Regional Research Center, Science and Education Administration, U . S . Department of Agriculture, 800 Buchanan Street, Berkeley, California 94710, U.S.A. G.E. HESSE, Institut fur Organische Chemie, Universitat Erlangen-Nurnberg, Henkestrasse 42, D-8520 Erlangen, German Federal Republic. G.H.

HIGGINS, Lawrence Livermore Laboratory (L-2091, P.O.Box 808, Livermore, California 94550, U.S.A.

E.C. HORNING, Institute for Lipid Research, Baylor College of Medicine, Houston, Texas 77030, U.S.A. M.G.

HORNING, Institute for Lipid Research, Baylor College of Medicine, Houston, Texas 77030, U.S.A.

C. HORVATH, Department of Engineering and Applied Science, Mason Laboratory, Yale University, New Haven, Connecticut 06520, U.S.A. J.F.K. HUBER, Institut fur Analytische Chemie, Universitat Wien, Wahringer Strasse 38, A-1090 Wien, Austria. A.T. JAMES, Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedfordshire MK44 lLQ, United Kingdom. J. JANAK, bstav Analytickk Chemie, EeskoslovenskH Akademie Vgd, Leninova 82, CS-66228 Brno, Czechoslovakia. R.E. KAISER, Institut fur Chromatographie, Postfach 1308, D-6702 Bad Durkheim 1, German Federal Republic.

A. KARMEN, Department of Laboratory Medicine, Albert Einstein College of Medicine, Yeshiva University, The Bronx, New York 10461, U.S.A. J.G. KIRCHNER, 1950 Old Dominion Drive, Dunwoody, Georgia 30338, U.S.A. J.J. KIRKLAND, Central Research & Development Department, Experimental Station, E.I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 19898, U.S.A. A.V. KISELEV, Laboratoriya Adsorbtsii i Khromatografii, Khimicheskogo Fakul'teta, Moskovskogo Gosudarstvennogo Universiteta imeni M.V. Lomonosova, 117234 Moskva, and Laboratoriya Khimii Poverkhnosti Instituta Fizicheskoi Khimii Akademii Nauk S.S.S.R., 117071 Moskva, U.S.S.R. E. s z . KOVATS, Laboratoire de Chimie-technique, Ecole Polytechnique FBdkrale de Lausanne, CH-1007 Lausanne, Switzerland.

E. LEDERER, Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, F-91190 Gif-sur-Yvette, France. M. LEDERER, Laboratorio di Cromatografia del C.N.R., Via

Romagnosi M A , R o d , Italy.

XI11

A. LIBERTI, Istituto di Chimica Analitica, UniversitP, Citta Universitaria, 1-00185 Romil, Italy. S.R. LIPSKY, Section Physical Sciences, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. J.E. LOVELOCK, Department of Cybernetics, University of Reading, Reading, Berkshire RG6 2AL, United Kingdom. A.J.P. MARTIN, Department of Chemistry, University of Houston, Houston, Texas 77004, U.S.A. S. MOORE, The Rockefeller University, 1230 York Avenue, New York, New York 10021, U.S.A.

H.W. PATTON, Eastman Chemical Products, Inc., P.O.Box 431, Kingsport, Tennessee 37662, U.S.A. C.S.G. PHILLIPS, Merton College, Oxford University, Oxford OX1 4JD, United Kingdom. J.O. PORATH, Institutionen for Naturvetenskaplig Biokemi, Uppsala Universitet, S-75123 Uppsala, Sweden.

V. PRETORIUS, Institute for Chromatography, University of Pretoria, Pretoria, Republic of South Africa. G.R. PRIMAVESI, 5 Denfield, Tower Hill, Dorking, Surrey RH4 2AH, United Kingdom. N.H. RAY, I.C.I. Corporate Laboratory, P.O.Box 11, The Heath, Runcorn, Cheshire WA7 4QE, United Kingdom. L. ROHRSCHNEIDER, Chemische Werke Huls, Postfach 1320, D-4370 Marl, German Federal Republic. K.I. SAKODYNSKII, Institut Fizicheskoi Khimii imeni Karpova, Ulica Obukha 10, 107120 Moskva, U.S.S.R. G. SCHOMBURG, Max-Planck-Institut fur Kohlenforschung, D-4330 Mulheim/Ruhr, German Federal Republic. G.-M. SCHWAB, Physikalisch-Chemisches Institut, Universitat Miinchen, D-8000 Munchen 2, German Federal Republic. R.D. SCHWARTZ, Pennzoil Company, P.O.Box 6199, Shreveport, Louisiana 71106, U.S.A.

C.D. SCOTT, Chemical Technology Divison, Oak Ridge National Laboratory, P.O.Box X, Oak Ridge, Tennessee 37830, U.S.A. R.P.W. SCOTT, Chemical Research Department, Hoffmann-La Roche Inc., Nutley, New Jersey 07110, U.S.A. G.T. SEABORG, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720, U.S.A. M.S. SHRAIBER, Khar'kovskii Nauchno-Issledovatel'ski KhimikoFarmatsevticheskii Institut, Khar'kov, U.S.S.R.

XIV L.R. SNYDER, Clinical Chemistry Department, Technicon Instruments Corporation, Tarrytown, New York 10591, U.S.A. E. STAHL, Pharmakognosie und Analytische Phytochemie, Universitat des Saarlandes, D-6600 Saarbrucken, German Federal Republic. W.H. STEIN, The Rockefeller University, 1230 York Avenue, New York, New York 10021, U,S.A. H.H. STRAIN, Four Seasons Retirement Home, 1901 Taylor Street, Columbus, Indiana 47201, U . S . A . F.H. STROSS, Department of Chemistry, Chemimetrics Laboratory, University of Washington, Seattle, Washington 98195, U.S.A. R.L.M. SYNGE, Agricultural Research Council, Food Research Institute, Colney Lane, Norwich NR4 7UA, United Kingdom.

R. TERANISHI, Westcrn Rcgional Research Center, U.S. Department of Agriculture, 800 Buchanan Street, Berkeley, California 94710, U.S.A.

J.J. VAN DEEMTER, Koninklijke/Shell Laboratorium, Badhuisweg 3, 1031 CM Amsterdam-Noord, The Netherland.

A.A. ZHUKHOVITSKII, Institut Stali i Splavov, Leninskii Prospekt 6, 117049 Moskva, U.S.S.R.

A. ZLATKIS, Department of Chemistry, University of Houston, Houston, Texas 77004, U.S.A.

1

EDWARD R. ADLARD

EDWARD RADCLIFFE ADLARD was born i n 1927, i n L i v e r p o o l , England. H e a t -

tended a l o c a l High School which had a considerable t r a d i t i o n i n s c i e n t i f i c e d u c a t i o n and a f t e r m i l i t a r y s e r v i c e he s t u d i e d a t Liverpool Univ e r s i t y , g r a d u a t i n g w i t h an Honours degree i n Organic Chemistry i n 1952. I n t h e same y e a r he j o i n e d t h e s t a f f of S h e l l Research L t d . , a t Thornton Research Centre n e a r Chester where he h a s worked e v e r s i n c e a p a r t from a y e a r i n 1968-1969 which he s p e n t as a v i s i t i n g s c i e n t i s t at Shell Development Company's r e s e a r c h labor a t o r y i n Emeryville, C a l i f o r n i a . M r . Adlard is t h e a u t h o r and coauthor of o v e r 20 p a p e r s . H e has been c l o s e l y a s s o c i a t e d w i t h t h e Chromatography Discuss i o n Group s i n c e i t s i n c e p t i o n i n 1957, s e r v i n g a s member of t h e Executive Committee s i n c e 1961, as Honorary S e c r e t a r y (1962-1965), Vice Chairman (1967-1968) and Chairman (1971-1973). H e r e c e n t l y r e t i r e d from t h e Committee and h a s had a honorary l i f e membership of t h e Group c o n f e r r e d upon him i n r e c o g n i t i o n of h i s o u t s t a n d i n g s e r v i c e . M r . Adlard has been involved i n gas chromatography s i n c e 1952 and pioneered i n a number of a s p e c t s of t h e t e c h n i q u e . H i s c u r r e n t i n t e r e s t s a r e i n t h e a p p l i c a t i o n of s e l e c t i v e d e t e c t o r s t o t h e a n a l y s i s of complex m i x t u r e s and t h e a p p l i c a t i o n of chromatography t o environmental a n a l y s i s .

I l e f t t h e U n i v e r s i t y o f L i v e r p o o l i n t h e summer of 1952 w i t h an honours d e g r e e i n o r g a n i c c h e m i s t r y and a p a s s d e g r e e i n p h y s i c s . The p a s s d e g r e e i n p h y s i c s came about a s a r e s u l t of t h e r e q u i r e ments imposed by t h e u n i v e r s i t y on s t u d e n t s who had been i n t h e f o r c e s . Although I and my e x - s e r v i c e c o l l e a g u e s r e g a r d e d t h e s e r e q u i r e m e n t s w i t h a somewhat j a u n d i c e d eye a t t h e t i m e , i t r e s u l t e d i n my r e c e i v i n g a b e t t e r e d u c a t i o n i n c l a s s i c a l p h y s i c s t h a n t h a t r e c e i v e d by many p r e s e n t - d a y c h e m i s t r y s t u d e n t s . The l a t t e r w i l l d i s c u s s t h e S c h r o d i n g e r wave e q u a t i o n o r t h e e l e c t r o n d i s t r i b u t i o n i n t h e boron h y d r i d e s a t l e n g t h , b u t a g l a z e d e x p r e s s i o n o f t e n a p p e a r s i n t h e e y e a t t h e mention o f B e r n o u i l l i ' s Theorem or P o i s e u i l l e ' s E q u a t i o n . I stress t h e p a r t p l a y e d by p h y s i c s i n my s c i e n t i f i c e d u c a t i o n s i n c e i t seems t o m e t h a t t h e advances made i n chromatography i n t h e last 25 y e a r s have had a s u b s t a n t i a l p h y s i c s and p h y s i c a l c h e m i s t r y c o n t e n t , and h i n d s i g h t makes m e g r a t e f u l f o r t h o s e irksome post-war u n i v e r s i t y r e g u l a t i o n s . Having s u r v e y e d t h e j o b market and h a v i n g r e c e i v e d an o f f e r from S h e l l R e f i n i n g and Marketing Company (as i t was t h e n ) , I took up a p o s t a t Thornton Research C e n t r e n e a r C h e s t e r i n t h e United Kingdom. E x a c t l y why I chose a r e s e a r c h environment i n which t o work is n o t now c l e a r t o m e , a l t h o u g h i t was a y o u t h f u l a m b i t i o n t o have a r e a c t i o n or an e q u a t i o n o r a p i e c e of equipment named a f t e r m e - a d i s t i n c t i o n which h a s so f a r e l u d e d me! I a m , however, c l e a r a s t o why I c h o s e Thornton i n p r e f e r e n c e t o o t h e r , s u p e r f i c i a l l y b e t t e r o f f e r s . The r e a s o n f o r my c h o i c e was t h e i m p r e s s i o n , conf i r m e d o v e r t h e y e a r s , t h a t t h e atmosphere and f a c i l i t i e s a t Thornton were s e c o n d t o none. A t Thornton I was a b l e t o c o n t i n u e my s c i e n t i f i c e d u c a t i o n : i n an e s t a b l i s h m e n t of around 1000 p e o p l e I soon r e a l i z e d t h a t t h e r e were e x p e r t s on s i t e from m o s t b r a n c h e s of t h e p h y s i c a l s c i e n c e s a n d t h a t few e x p e r t s c a n resist an a p p e a l f o r a d v i c e . My f i r s t few months a t Thornton w e r e s p e n t on o r g a n i c s y n t h e s i s . But my s u p e r v i s o r s came t o t h e c o n c l u s i o n t h a t t h i s was n o t my f o r t e . A t t h a t t i m e t h e r e was a c o n s i d e r a b l e i n t e r e s t i n a l t e r n a t i v e anti-knock a d d i t i v e s t o t h e l e a d a l k y l s (how t h e wheel t u r n s f u l l c i r c l e ! ) . I n p a r t i c u l a r t h e r e was i n t e r e s t i n t h e p r e p a r a t i o n and p r o p e r t i e s of f e r r o c e n e and o t h e r m e t a l l i c c y c l o p e n t a d i e n e d e r i v a t i v e s , and I was g i v e n t h e job of s y n t h e s i z i n g f e r r o c e n e by t h e r e a c t i o n of p o t a s s i u m c y c l o p e n t a d i e n y l w i t h f e r r i c c h l o r i d e . T h i s work r e s u l t e d i n my d i s c o v e r i n g t h a t p o t a s s i u m c y c l o p e n t a d i e n y l i s s p o n t a n e o u s l y inflammable and i n a s h o r t t i m e caused m e t o b e nicknamed Prometheus. The t a r r y mess t h a t a c c o u n t e d f o r most of my r e a c t i o n p r o d u c t needed extensive p u r i f i c a t i o n before anything with a melting point approximating t o t h a t of f e r r o c e n e c o u l d b e o b t a i n e d , and I e v e n t u a l l y d e c i d e d t o u s e column chromatography w i t h alumina. My e f f o r t s a t chromatography were more s u c c e s s f u l t h a n my e f f o r t s a t s y n t h e s i s and r e s u l t e d i n a few m i l l i g r a m s of p u r e f e r r o c e n e . T h i s work c o i n c i d e d w i t h two o f t h e e a r l i e s t p u b l i c a t i o n s on gas chromatography a t a n i n t e r n a t i o n a l c o n g r e s s on a n a l y t i c a l chemi s t r y s p o n s o r e d by t h e S o c i e t y f o r A n a l y t i c a l Chemistry i n Oxford i n September 1952, which w a s a t t e n d e d by s e v e r a l Thornton p e r s o n n e l .

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F i g . 1.1. One of o u r e a r l y gas chromatographs b u i l t i n 1953-1954. I t p r o b a b l y r e p r e s e n t s one of t h e f i r s t dozen GCs i n t h e w o r l d . Under t h e bench can j u s t be d i s c e r n e d t h e l a r g e l e a d - a c i d b a t t e r y f o r t h e k a t h a r o m e t e r power s u p p l y and t h e vacuum pump which sucked t h e c a r r i e r gas through t h e column ( t h i s b e i n g t h e s t a n d a r d t e c h n i q u e u n t i l t h e l a t e r 1950s). Note a l s o t h e b r i c k under t h e column f u r n a c e t o g i v e an e l e g a n t h e i g h t a d j u s t m e n t ; i n t h o s e days l a b j a c k s were unknown i n t h e UK.

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I t a l s o c o i n c i d e d w i t h t h e s t a r t - u p o f a S h e l l P l a t f o r m e r u n i t des i g n e d t o produce mixed x y l e n e s t o b e u s e d by a major UK chemical company t o e x t r a c t p-xylene f o r t h e manufacture o f t e r y l e n e . T h i s company wished t o know t h e c o m p o s i t i o n of t h e x y l e n e s u p p l i e d t o

F i g . 1.2. Summary page of t h e f i r s t Thornton i n t e r n a l r e p o r t i s s u e d i n February 1955 on "the a p p l i c a t i o n of g a s / l i q u i d p a r t i t i o n chromatography t o a n a l y s i s . ' I them and i t was t h e i r s u g g e s t i o n t h a t t h e new t e c h n i q u e of gas chromatography b e u s e d t o g i v e t h e t o l u e n e , 0-xylene and C 9 a r o m a t i c s c o n t e n t s of t h e p l a n t stream. S i n c e t h e s e p a r a t i o n o f m- and px y l e n e by GC was t o be a c h i e v e d o n l y c o n s i d e r a b l y l a t e r , t h e s e compounds were r e s o l v e d by IR s p e c t r o s c o p y . Faced w i t h my i n a d e q u a c i e s a s a s y n t h e t i c o r g a n i c chemist and my a p p a r e n t p o t e n t i a l as a c h r o m a t o g r a p h e r , t h e powers t h a t be d e c i d ed t h a t I s h o u l d work on t h e new t e c h n i q u e under B . T . ( B i l l ) Whitham an a s s o c i a t i o n t h a t was t o l a s t f o r twenty y e a r s . Some t i m e around t h e middle o f 1953 I a t t e n d e d t h e f i r s t m e e t i n g e v e r d e d i c a t e d t o gas chromatography. T h i s m e e t i n g w a s a g a i n o r g a n i z e d by t h e S o c i e t y for A n a l y t i c a l Chemistry and took p l a c e i n Ardeer i n S c o t l a n d . I t s t a n d s o u t c l e a r l y i n my mind l a r g e l y b e c a u s e of t h e

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F i g . 1 . 3 . A more advanced apparatus made by t h e Applied P h y s i c s D i v i s i o n i n Thornton. I n s p i t e of t h e f a c t t h a t t h e y o u t h f u l author i s shown w i t h s y r i n g e a t t h e ready, i t was never used a t Thornton, b e i n g made s p e c i f i c a l l y for x y l e n e a n a l y s i s a t t h e adjacent r e f i n e r y .

6 appearance of a n o t h e r keen young man by t h e name of R.P.W. S c o t t , who i n s i s t e d on t e l l i n g everyone about h i s wonderful new f l a m e thermocouple d e t e c t o r . 1956 marked t h e F i r s t I n t e r n a t i o n a l Symposium on Vapour Phase Chromatography ( s i c ) o r g a n i z e d under t h e a u s p i c e s o f t h e Hydrocarbon Research Group o f t h e I n s t i t u t e of P e t r o l e u m . Out of t h i s meeting a r o s e t h e G a s Chromatography D i s c u s s i o n Group and a s e r i e s o f I n t e r n a t i o n a l Symposia now a p p r o a c h i n g t h i r t e e n i n number. I t a l s o exp l a i n s why t h e G a s Chromatography D i s c u s s i o n Group was a s s o c i a t e d w i t h t h e I n s t i t u t e o f P e t r o l e u m f o r many y e a r s . By t h e l a t e ~ O ' S , t h a n k s t o t h e work o f A . J . Davies and h i s c o l l e a g u e s o f t h e Applied P h y s i c s D i v i s i o n s a t T h o r n t o n , w e were equipped w i t h a chromatograph c o n s i d e r a b l y s u p e r i o r i n performance t o any commercially a v a i l a b l e f o r a number o f y e a r s t o come. The column oven had f o r c e d a i r c i r c u l a t i o n and w a s c a p a b l e of r i s i n g i n t e m p e r a t u r e by 20 OC/min. The t e m p e r a t u r e c o n t r o l was 2 0 . 5 O C a t 100 O C and 2 1 O C a t 300 O C . S i n c e t h e oven was i n t e n d e d f o r Utube columns and w a s o v e r t h r e e f e e t i n h e i g h t , t h e s e performance f i g u r e s s p e a k volumes f o r i t s d e s i g n and c o n s t r u c t i o n . The t h e r m a l c o n d u c t i v i t y c e l l d e t e c t o r w a s o p e r a b l e up t o 300 OC and was l e a k f r e e i n t h e high-vacuum sense o f t h e word. The p a p e r d e s c r i b i n g t h i s a p p a r a t u s ( I ) shows a v e r y e a r l y example o f t e m p e r a t u r e programming. The y e a r 1958 w a s n o t a b l e f o r t h e f i r s t p a p e r s on c a p i l l a r y columns which w e t r i e d t o make from c o p p e r t u b i n g , w i t h s i n g u l a r l y poor r e s u l t s : n o t u n t i l w e f o l l o w e d S c o t t i n t h e u s e o f nylon t u b i n g d i d w e a c h i e v e s u c c e s s . Although nylon h a s obvious l i m i t a t i o n s a s a column m a t e r i a l , i t i s worth p o i n t i n g o u t i n t h e s e days when g l a s s c a p i l l a r i e s a r e back i n f a s h i o n t h a t w e w e r e a b l e r e a d i l y t o a c h i e v e e f f i c i e n c i e s of o v e r 4000 p l a t e s p e r metre w i t h h e p t a n e / d i n o n y l p h t h a l a t e on nylon columns. 1958 was a l s o n o t a b l e f o r m e i n t h a t I f i r s t m e t P r o f e s s o r J . E . Lovelock when h e gave an a c c o u n t o f t h e argon d e t e c t o r a t a meeting of t h e Gas Chromatography D i s c u s s i o n Group a t Cambridge. L a t e r i n my c a r e e r I was t o h a v e t h e p r i v i l e g e of working i n c o n j u n c t i o n w i t h J i m Lovelock on a p p l i c a t i o n s o f t h e e l e c t r o n c a p t u r e d e t e c t o r . The e a r l y 6 0 ' s saw t h e e s t a b l i s h m e n t of t h e flame i o n i z a t i o n d e t e c t o r a s p e r h a p s t h e most i m p o r t a n t weapon i n t h e hydrocarbon a n a l y s t ' s armoury. I t s h i g h s e n s i t i v i t y e n a b l e d sample s i z e s t o b e reduced by two o r d e r s o f magnitude and t h i s i n t u r n l e d t o s m a l l e r d i a m e t e r , more l i g h t l y l o a d e d columns and t h e a b i l i t y t o t a c k l e h i g h e r and h i g h e r - b o i l i n g m i x t u r e s . T h i s p e r i o d s a w us o c c u p i e d a t Thornton i n s e v e r a l d i s t i n c t d i r e c t i o n s - on t h e one hand w e were p u s h i n g on w i t h t h e a p p l i c a t i o n o f GC t o more and more i n t r a c t a b l e m i x t u r e s , and on t h e o t h e r B i l l Whitham, Abid Khan and myself were busy u s i n g GC t o i n v e s t i g a t e s o l u t i o n phenomena ( 2 ) . Low-boiling complex hydrocarbon m i x t u r e s s u c h a s p e t r o l e u m s p i r i t s were, by now, r e a d i l y r e s o l v e d w i t h c a p i l l a r y columns, b u t i t must be remembered t h a t t h i s w a s t h e e r a i n which i d e n t i f i c a t i o n o f GC e f f l u e n t s by mass s p e c t r o m e t r y was i n i t s i n f a n c y and d a t a h a n d l i n g equipment c o n s i s t e d of e i t h e r a r u l e r o r an a n a l y t i c a l b a l a n c e t o weigh t h e c u t - o u t peaks! Under t h e s e c o n d i t i o n s h i g h - r e s o l u t i o n columns w e r e

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Fig. 1.4. Equipment from the early 19708, f o r oil spill identification, fitted with an FID and a flame photometric detector. Although somewhat neater and more compact than the apparatus shown in Fig. 1, there is a distinct family resemblance in the mass of trailing cable and pipework.

o f t e n a p o s i t i v e embarrassment, and t h i s may b e one o f t h e r e a s o n s why c a p i l l a r y columns took so l o n g t o come i n t o common u s e . I n a d d i t i o n t o t h e work a t Thornton I had t h e o p p o r t u n i t y , r a r e f o r a petroleum c h e m i s t , t o c o l l a b o r a t e w i t h t h e Research Department of A n a e s t h e t i c s o f t h e Royal C o l l e g e o f Surgeons o f England. The o r i g i n a l o b j e c t i v e s l a i d down f o r t h i s work w e r e t h a t w e s h o u l d produce a r a p i d 3 c h e a p and s i m p l e a p p a r a t u s f o r t h e a n a l y s i s of r e s p i r e d g a s e s from p a t i e n t s undergoing s u r g e r y . W e w e r e , a l a s , n e v e r a b l e t o meet t h e l a s t two o b j e c t i v e s b u t w e d i d manage t o s e p a r a t e oxygen, carbon d i o x i d e and n i t r o u s o x i d e i n one m i n u t e , a c o n s i d e r a b l e achievement a t t h a t t i m e ( 3 ) . A p e r i o d o f c o n s o l i d a t i o n was f o l l o w e d by s e v e r a l y e a r s o f f r u i t f u l c o l l a b o r a t i o n w i t h J i m Lovelock. The outcome o f t h i s work was a j o i n t p a p e r w i t h A . J . Davies and A l b e r t Evans on t h e u s e o f m i x t u r e s o f compounds w i t h h i g h e l e c t r o n a f f i n i t i e s a s t r a c e r s i n a manner analogous t o t h e u s e o f r a d i o a c t i v e t r a c e r s ( 4 ) . I do n o t know i f t h e t i t l e of t h i s p a p e r , "An a p p a r a t u s f o r t h e d e t e c t i o n of i n t e r f a c e s between p r o d u c t s i n p i p e l i n e s " , m i s l e d p e o p l e o r whether t h e f a c t t h a t i t was p u b l i s h e d i n a book w i t h a s m a l l c i r c u l a t i o n meant t h a t very few p e o p l e e v e r knew o f i t . Whatever t h e r e a s o n , t h i s p a p e r , which I r e g a r d as p e r h a p s t h e b e s t I have e v e r p u b l i s h e d , rec e i v e d n o t one r e q u e s t f o r a r e p r i n t w h i l s t less worthy p a p e r s o f mine, b e f o r e and a f t e r t h i s , seem t o be i n c o n s i d e r a b l e demand. I n 1968-1969 I s p e n t a v e r y happy s a b b a t i c a l y e a r a t S h e l l Development Company's l a b o r a t o r y a t E m e r y v i l l e , C a l i f o r n i a . During my s t a y t h e r e I t r i e d t o make a d i r e c t measurement o f t h e c h a r g e c a r r i e d by a s t r o n g l y e l e c t r o n - c a p t u r i n g s p e c i e s , s i n c e under t h e r i g h t c o n d i t i o n s t h e e l e c t r o n c a p t u r e d e t e c t o r c a n behave coulomet r i c a l l y , i . e . one mole o f a compound s h o u l d c a r r y one f a r a d a y . T h i s work was n o t s u c c e s s f u l b u t I was a b l e , t o my own s a t i s f a c t i o n , t o d e m o n s t r a t e t h e phenomenon o f h y p e r c o u l o m e t r i c r e s p o n s e , l a t e r desc r i b e d by o t h e r w o r k e r s , and t o show t h a t some compounds, C C l 4 i n p a r t i c u l a r , c o u l d account f o r two e l e c t r o n s p e r m o l e c u l e . Although t h e work d i d n o t r e a c h a p u b l i s h a b l e s t a t e , J i m Lovelock g e n e r o u s l y i n c l u d e d my name a s a c o - a u t h o r i n a l a t e r p a p e r on t h e t o p i c ( 5 ) . E a r l i e r I mentioned GC/MS. W e a c q u i r e d a GC/MS i n 1963 and f o u g h t i t f o r o v e r t e n y e a r s b e f o r e we f i n a l l y p e n s i o n e d i t o f f a y e a r o r two ago. L e s t anyone m i s u n d e r s t a n d m e a t t h i s p o i n t l e t m e s t a t e a t once t h a t I t h i n k t h e p r e s e n t g e n e r a t i o n GC/MS i n s t r u m e n t s i n c o n j u n c t i o n w i t h a computer r e p r e s e n t t h e most powerful combinat i o n a v a i l a b l e , e s p e c i a l l y f o r t h e a n a l y s i s o f complex m i x t u r e s o f unknown compounds; b u t W/MS w i t h o u t a computer i s l i k e H a m l e t without the Prince. The 7 0 ' s h a v e s e e n chromatography a p p l i e d i n c r e a s i n g l y t o e n v i ronmental p r o b l e m s , a n d , f o l l o w i n g t h e f a s h i o n , w e have i n v e s t i g a t e d the use of s e l e c t i v e d e t e c t o r s f o r t h e i d e n t i f i c a t i o n of o i l s p i l l s (6) and t h e i n v e s t i g a t i o n o f o t h e r c o n s e r v a t i o n problems. 1972 marked t h e l a s t i n t e r n a t i o n a l symposium o r g a n i z e d by t h e Gas Chromatography D i s c u s s i o n Group ( o f which I was t h e n Chairman) i n c o n j u n c t i o n w i t h t h e I n s t i t u t e o f P e t r o l e u m , h e l d i n Montreux, S w i t z e r l a n d . I n 1973, s t i l l d u r i n g my term of o f f i c e a s Chairman,

9 t h e Group d e c i d e d w i t h some r e l u c t a n c e t o s e v e r i t s a s s o c i a t i o n w i t h t h e I n s t i t u t e o f Petroleum and become a completely independent body. I t is p l e a s i n g t o r e p o r t t h a t t h e Group (now t h e Chromatography D i s c u s s i o n Group) has s u r v i v e d t h e change and has m a i n t a i n e d i t s world-wide membership a t around 700 p e o p l e . Coming up t o t h e p r e s e n t , w e have now a r r i v e d a t t h e day of t h e f u l l y a u t o m a t i c , c o m p u t e r - c o n t r o l l e d GC which can t u r n o u t a dozen 250 peak g a s o l i n e chromatograms e v e r y 24 h o u r s - p r o v i d e d , of c o u r s e , t h a t someone remembers t o check t h e c a r r i e r gas s u p p l y , t o p up t h e l i q u i d n i t r o g e n c o o l a n t , change t h e c a s s e t t e o f magnetic t a p e and e n s u r e t h a t t h e r e is s u f f i c i e n t c h a r t p a p e r . When a l l t h i s h a s b e e n done i t i s , p e r h a p s , c o m f o r t i n g t h a t t h e r e c o r d e r pen s t i l l r u n s d r y a t a c r u c i a l p o i n t j u s t a s i t u s e d t o do i n t h e h e r o i c d a y s : pzus ga

change, pZus c ' e s t Za m&me chose. A f t e r 25 y e a r s of i n t e n s i v e development one might b e excused f o r t h i n k i n g t h a t l i t t l e remains t o be a c h i e v e d . I n e v i t a b l y " d i s c o v e r i e s " a r e now c r o p p i n g up f o r t h e second o r t h i r d t i m e round, and when I a t t e n d meetings I s i t w i t h e q u a l l y e l d e r l y f r i e n d s and a c q u a i n t a n c e s who m u t t e r "David X d i d t h a t 1 5 y e a r s ago", o r "We've been d o i n g t h a t f o r y e a r s b u t n e v e r t h o u g h t i t worth p u b l i s h i n g " . I n s p i t e o f t h i s , new developments a r e s t i l l t a k i n g p l a c e a s w i t n e s s e d by t h e r a p i d growth of High Performance L i q u i d Chromatography (HPLC) i n t h e l a s t f i v e y e a r s and t h e r e v i v a l of i n t e r e s t i n g l a s s c a p i l l a r y columns. HPLC s t i l l a w a i t s t h e development o f a u n i v e r s a l d e t e c t o r of h i g h s e n s i t i v i t y , and t h e new c o a t i n g t e c h n i q u e s f o r g l a s s c a p i l l a r i e s s h o u l d encourage someone t o take up D e s t y ' s e l e g a n t work i n which he used very narrow b o r e t u b i n g t o o b t a i n r a p i d s e p a r a t i o n o r u l t r a h i g h r e s o l u t i o n (7) I t may s u r p r i s e t h e u n i n i t i a t e d t o c o n f e s s t h a t even a f t e r 25 y e a r s t h e s i g h t o f a r e c o r d e r pen w h i z z i n g up and down a s i t f o l l o w s t h e c a p i l l a r y column s e p a r a t i o n o f a complex m i x t u r e s t i l l g i v e s g r e a t s a t i s f a c t i o n . To my Company and t o my chosen d i s c i p l i n e I owe t h e f a c t t h a t I have journeyed from Samarkand t o San F r a n c i s c o and made many f r i e n d s i n d i f f e r e n t p a r t s o f t h e w o r l d . L i m i t a t i o n s o f s p a c e (and a f a i l i n g memory) have p r e v e n t e d m e from naming more t h a n a f e w . To them, t o t h e unnamed m a j o r i t y and t o A . J . P . M a r t i n I s h o u l d l i k e t o t a k e t h e o p p o r t u n i t y t o s a y "thank you" and t o conclude by a s k i n g L e s l i e E t t r e and A 1 Z l a t k i s t o c o n s i d e r me for i n c l u s i o n i n t h e second e d i t i o n of t h i s book i n a n o t h e r 25 y e a r s t i m e .

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REFERENCES 1 K . Ashbury, A . J . Davies and J.W. D r i n k w a t e r , A n a l . Chem. 29 (1957) 918. 2 E . R . Adlard, M . A . Khan and B . T . Whitham, i n Gas Chromatography 1962 (Hamburg Symposium), M. van Swaay, e d . , B u t t e r w o r t h s , London, 1962, pp. 84-101. 3 E . R . Adlard and D . W . H i l l , Nature 186 (4730) (1960) 1045.

4

5

6 7

E . R . Adlard, A . J . Davies and A.Evans, i n GQS Chromatography 1968 (Copenhagen Symposiwn), C.L.A. Harbourn, e d . , I n s t . of Petroleum, London, 1969, pp. 170-184. J.E. Lovelock, R.J. Maggs and E.R. Adlard, A n d . Chem. 43 (1971) 1962. E.R. Adlard, L.F. Creaser and P.H.D. Matthews, AnaZ. Chem. 44 (1972) 6 4 . D.H. Desty, A . Goldup and W.T. Swanton, i n Gus Chromatography (1961 Lansing sympos~um),N . Brenner, J . E . Callen and M . D . Weiss, e d s . , Academic P r e s s , New York, 1962, pp. 105-135.

11

HENDRIK BOER

HENDRIK BOER was born in 1921, in Amsterdam, The Netherlands. He studied at the Fdunicipal University of Amsterdam where he received his doctorate (cum laude) in 1949. In the same year, he joined the Koninklijke/Shell Laboratorium, in Amsterdam. Presently, he is a senior research chemist at this laboratory. In 1956 and 1957 he worked for Shell in the United Kingdom Atomic Energy Authority Wantage Radiation Laboratory, in England. Dr. Boer is the author and coauthor of some 30 scientific papers in the field of ozonolysis, ozonometry, selective hydrogenation, gas chromatography and instrumentation. He developed a stable electrolytic generator for concentrated ozone, which enabled both the study of ozone reaction kinetics and the titration of olefinic unsaturation. Ozonolysis, selective hydrogenation by calcium hexammine - for which a novel method was developed whereby the use of liquid ammonia was obviated - and chromatography were applied to oil constitutional analysis. Via some aspects of radiation chemistry, he entered the field of oil process research. His work in this area quite soon shifted towards instrumentation, automation, data handling and the resolution of special analytical problems. Dr. Boer's involvement in chromatography started in 1952; it involved the development of special instruments and novel techniques, such as e.g. multidimensional gas chromatography.

12 Although my first experience with chromatography dates from 1950, to me the real "flash" came in the fall of 1952. Being involved in oil constitution research, I was working on an improved method for determining the average alkyl substitution pattern of monoaromatic fractions. The method I had in mind comprised ozonolytic and oxidative fission of the aromatic bonds, by which the aromatic carbon would be converted into a carboxyl group, whilst the alkyl substituent should remain intact. From an analysis of the resulting mixture of lower fatty acids, the percentages of the various alkyl substituents had to follow. Mass spectrometric analysis could provide these data, but with a rather unsatisfactory accuracy. At that very moment by boss - K. van Nes, a well known expert in classical oil constitution analysis - returned from a visit to Oxford and informed me of the historical lecture by A.T. James and A.J.P. Martin that started the gas chromatographic era. The detailed information that this procedure could provide in the analysis of the lower fatty acids looked like a godsend. So I happily decided to try and duplicate their results. Viewed in retrospect, the fact that I so whole-heartedly entered the GC field, cannot be ascribed just to the feeling of satisfaction from having been presented with the solution to a difficult analytical problem. It is perhaps more appropriate to refer to the happening around my silver jubilee at our laboratory, in 1973, when my colleagues awarded me an honorary engineer's degree in "chemical gadgeteering", albeit from the Technical University of "Obscurodam". .. Indeed, immediately after having made myself familiar with the new technique, via a copy of the apparatus described by James and Martin, the gadgeteering field was thrown wide open, and I soon had built a dual setup of different layout, with different detection systems (including coulometric titration and electrolytic conductivity), a modified column packing, and so on and so forth. In due course the ozonolysis/GC technique was worked out successfully, whereafter the results were safely buried in the Proceedings of the World Petroleum Congress 1955 ( 2 ) . . . Since present day capillary column analysis allows resolution of components upto well within the Cll aromatics, the method is obsolete anyhow, and thus it may rest there in peace. The same holds for the chemically quite interesting analysis of benzothiophenes and diphenylalkanes ( 2 , 3 ) , for which contemporary chromatographic techniques also seem indicated. By the end of 1952 two more groups at our laboratory had entered the field of practical GC: A.I.M. Keulemans and A . Kwantes for liquid hydrocarbon analysis, and F. van der Craats and G.W.A. Rijnders for gas analysis; while J.J. van Deemter soon provided a theoretical basis by means of his well known equation. At first unaware of one anothers activities, we soon started a fruitful exchange of ideas. For me this meant a gradual shift of interest from functional compounds such as acids and ketones, to hydrocarbons. Soon I was working with a borrowed katharometer, leak-proofed by means of cellon sealant, and carefully heated on a hot plate controlled by a "Simmerstat". Then later, being after all an organic chemist, I started synthesizing polar liquid phases, which led to a short evaluation of p-nitroaniline picrate, amongst others. I still remember the disbelief of some

13

Ffg.2.1. Dual, vapourJacketed gas chromatographs with electrolytic conductivity detection (1953). For details see ( I ) .

reputed overseas confreres when in a discussion I referred to the quite abnormal elution sequence of the C8-Cg aromatics on such a highly polar liquid phase. And when, on another liquid phase, an ester of dinitrodiphenic acid, I managed to analyse high boilers such as octahydrophenanthrenes and anthracene, a member of the managing board came down in person to watch the marvel, and then solemnly declared that the technique now surely had come of age. Apparently he did not in the least mind the smell of the nitroester 0 being stripped off at the rather high column temperature of 255 C. Those were the days ... Although by that time our instrument shop could make remarkably good katharometers, there was clearly room for other detection tech-

14

Fig. 2 . 2 . Exploratory stages of the 8-ray ionisation detector (1954). For the final concept see ( 4 ) . niques. Martin's gas density balance (GDB) being considered as a somewhat mystic gadget, I turned my attention to the "8-ray ionization cell", which had been patented by our Emeryville colleagues as a device for gas analysis. This instrument was now modified for use in GC detection by both Emeryville and Amsterdam groups, independently. During its development Archer Martin paid a visit to our laboratory. It was a great experience to observe how he immediately got carried away by the sight of the various bits and pieces as well as a few chromatograms, completely forgetting the member of the board who accompanied the honoured Nobel laureate on his guided tour. Later, in the fall of 1955, I paid a return visit to Martin's Mill Hill laboratory where I was introduced to a young physicist, Jim Lovelock; there and then in a most gentlemanlike manner Archer asked me if I had ever considered means other than @-rays for the ionization of organic molecules, such as excited argon atoms. I answered, truthfully, that being an organic chemist, such odd things had never occurred to me; in my opinion this answer cleared the way for the development of Lovelock's admirable family of super-sensitive ionization detectors. It is perhaps not widely known that Martin himself is a gifted gadgeteer. This is probably best exemplified by the fact that he constructed the first GDB himself - quite skillful job - and that the first instrument firm that attempted to start series production, was faced with considerable constructional problems. I recall how Martin vented his annoyance by stamping his foot and uttering a few pet names for those "incompetent professionals". In my notes on that 1955 visit to England I also find the following reference regarding a visit to Professor Norrish's laboratory at Cambridge: "I met a bright young chap, named Howard Purnell; since

15 Prof. Norrish thinks that VPC is a by-path only, Purnell has to work with rather primitive VPC-equipment and is allowed only marginal time to refine it". From the fact that in 1977 Purnell received the Tswett Chromatography Medal, I have to conclude that the "chap" has since overcome these difficulties. At the memorable 1956 London Symposium my version of the @-ray ionization detector - nowadays referred to as cross-section detector was disclosed ( 4 ) . One month earlier, at the National American Chemical Society meeting, in Dallas, Texas, my Emeryville colleagues had presented their - quite differently designed - baby for baptism (5). I recall a funny incident when I had to show to the Customs officer the model that I had brought for demonstration purposes. Apparently, his major concern was how to classify the instrument. Finally, after much discussion, to his - and my - relief he formulated its purpose as corresponding with that of the violin of a violin player . . . So, at the symposium, I had to try playing first fiddle: Returned home from the symposium, we had some discussions - with Lou Keulemans and others - on the possibility of studying reaction kinetics by means of a catalyst-filled GC column. I disagreed with the idea and argued that an independently heated, small catalyst bed, followed by a normal GC column, would be more appropriate. In order to demonstrate my point, I made a glass microreactor - glassfilled it with Pt/A1203 blowing being part of my gadgeteer's outfit catalyst, connected it at the column inlet, and in no time produced a substantial amount of data on hydrogenation and dehydrogenation reactions. Since I had to leave in a few weeks time for a prolonged stay at Wantage Radiation Laboratory (WRL) of the United Kingdom Atomic Energy Authority, I passed the promising tool on to Keulemans, who later published a paper with €IVoge . (6) on the novel technique. I learned from that paper that P. Emmett had conceived a similar idea.. . Wantage Radiation Laboratory was devoid of gas chromatographs. Consequently, I set one up - comprising, of course, a cross-section detector - for my own use. It was regarded with some distrust, although I repeatedly emphasized its analytical power. In addition I demonstrated that in combination with a pre-column reactor filled with a hydrogenation catalyst, a gas chromatograph was a powerful instrument for assessing the carbon skeleton configuration of functional compounds. A few years later, following the publications of M. Beroza, this technique became widely known as "reaction chromatography". Eventually, one of my colleagues at WRL challenged me to verify my bold claims and handed me a weighed-in mixture of two methoxy-toluenes. Half an hour later he had to admit that my results were correct to within 0.2 %. From that day on WRL considered GC a must. I returned to Amsterdam in 1958 and soon after got involved in oil process research. By that time there was a growing awareness of the need for efficiency in this time and manpower-consuming branch of research. This pertained to improved instrumentation and automation in general, and t o analysis in particular. For a gadgeteer this was a most gratifying challenge, and I am still most grateful to the

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16 management of the time in that they cleared the way for me. The time I could devote to chromatography since then has been concerned with the following main topics: preparative-scale GC, multidimensional GC, process GC, capillary GC, and data processing. In prep GC I intentionally kept away from the large-diameter column approach recommended by my colleagues Huyten and Rijnders, and instead conceived a rigidly automated, repetitive instrument, using relatively narrow, efficient columns ( 7 , 8 ) . We did some fine work with it, using it for both oil constitution purposes and in the preparation of highly pure reference compounds. Keulemans - who at that time had already left for Eindhoven Technical University - was so impressed that, with his usual enthusiasm and charm, he widely advocated the instrument as the only sensible way of doing high-performance preparative work. He followed his own advice by asking - and getting - permission to install a dual unit of improved design at his Institute in Eindhoven. Then came the Rome 1966 Symposium. Upon my arrival there Ray Scott rushed to me, exclaiming: "Henk, that's a lovely piece of apparatus of yours! Wish I could afford to buy it!" I must have looked rather sheepish, for I had no idea what he was talking about. The next day the Philips stand provided the answer: there stood my pet-prep GC, thoroughly acknowledged on an accompanying, king-size wall board. It was a nice surprise, it was a fair sight, but. . . there was a little problem: the improvements I had made - that had never been published were there!' My poor friend Ron Evans - at that time manager of Philips Chromatography - was terribly embarrassed and swore that this was mere coincidence ... However it may be, his apologies on behalf of Philips were accepted and the incident was closed. Unfortunately - for in my opinion this was certainly the most versatile and sophisticated prep GC commercially available at that time - the terms of the soon following merger betwecn Philips and Pye left no room for this instrument to be marketed. My work with prep GC had led to my first qualitative experiments in the field of multidimensional GC (MDGC). The real breakthrough in oil analysis came, however, when the collection and re-injection of hydrocarbon fractions was made quantitative and automatic on an analytical scale ( 9 ) . This was gadgeteering at its best! The most sophisticated, 4-column instrument we designed in those days, was run fully automatically from an 87-step, 24-function, decision-taking programmer. Amongst other things it could perform the analysis of a heavy naphtha for n-paraffins, isoparaffins, 5-ring naphthenes, 6-ring naphthenes, and aromatics, for every carbon number. By adding an off-line capillary column as another dimension, dazzling possibilities for quantitative component analysis were realized and briefly explored. To my knowledge, the separating power of this set-up has never been equal led. I have to admit that the above analytical tool was not exactly a routine instrument that could be given into the hands of refinery operators. Bringing the important PONA-by-GC analysis into the realm of the routine laboratory had to await the discovery by J.V. Brunnock and L.A. Luke of the fancy paraffin/naphthene separation on 13X molecular sieves. The reaction of Dennis Desty to my hint, during a

17 private chat, that I was able to analyse naphtha to the detail described in the previous paragraph - being a gentleman he immediately stopped me and asked me not to tell him any details - has later made me speculate that this very chat triggered the publication of Brunnock and Luke's results. However it may be, their disclosure enabled us to conceive a relatively simple, automatic, MDGC naphtha analyser later marketed as the Packard-Becker type 411 - which since then has found worldwide acceptance for naphtha specification analysis. It is only one example indicating that MDGC is going to stay. In my Montreux paper ( 9 ) I indicated the feasibility of employing LC as a heading dimension for MDGC. With the higher boiling hydrocarbon fractions, in particular, where type-selectivity with LC is usually better than with GC, this technique has allowed us to cope with a number of rather demanding analytical problems.

Fig. 2.3. Glass capillary column assembly, with capillary dipper injector and a very simple, homemade flame-ionisation detector (1959).

As far as on-line process analysis by GC is concerned, this is certainly a most gratifying field for the gadgeteer. I still find great fun in bargaining with the process researchers on the minimum amount of analytical information they really want, and then to conceive an analyser that will fit into the set-up as a whole. The first chromatogram from any novel analyser is to me still a special sensation. A considerable number of automatic "special purpose" GC analysers have found their way into the process research laboratory, and the demand still does not lessen. The fascinating possibilities offered by both miniaturization and the application of microprocessors in this field would merit a separate paper.

18 In capillary GC - apart from the fascinating applications - the gadget that has impressed me most is the Desty glass-drawing machine. For a couple of years my own version has greatly contributed to the success of the regular open days at our laboratory: the ladies especially, were obviously delighted to receive a wobbly section of fragile glass spiral. These spirals were invariably placed very careI don't dare to think what remained when fully in their handbags they arrived back home. .. At the Rome 1966 Symposium I had the pleasure of starting a discussion on peak integration. In the introduction I said: "In the early days of GC the separation process itself was so fascinating that the real hobbyist spent hours and hours watching the recorder pen moving across the paper. If one was interested at all in quantitative results, reading the counter of an Electromethods low-inertia motor in the meantime was no imposition. Even cutting out the peaks with a pair of scissors was practiced, this physical contact with the beautiful shapes we had produced being a gratifying excuse for this rather childish occupation". How times have changed! From a pair of scissors to an on-line computer in 25 years. .. I would like to mention two of the many integration techniques I have had pleasure to play with. The first one, the "peak area to peak height converter" ( 1 0 ) demonstrates how charmingly simply instrumented a simple concept can be. Before the advent of the GC computer, this device was quite useful in providing remarkably accurate data handling and reduction with process-type chromatographs. The second one, an electromechanicallyautomated version of the analog computer circuit originally suggested by C.L.A. Harbourn at the 1959 Informal Symposium in Bristol, demonstrates how impressively involved another simple concept has been made ( 1 1 ) . This fine piece of workmanship was accommodated in a

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Fig. 2 . 4 . Peak area/peak height converter, yielding response-corrected peak areas for up t o 12 components (1963).

19

Fig. 2.5. Prototype of a calculating integrator (electromechanical part) for up to 15 components (1961). Perspex, dust-protective coffin and aptly nicknamed '%now White". Together with a solid amount of electronics, it would provide response-corrected, normalized percentages for up to 15 components. Is she not a beauty? Could computers ever radiate such appeal? Contemplating that less than 20 years ago this concept had been considered a fairly realistic approach to GC data handling, one should be duly impressed by the rapid evolution of this technique. For the time being my story ends here. According to my wife I am a hobbyist who is being paid for having fun. In a way she is right. I fully agree with honouring Tswett for opening the field of chromatography, but as far as the gadgeteer's fun is concerned, I feel more particularly thankful to Martin and James, further to my many friends and fellow-hobbyists all over the world, and last but not least to my co-workers, whose names have appeared in my various pub1ications. REFERENCES 1 H. Boer, Proc. 4th World Petroleum Congress, Section V/A, Paper 1. 2 H. Boer, J . I n s t . Petrol. 46 (1960) 234. 3 H. Boer and P.M. Duinker, J . I n s t . Petrol. 47 (1961) 314, 4 H. Boer, in Vapour Phase Chromatography ( 1 9 5 6 London Symposium), D.H. Desty, ed., Butterworths, London, 1956, pp.169-184. 5 C.H. Deal, J.W. Otvos, V. N. Smith and P.S. Zucco, 129th National Am. Chem. Soc. Meeting, Dallas, Tex., A p r i l 9-13, 1956; Anal. Chem. 28 (1956) 1958. 6 A.I.M. Keulemans and H.H. Voge, 1 3 3 r d Nat. Am. Chem. Soc. Meeting, Sun Francisco, Calif., A p r i l 1958; J . Phys. Chem. 6 3 (1959) 476. 7 H. Boer, J . Scient. I n s t r . 41 (1964) 365. 8 H. Boer, J . A p p l . Chem. 14 (1964) 275. 9 H. Boer, in Gas Chromatography 1972 (Montrem Symposiwn), S.G. Perry, ed., Applied Science Publishers, Barking, 1973, pp.109-132. 10 H. Boer, Chromatographia 2 (1969) 118. 11 H. Boer, A.Schuringa and K. Kampman, B r i t . Pat. 974 964.

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21

ERIKA CREMER

ERIKA CREMER was born in 1900, in Munich, Germany, into a family of scientists: her great-grandfather, grandfather and father were university professors and so are her two brothers. Thus, it is natural that she also chose a scientific career. She studied at the University of Berlin and received her Ph.D. in 1927, under Bodenstein. In the following 12 years, she was associated with the Kaiser-Wilhelm-Institut in Berlin, the Universities of Freiburg, Munich and Kiel, and the Physikalisch-Technische Reichsanstalt, working with Bonhoeffer, v. Hevesy, Polanyi, Fajans and Otto Hahn. She received her habilitation in 1938 from the University of Berlin and in 1940, was appointed a Dozent at the University of Innsbruck with the assumption that at the end of the War, "when the men return", she would have to give up her position. However, fate decided differently In 1945, Dr. Cremer was appointed as the head of the Institute of Physical Chemistry at Innsbruck University, assuming full professorship in 1951. At present, she is professor emeritus of the University. Dr. Cremer is the author of a large number of papers on various physico-chemical questions and chromatography. In 1959, she translated Keulemans' book on Gas Chromatography into German, adding to it a supplement on gas-adsorption chromatography. She received an honorary doctorate from the Technical University Berlin and is a corresponding member of the Austrian Academy of Sciences. Dr. Cremer is the recipient of the Wilhelm Exner Medal, the Prechtl Medal of the Technical University of Vienna, the Erwin Schrodinger Prize of the Austrian Academy of Science, and the M.S. Tswett Chromatography Medal. In 1978, the President of the Austrian Republic awarded her the first class cross of the Austrian Order for Science and Art. Dr. Cremer's activities were related to a number of basic investigations in physical chemistry, catalysis and adsorption. Her work in gas adsorption chromatography began in 1944 and continued in the years after the end of the Second World War, under very difficult conditions. Dr. Cremer also pioneered in the investigation and development of selective detectors.

....

22 It was in a lecture by G. Hesse in Munich, about 1935, that I heard of chromatography for the first time and saw the first "rings" traveling through a column. At that time I was interested in adsorption in connection with reaction kinetics and catalysis. I had studied the heterogenous conversion of 0- to p-hydrogen on solid oxygen. The kinetics could be understood if one assumed that the reaction took place in an adsorption layer. Using the Langmuir isotherm it was possible to calculate the difference in the heats of adsorption of both spin modifications. A little later (1938) I tried to combine the two most used adsorption isotherms, called after Langmuir and Freundlich. The latter was explained as a resulting isotherm of superimposed Langmuir isotherms, due to adsorption centers whose frequency exponentially diminish with the energy. Experimental results were explained by assuming a frozen thermal equilibrium. In the years between 1935 and 1940 many experiments were started on the use of adsorption for the separation of gases. Peters separated the rare gases by adsorption and pumping off at reduced pressure and different temperatures. This was followed by a variety of adsorption and desorption methods developed and utilized in Germany mainly by Eucken's school in Gottingen. This work was summarized in several treatises by Wicke ( I ) . Concerning gas chromatography, however, most physical chemists of that time shared the sceptical opinion that the use of the chromatographic method with a gas as the means of elution seems to be almost without any prospect owing to the mixing in the direction of flow. In the first years of the forties several remarkable achievements were reported in chromatography: Hesse and coworkers used a carrier gas combined with separation by distillation and adsorption while Martin and Synge developed a new kind of chromatographic technique, liquid-liquid-partition chromatography (LLC). From that time (1941) on, a sharp difference was made between adsorption and partition chromatography; a sort of rivalry began between the two methods where sometimes the one, sometimes the other was more successful. These events took place during World War I1 when scientific communication through boundaries was very difficult if not impossible and the different laboratories in which chromatography was developed knew nothing of each other. This was particularly true of the activities of Damkohler who also came from Eucken's school and carried out (together with Theile) experiments which may be termed as a variation of Tswett's method. They did not work in a university laboratory but in an industrial institute dealing with engine research. They separated methanol and ethanol as well as cyclohexane and benzene on an adsorbent, using a carrier gas; the breakthrough was signalled by a detector. They also tried "gas-liquid partition chromatography'' by loading the adsorbent with glycerol. The experiments were finished in 1942; however, a short communication was published only in 1943 and a detailed one in 1944 ( 2 ) . These papers remained (even for us) unknown at that time as they were - due to the political conditions - not reported in reference journals. The work of Damkohler and Theile, although chromatography in principle, was not carried out from the point of chemical analysis

23 and particularly not as microanalysis. The detector served only to "indicate the breakthrough of a component", i.e., to indicate when a separation was finished. The concentration profiles were very broad and irregular, caused by diffusion, condensation and column overloading and thus, an analytical evaluation was not possible. As expressed by Neufeld ( 3 ) , "at these preparative dimensions the efficiency of the method in the milligram region had to remain hidden". Coming back to my own activities, in 1941 I was involved in kinetic measurements on the hydrogenation of acetylene. In this connection the need for a quick and exact method of analysis of the two hydrocarbons, acetylene and ethylene, arose and as we were also occupied with investigations on the adsorption processes, it was evident to try separation by adsorption. Therefore, A. Kunte, one of the students at the University of Innsbruck, measured the adsorption data for these two substances, taking charcoal as the adsorbent. The introduction of the final written form of his thesis ( 4 ) - which was started about 1942 and finished in 1944 - emphasized the special aim of the investigations: to find a method for the analysis of acetylene and ethylene, based on the difference of their adsorption strength. This aim, however could not be reached and no marked difference was found in the adsorption heats on charcoal; at least it was too small to give any hope of obtaining a rapid separation by the methods known at that time. A special impulse to try it with chromatography most certainly came from the book by Hesse published in 1943 ( 5 ) : the "chromatographic stepladder" seemed to be very attractive for the investigation. I expected that it would be possible to measure the height of steps quantitatively and to bring this value into connection with the adsorption heat, especially in the gas phase where the solvent does not cause any disturbance. The simple picture of vessels, floating on a river and being stuck from time to time on the shore leads to the relationship that the difference in the adsorption heats of two chromatographically separated Substances is proportional to the logarithm of the quotient of the traveled distance (respectively the reciprocal quotient of the time needed for it). By this the height of the ladder steps was fixed. I sent a short note on these calculations to the editors of the journal Die Natumissenschaften, an November 29, 1944. However, although I received the proofs, the paper was not published, due to the events at the end of the War; this paper was finally published recently together with its history (6). Still, the considerations summarized in this note represented the theoretical starting point of the theses of R. Knopfler (1946), F. Prior (1947) and R. Miiller (1950) In December 1944 Innsbruck suffered its worst air raid and our Institute was also heavily damaged; we had to move to temporary quarters, in the laboratories of the D. Swarovski Glass Factory, in Wattens, the lower Inn valley. We had to work on a reduced scale and it was not possible to build any new apparatus which would have been necessary for experiments in gas chromatography. However, the early conception about the steps of the chromatographic ladder and the heats

.

of adsorption was also valid in the already well-established liquidsolid chromatography. Therefore, Reingard Knopfler used this technique in investigating the travel velocity of chromatographic rings. The velocity of the rings was tested with azo-dyes as a function of flowvelocity, concentration and temperature, with solvents of different elution power such as hexane, petrol ether, gasoline, carbon tetrachloride, benzene, toluene and xylene. The velocity of the "rings" increased in the order of three times more than that obtained with hexane. Also, differences of adsorption heats of the dyes on A1203 and of the used elution liquids were calculated and a quantitative step-ladder could be fixed. The thesis was finished in January 1946

(7). In November 1945 Fritz Prior, a young man who was just appointed as a high school teacher at the Paulinwn, in Schwaz, not far from Innsbruck, came to me and expressed his interest in finishing a Ph. D. thesis under my supervision; since the physics laboratory of his school was in a good shape, he wanted to carry out the investigations there. We discussed the possibility of gas chromatography as the subject of the thesis. Parts of Kunte's apparatus still existed particularly the thermal conductivity cell he had used for the measurements although it had no wire in it any more - and it could be supplemented with equipment in the school's laboratory; thus, we could soon put together a system containing all the basic elements of a gas chromatograph: an apparatus to generate the carrier gas, a device for sample gas inlet, a column filled with adsorbent and finally, a selfconstructed thermal conductivity cell with a Wheatstone bridge. Most importantly, I could find a piece of the Pt-resistance wire of 7 Dm diameter (a so-called Wollaston wire) which we had used to build the original thermal conductivity cell of Kunte. I gave this to Prior, saying: "be careful because if this wire burns through, the whole work will not be possible!" Readers might not remember the conditions we had at that time. There was simply no possibility of buying these things and also, we had no money. The Institute had no financial support and we had to work with our old equipment, resurrecting it from the ashes and fixing it. Thus, the success of our first attempt to analyze substances by gas chromatography was really hanging on a thin thread! The first gas chromatogram already obtained in 1946 was very simple and at that time,analytically uninteresting: it showed the peaks of air and carbon dioxide, separated on charcoal. Still, it showed good separation and peak shape with definite maxima which could be precisely evaluated. The only problem Prior had was with the baseline; however it is clear from Fig. 3.1 (the chromatograms of two experiments with drifting baseline are shown) that the time of the breakthrough of the maximum is not influenced by the drift of the baseline. Besides air-carbon dioxide Prior also investigated the separation of carbon dioxide and acetylene on silicon dioxide. The separation was incomplete, nevertheless, one could obtain under certain conditions reproducible values for the heats of adsorption. Encouraged by these results, we decided to try the solution of the problem I considered

25

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Fig. 3.1. The first gas chromatogram: air and separated on charcoal. From the thesis of F. Prior (8).

C02,

in 1941-1944: the qualitative and quantitative analysis of acetylene and ethylene. I jokingly told Prior (he remembers it well to this day): "If you succeed in the chromatographic analysis of these two substances you may finish your thesis." I thought that the solution would be difficult but Prior was very optimistic. On a beautiful day in early Spring 1947, Prior made the deciding experiment and succeeded in getting two clearly separated peaks. In accordance with my promise, he was allowed to finish his thesis in. May 1947 (8). It closes with the following statement: "Besides this qualitative analysis, quantitative analysis may also be performed as the height of the response at identical conditions depends only on the amount of the gas. Therefore, one could perform - especially in microanalysis - a very quick and very exact quantitative determination of gases." This thesis represented the first publication of gas chromatograms which could be evaluated analytically. In Prior's work we used silica gel with medium grain sizes, dried at 22OoC. When continuing the experiments as part of R. Miiller's thesis ( 9 ) , we used a so-called "Blaugel" , having small grains which was practically "air moist. The profiles obtained for carbon dioxide had a nearly ideal shape and we could also demonstrate the proportionality of the amount of gas with the detector response. We could also separate multicomponent mixtures: the lower part of Fig. 2 shows the separation of nitrogen, ethylene and acetylene. I showed this chromatogram at international meetings held in 1950 in Marburg and Grai. In our early work - investigations of Prior and Miiller - the detector response was not recorded automatically as is done in present-day systems, but simply read point-by point on a galvanometer. I'

26

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Fig. 3.2. Chromatograms from the thesis of R. Muller (9). "Abb. 24.": peaks of C02 on silica gel. "Abb. 10.": separation of N2, C2H4 and C2H2 on silica gel.

In 1949-1952 short reports were published ( 1 0 ) of the paper discussing the results of Prior's work which was first presented at a meeting in Linz, in 1949, but, by a regrettable delay, the detailed work as well as a summary presented as a remark at the May 19, 1950 meeting of the BunsengeseZZschaft in Marburg devoted specially to chromatography were published only in January 1951 in the Z e i t s c h r i f t f u r Elektrochemie und angmandte physikalische Chemie ( 12,121 Two papers by Cremer and Muller followed these publications in the same year ( 1 3 , 1 4 ) . In these four publications the difference between measured and adjusted retention time was clearly emphasized from the beginning. Differences of adsorption heats are given, e.g., the height of the ladder-"steps" in logarithmic scale, which later became the basis of the retention index. We used the new technique not only for separation and analysis but also for the determination of the heat of adsorption. The relationship between the log of the adjusted retention time and the reciprocal of the absolute column temperature was found to be linear. Also, the proportionality between the peak width at half height and the retention time was proved experimentally (the factor of proportionality is a measure of the quality of separation). The introduced sample amounts varied between 10 and 0.5 mg per peak, the limit of detection being in the order of 10-5g; however, it was emphasized that this is not the theoretical lower limit of detection. Great progress was also made in the speed of analysis: while in the experiments of Damkohler and Theile, hours have passed between the breakthrough of the separated substances, we only needed minutes and later, this could further be reduced by a factor of ten. The papers already used the expression "chromatographic

.

27

spectrum" and recommended for quantitative evaluation the calculation of peak area as peak height multiplied by the peak width at half height. We have also employed a selective detector. Our work was first followed by JanAk in Czechoslovakia who utilized it very successfully in petrochemistry. In 1952, the famous paper of James and Martin on gas-liquid (partition) chromatography (GLC) was published. This technique has advantages over gas-solid (adsorption) chromatography (GSC) which are well known from liquid-liquid chromatography (LLC) and is specially valuable if we have to deal with molecules of medium size and with sample amounts ranging between a milligram and a microgram. However, gas-solid chromatography has also been continuously improved and its range of application widened. New solid stationary phases with a very regular or a specially modified surface were developed and in the case of medium size molecules and amounts, there is really no separation problem which could not be solved by either technique. At high temperatures the liquid stationary phases "bleed" and one is forced to use GSC; GSC is also preferable at very low temperatures where no adequate liquid stationary phase is available. I would only like to refer here to the porous-wall glass capillary columns of Mohnke and Saffert ( 1 5 ) with which one can even separate hydrogen isotopes! The spin isomers ortho- and para-hydrogen can also be separated on molecular sieves ( 2 6 ) and there is no difficulty in obtaining pure o-hydrogen which is not stable at any temperature. If the adsorbent is simultaneously also a catalyst for the conversion, one obtains distorted profiles with a bridge, from which one may calculate (with the help of a computer) the velocity constant ( 1 7 ) . Even without reaction, the state of the surface influences the retention time and the shape of the peaks. One may calculate from the chromatogram not only the energies which control the chromatographic process but also the adsorption isotherms; this was demonstrated in the Ph.D. thesis of J.F.K. Huber, my pupil who is at present professor at the University of Vienna ( 1 8 ) , and in subsequent work ( 1 9 ) . From the chromatograms one may also calculate the size of the surfaces ( 2 0 ) . If the surface of a Pt-catalyst is poisoned with HzS, the poisoned surface will be proportional to the reduction of the retention time. With the help of this surface-covering process, one has another possibility for determining the size of the whole surface ( 2 2 ) . Roselius, another pupil of mine, carried out investigations on catalysts in connection with his Ph.D. thesis work and measured the retention time of C02 on silica gel having a water content between nearly zero and 24% (22,23). He found a constant shift from GSC to GLC and also observed a very interesting effect when changing the carrier gas (24, 2 5 ) . For example, analyzing xenon on charcoal using hydrogen as the carrier gas, an extremely asymmetrical peak is obtained with a retention time of 150 min; on the other hand, in using carbon dioxide as the carrier gas, xenon elutes from the column in only 10 min and displays a very symmetrical peak. This effect can be explained by assuming the formation of an adsorption layer of C02 on which the separation takes place.

28

Fig. 3.3. Chromatogram from the thesis of E . Seidl showing the separation of amino acids ( 5 0 pg) on a thin layer of In203 (28). Reference sample: 40 pg prolin. Autoradiograms and their evaluation by a scanning photometer. START FRONT

START

FRONT

For the scientists familiar with adsorption phenomena, a fixation of molecules on a solid surface is “adsorption” as long as the vapor pressure of the adsorbate is smaller than that of the pure liquid. I therefore emphasized as early as 1959 that, if partition of foreign molecules takes place in an adsorption layer one should speak of adsorption-layer chromatography” (20). In many cases, adsorption and partition chromatography coincide: a continuous transition exists from one technique to the other; the smaller the amounts to be analyzed with a given detector, the smaller the layer must be and the more will partition chromatography approach adsorption chromatography. At least, there was an absolutely unexpected demand for LSC. In the efforts to improve paper chromatography, two-dimensional plates were prepared using powdered adsorbents and thin-layer chromatography, already introduced in 1938 by Ismailov and Shraiber, started to gain once more in application and importance. Stahl and his school succeeded in making the method very reproducible and superior to paper chromatography. With modern thin-layer chromatography, one can analyze sample components in the sub-nanogram region; furthermore, vacuum-evaporated films with a thickness of only 1-2 I-\mcan be used as the stationary phase (26,27). E . Seidl, one of my pupils, showed in her Ph.D. thesis the application of an In203 film for this purpose with radioactive-labelled substances ( 2 8 , 2 9 ) . The limit of detection - 10-14g) depends only on the detector (30). Similarly, layers of MgO obtained through the condensation of MgO smoke, or layers of oxidized aluminum are also suitable as solid phases for samples in the subnanogram range ( 3 2 ) . REFERENCES

1 E. Wicke, KoZZoid Z. 86 (1939) 259; 90 (1940) 156; 93 (1940) 129. 2 G. Damkohler and H. Theile, Chemie 56 (1943) 353; Beih. 2. Ver. dtsch. Chemiker No. 49 (1944).

3 S. Neufeld, Chronologie Chemie 1800-1970, Verlag Chemie, Weinheim, 1977. (See note at the end of this reference list)." 4 A, Kunte, Adsorption und Desorption von Acetylen und Athylen an aktiver Kohle. Diploma Thesis, Faculty of Natural Sciences, University of Innsbruck, 1944; partly published: Monatsh. Chem. 77 (1946) 126. 5 G. Hesse, Adsorptionsmethoden i n chemischen Laboratorium m i t

besonderer Beriicksichtigung der chromatographischen Adsorptionsanazyse (Tswett-Analyse). W. de Gruyter & Co., Berlin, 1943. 6 E. Cremer, Chromatographia 9 (1976) 364. 7 R. Knopfler, Untersuchung uber Chromatographie. Doctoral Thesis, Faculty of Natural Sciences, University of Innsbruck, January 1946. 8 F. Prior, uber die Bestimmung der Adsorptionswarmen von Gasen und Diimpfen unter Anwendung der chromatographischen Methode auf die Gasphase. Doctoral Thesis, Faculty of Philosophy, University of Innsbruck, May 1947. 9 R. Muller, Anwendung der chromatographischen Methode zur Trennung und Bestimmung kleinsten Gasmengen. Doctoral Thesis, Faculty of Philosophy, University,of Innsbruck, May 1950. 10 E. Cremer and F. Prior, Meeting of the Verein lfsterreichischer Chemiker, Linz, May 1949; u s t e r r . Chem. Ztg. 50 (July 1949) 161; Angew. Chem. 62 (1950) 576 (abstracts). 11 E. Cremer, Z. Elektrochem. 55 (1951) 65 (discussion remarks). 12 E. Cremer and F. Prior, Z. Elektrochem. 55 (1951) 66. 13 E. Cremer and R. Muller, Mikrochem./Mikrochim. Acta 36/37 (1951) 553. 14 E. Cremer and R. Muller, Z. EZektrochem. 55 (1951) 217. 15 M. Mohnke and W. Saffert, in Gas Chromatography 1962 (Hamburg Symposium), M. van Swaay, ed., Butterworths, London, 1962, pp. 216-224. 16 E. Cremer, L. Bachmann and E. Bechtold, J . Catalysis 1 (1962) 113. 17 E. Cremer and R. Kramer, J . Chromatogr. 107 (1975) 253. 18 J.F.K. Huber, uber die Eluierungs-Gas-Festkorper-Chromatographie und ihre Anwendung zur Bestimmung von Adsorptionsisothermen. Doctoral Thesis, Faculty of Philosophy, University of Innsbruck, April 1960. 19 E. Cremer and J.F.K. Huber , in Gas Chromatography (1961 Laxsing Symposium), N. Brenner, J.E. Callen and M.D. Weiss, eds., Academic Press, New York, 1962, pp.169-182. 20 E. Cremer, Angew. Chem. 71 (1959) 512. 21 E. Cremer, Z. Anal. Chem. 170 (1959) 219. 22 L. Roselius, Uber die Anwendung der Gas-Chromatographie zur Bestimmung der Adsorptionsenergien von Gasen und zur Untersuchung der adsoptiven Eigenschaften von Adsorbentien und Katalysatoren. Doctoral Thesis, Faculty of Philosophy, University of Innsbruck, June 1957. 23 E. Cremer and L. Roselius, Angew. Chem. 70 (1958) 42. 24 A.I.M. Keulemans, Gaschromatographie. Translated and supplemented by E. Cremer, Verlag Chemie, Weinheim, 1959; Suppl. 11, p.194. 25 E. Cremer, Z. Elektrochem. 69 (1965) 802.

30 26 E. Cremer and H. Nau, N a t u m i s s . 55 (1968) 651. 27 E. Cremer, Th. Kraus and H. Nau, Anal. Chem. 245 (1969) 37. 28 E. Seidl, Die stationare und die mobile Phase in der Dunnfilm-

z.

Chromatographie, Trennungen und Nachweis radioaktiv markierter Verbindungen. Doctoral Thesis, Faculty of Natural Sciences, University of Innsbruck, June 1971. 29 E. Cremer and E. Seidl, Chromatographia 3 (1970) 137; Monatsh.

Chem. 101 (1970) 1614. 30 E. Cremer, J . Gas Chromatogr. 5 (1967) 329. 31 E. Cremer, F. Deutscher, P . Fill and H. Nau, J . Chromatogr. 48 (1970) 132. Note: in the book of Neufeld (ref. 3), my first paper on chromatography is incorrectly indicated; my first publications were actually from 1951 (see refs. 11-13).

31

DENIS H. DESTY

DENIS HENRY DESTY was born in 1923 at Southampton, Hampshire, United Kingdom. His school days were at Taunton's School, Southampton and he went up to University College, Southampton to read for an Honours Degree in Chemistry in 1941. After one year he volunteered for service with the Royal Air Force and served as a Signals Officer in the United Kingdom and India until the end of the War. After demobilisation in 1946 he returned to University College and completed his degree in 1948. Since then he has been employed at the British Petroleum Research Centre continuously for 30 years with a succession of positions first as technologist, then group leader, senior chemist and finally research associate, special projects. In 1978 he has been appointed Visiting Professor at the University of Surrey in the Department of Chemical Engineering. Mr. Desty is the author and coauthor of a number of publications and patents. He was twice the editor of the proceedings of international gas chromatography symposia (1956 London and 1958 Amsterdam). He organised the Gas Chromatography Discussion Group in the United Kingdom and served as its chairman for a number of years. His contributions were recognized in 1970 by the award of a special parchment to him by the Group. In 1974, he was one of the first recipients of the M.S. Tswett Chromatography Medal. In addition to chromatography, Mr. Desty's activities concerned a number of fields related to the petroleum industry. In the combustion field he originated a number of new burners and one of these devices, the Coanda Burner, became the basis of a considerable effort to develop very large units for the disposal of waste gases arising from petroleum production. This work has now come to fruition in the form of a commercial company which supplies equipment on an international basis. Another new idea created by Mr. Desty was in a completely different area, that of oil pollution on the sea. He devised a novel approach employing floating booms on the open sea to trap, contain and recover oil spillages. This work came to fruition in the early 1970's with a complete system concept, after a long series of very laborious and dif-

f i c u l t s e a t r i a l s . A commercial company h a s now emerged f o r t h e e x p l o i t a t i o n of such f a c i l i t i e s and a g a i n o p e r a t e s on an i n t e r n a t i o n a l b a s i s . I n t h e l a s t few y e a r s he h a s o r i g i n a t e d a major p r o j e c t on wave calming i n r e s t r i c t e d a r e a s o f t h e open s e a and is a c t i v e l y concerned w i t h t h e development of new p r o p u l s i o n methods f o r b o t h a i r b o r n e and seaborne c r a f t Mr. Desty became one o f t h e o r i g i n a l s m a l l group of p i o n e e r s i n g a s - l i q u i d chromatography s t i m u l a t e d by t h e o r i g i n a l work of M a r t i n and James. H e i s p a r t i c u l a r l y a s s o c i a t e d w i t h t h e emergence o f c a p i l l a r y columns a s a p r a c t i c a l t o o l i n t h i s f i e l d w i t h t h e i r o u t s t a n d i n g p e r formance i n r e s p e c t o f h i g h column s e p a r a t i o n e f f i c i e n c i e s and v e r y rapid separation. Mr. D e s t y i s an e x t r a o r d i n a r y i n v e n t i v e man. Not o n l y does h e have a s e c u r e g r a s p and g r e a t u n d e r s t a n d i n g of p h y s i c a l c h e m i c a l and engine e r i n g p r i n c i p l e s , b u t h e is a l s o a b l e t o t h i n k of p r a c t i c a l a p p l i c a t i o n s ; even more r a r e , he h a s t h e o r g a n i s a t i o n a l f l a i r , t e c h n i c a l a b i l i t y and p e r s o n a l i t y t o b e a b l e t o push h i s i d e a s t h r o u g h e v e r y s t a g e l e a d i n g t o t h e f i n a l p r o d u c t i o n o f a working d e v i c e .

.

My first contact with gas chromatography came in the middle of 1949 when, while attending a small scientific exhibition in London, I saw an exhibit describing a new technique called gas displacement chromatography prepared by its originator C . S . G . Phillips. I must confess that it did not, at the time, seem very significant in the context of the sophisticated equipment for hydrocarbon gas analysis using thermal displacement from carbon, pioneered by Turner, which had appeared on the scene somewhat earlier. Work on established methods of separation for complex liquid petroleum mixtures such as high efficiency distillation, urea adduction, solvent extraction, crystallisation and adsorption chromatography much occupied me over the next two years. It was slow and tedious work with hundreds of fractions which represented a major storage problem. One hot afternoon in the early summer of 1952 there arrived in the mail a small envelope which contained a chromatogram from a simple pen recorder and a short letter requesting samples of pure hydrocarbons for evaluation of a new separation technique, gas-liquid chromatography. The letter was signed by A.J.P. Martin at the National Institute of Medical Research at Mill Hill, London. In order to whet our appetite he indicated that the recording was that of the separation of a milligram or so of gasoline from the car of his collaborator A.T. James. It had been obtained in about 30 minutes and, even from a superficial view, a separation had been achieved similar to that obtained on a 100 plate distillation column over perhaps a month. As you can imagine the impact both on myself and my old boss, S.F. Birch, was fairly spectacular. We immediately rang the Institute at Mill Hill and it soon emerged that Martin had very recently designed and constructed a very sensitive omniverous vapour detector, which allowed the detection of hydrocarbons for the first time. This combined with a simple packed four foot glass tube as the column, both contained in a vapour thermostat enclosure, seemed to be the major components of what was evidently a simple apparatus. Having hastily fixed an appointment for the next day, we dashed over to Mill Hill with suppressed excitement and met Martin and James, who seemed somewhat surprised that we should arrive so quickly after the despatch of their letter. Birch and I were even more impressed by the physical equipment and decided on-the-spot to reproduce the Martin and James densitybalance equipment immediately. My old friend Barry Whyman, who unfortunately died suddenly a few years ago, started on the job the next day and within three weeks we had everything complete with the exception of the very fine differential copper/constantan thermocouple, which was the heart of the device. Whyman, after two weeks of abortive attempts to duplicate Martin's elegant technique for making two junctions 1/8 inch apart in one thou wires, eventually succeeded just before he became convinced it was impossible. We proudly demonstrated our complete apparatus to all our colleagues and invited A.J.P. Martin over to inspect the polished-up assembly. As an imminent Nobel nrize winner he was, of course,

34 e s c o r t e d i n t o o u r l a b o r a t o r y by t h e u s u a l r e t i n u e o f s e n i o r management VIPs and t h e y were a s t o u n d e d when h e d r o p p e d o n t o h i s k n e e s , t o o k a v e r y close l o o k a t t h e t h e r m o c o u p l e m o u n t i n g c a p s o n t h e dens i t y b a l a n c e and announced v e r y c l e a r l y t h a t w e had c h e a t e d . W e h a d ,

? i x , 4.1. G a s d e n s i t y b a l mce apparatus i n i t s origi n a l form (1952-1953).

w i t h o u t r e a l i s i n g t h a t w e were d o i n g so, as t h e t h e r m o c o u p l e c a p s had been made from two p i e c e s o f c o p p e r s o l d e r e d t o g e t h e r i n s t e a d of t u r n i n g them from o n e p i e c e o f c o p p e r . Humbly w e h a d t o admit t h a t t h e r e was a s i g n i f i c a n t s t r a y e m f , which r u i n e d t h e b a s i c l i n e s t a b i l i t y . We had t h e r e f o r e t o f a c e UD t o a r e c o n s t r u c t i o n o f t h e

35

Fig. 4 . 2 . D.H. Desty (in lab coat) operating the density balance instrument to some visitors, in 1952. Second from right: Dr. S.F. Birch. thermocouple and a repetition of the extremely tedious and delicate mounting procedure. Now that this first copy of Martin's original apparatus was performing satisfactorily, new vistas of work appeared before us to determine the retention data of all the 500 or so hydrocarbons in the bank of pure samples we had accumulated at Sunbury over the previous 30 years. The non-polar stationary phase we chose was. n-hexatriacontane and the polar phase benzyl diphenyl, as used by Martin and James. We were the first to publish such a complete double logarithm plot of retention data for low molecular weight hydrocarbons on these two phases at 7 8 0 C ( I ) . Around this time the first formal meeting on gas chromatography took place at the I.C.I. Explosives Division at Ardeer in Scotland. On the way up I persuaded R.P.W. Scott, then a young technologist with Benzole Producers Research Laboratories, to overcome his natural diffidence and ask for time to describe his newly created hydrogen flame temperature detector. This made considerable impact and we both had some stimulating discussions with Keulemans from Shell, Amsterdam, who was self-evidently the man with the most experience there. It was a memorable first meeting with an aura of gathering expectation and excitement.

The next two years were a hectic exploration of this magnificent new technique and we developed methods for gas analysis using Janhk's very simple micronitrometer equipment, the rapid separation of liquid petroleum fractions using the gas density balance at operating temperatures up to 25OoC and constructed a preparative scale unit with a one-inch column capable of separating a few millilitres of sample. Up until this time in 1954 most, if not all, of the workers on gas-liquid chromatography were in the United Kingdom or at the Shell Laboratories at Amsterdam. The following year Birch decided to make a tour of the U.S.A. to spread the gospel amongst American petroleum company laboratories and his visit created much interest there. Apparently the technique came as a complete surprise even to Rossini of API Research Project 6. They had separated, after 30 years work, less than 100 hydrocarbons from a special barrel of Ponca City crude taken as the basic sample for systematic analysis very early in the programme. Their vast array of fractionation columns, solvent extracting equipment and large absorption columns seemed suddenly likely to become completely obsolete, as did in fact happen over the next few years. When Birch returned two further decisions were taken: first I was to visit the U.S.A. on an extended tour during 1956, and second we would organise a Formal Symposium in the autumn of that year in London, under the auspices of the Institute of Petroleum. I soon found myself drafted as the editor and faced the,prospect with some trepidation. The Spring of 1956 came and I set off to America for the first time with an itinerary to visit 21 laboratories, attend two Symposia and present a paper, over a period of six weeks. My ticket was about half an inch thick and covered a total distance of about 12,000 miles. Arising from Birch's earlier visit the American Chemical Society had decided to organise a small Symposium on the technique at their National Meeting at Dallas and I went there first. The morning of the meeting came and the room allocated on an upper floor of the hotel was suitable for the accommodation of about 250 participants. Over 800 turned up and there was a grand panic trying to reorganise the proceedings in the Main Ballroom instantaneously, Eventually the session started and the air of excitement was something which I have never subsequently experienced at any Symposium. Every slide presented was accompanied by a flurry of activity all over the hall as participants stood up with cameras, equipped with high-speed film, and snapped everything vaguely relevant. Private discussion went on continuously during the whole meeting and I became quite hoarse and confused trying to remember what I had said when and to whom. The rest of the trip was almost a nightmare darting by air from one city to another. Fortunately while visiting AMOCO Research Laboratories at Whiting, Indiana, about half way through the trip I met John Winters and he was kind enough to invite me to his home for the weekend. We have remained close friends over the whole of the subsequent 2 0 years and have both maintained an active interest in gas chromatography.

37 The few months between my return from the U.S.A. and the First International Symposium on "Vapour Phase Chromatography" were a period of difficult struggles trying to sope with the organisation of this pioneering meeting on an entirely new subject, while at the same time keeping the momentum of our own research effort going. It was a formidable task and I soon had a full filing cabinet of correspondence with authors and members of the Organising Committee. The members of the latter were a small group of pioneers from Europe and included personalities such as Keulemans, Phillips, Martin and James, who were to become household names in the field a few years later. Summer came eventually and the Symposium went off very well with an attendance of around 600. Apart from the formal papers, which were surprisingly wide in scope creating much active discussion, there was a considerable debate about the formal name to be adopted for the technique. With some embarrassment I had to accept that the name Vapour Phase Chromatography used for the Symposium was changed to Gas-Liquid Chromatography, which emerged as the most accepted title, allowing all other forms of chromatography to be described in a systematic nomenclature. The task of editing the Proceedings proved even more formidable than the business of organising the Symposium itself. We had made a decision to include a verbatim record of the discussion and in spite of this difficult job aimed to publish the volume before the end of the year. This was accomplished early in 1957 and the book became the pattern in which many subsequent Symposia were recorded (2). After much discussion amongst the members of the Organising Committee of the 1956 Symposium we all agreed that it seemed worthwhile to form a Special Group to organise subsequent activity in the gas chromatographic field. For the next two years myself and a colleague, Mr. C. Harbourn, ran the Group with a very informal structure and we had to prepare and reproduce, during this period, hundreds of copies of the initial newsletter for circulation. In addition we organised a number of informal meetings in the United Kingdom at which members of the Group could describe their contributions to experimental techniques which began to emerge very rapidly. By 1958 the Gas Chromatography Discussion Group had several hundred informal members to whom relevant documents were circulated and it became necessary to organise a more formal structure with a constitution and an Annual General Meeting. The Group was almost overwhelmed at this stage by applicants who wanted to participate in its activities and soon had a formal membership of around one thousand. Mr. Knapman undertook the onerous task to edit a compilation of references with abstracts of papers, which were beginning to emerge in the literature in a very rapid cauliflower expansion way. He assembled a group of volunteers around him who prepared all the extracts, on a voluntary basis, and the Institute of Petroleum, with whom the Group was associated at that time, published these on a quarterly basis. During this period the impact of the visits by Birch and myself to the U.S.A. had made a considerable impression and the first instrument company to actively proceed with the design, manufacture

38 and sale of a commercial instrument, was Perkin-Elmer. They achieved an almost unbelievable schedule of producing this instrument, the Model 154, over only 8 months and laid a path which was to be actively pursued by other instrument companies all over the world for 20 years The Instrument Society of America decided to organise formal Symposia in the U . S . A . and held two very successful meetings at East Lansing in Michigan at which both Martin ( 3 ) and James (4) told successively the story of the origination of gas chromatography. The two accounts are both fascinating reading on a comparative basis as they both told the same basic story but with quite different personal viewpoints. Meanwhile our own work at Sunbury was proceeding apace with both basic technique development and a variety of applications to the problem of resolving complicated petroleum products. We pioneered the use of automatic integrators to obtain reproducible precise quantitative results and initiated the beginning of process gas chromatographic monitors for use in refinery operations. In spite of the fact, however, that Martin had mentioned the possibility of micro-bore columns at the 1956 Symposium, with considerable potential advantages in improving the column efficiency, we did not appreciate the significance of his suggestion and pressed on with conventional 6-mm diameter packed columns. R.P.W. Scott at Benzole Producers, near London, pushed such columns to their ultimate efficiency producing over 30,000 plates ( 5 ) . His column would almost completely resolve the very close boiling mixture C7 and C B paraffin hydrocarbons. The time came, in 1958, for the next formal Symposium to be organised by the Gas Chromatography Discussion Group and once again I was pressed into becoming the editor of this Symposium which was to be held in Amsterdam. As soon as the papers started to come in it became clear that the forthcoming Symposium was likely to become a really outstanding meeting. Amongst many other fascinating contributions, Golay submitted a mathematical paper on a new concept, that of open-tube capillary columns, without any packing, but having instead a retentive layer of liquid on the inside wall of the tube. Editing and printing of this paper presented me with an extremely difficult task which so occupied me that I failed to appreciate the truly basic significance of his concept. Several hundred participants eventually assembled in the beautiful City of Amsterdam and, during the Proceedings, several papers made immense impact. McWilliam's description of the hydrogen flame ionisation detector from Australia produced an immediate appreciation of the value of this new detector concept (6). Its sensitivity and immense linear range were almost unbelievably striking and it is unfortunate that the paper (7) published by Pretorius, some months earlier, describing the basic concept, did not enjoy the recognition it deserved. Nevertheless the McWilliams paper, coupled with the first description of the argon ionisation detector by Lovelock in the context of a whole family of similar devices, opened up completely new prospects of new detectors with sensitivities several orders of magnitude higher than established detectors. In addition the fact that they had extremely small volumes was subsequently to be of major significance.

39 As I had expected the presentation by Golay (8) was totally beyond almost all of the participants of the Symposium, but fortunately in his additional comments he produced the first experimental chromatograms from his new capillary columns, which produced an immediate gasp of surprise from the audience. He had separated the xylene isomers with a non-selective stationary phase with a column which achieved over 50,000 theoretical plates. This column performance was so much better than the best packed columns that it seemed almost inconceivable. It was not until many years later that it became clear to me that these striking chromatograms were in fact produced by his young colleague at Perkin-Elmer, Dick Condon, and, even now with the perspective of history, it appears that this effort should have received more recognition with his name. The technical proceedings were capped by a magnificent reception at the Rijksmuseum and we all spent the evening strolling around enthralled by the splendidly impressive pictures. In spite of the fact that the Symposium was exceptionally successful we found it almost unbearable waiting to get back to the laboratory at home. Within hours of returning we were engaged in a ferocious discussion with Whyman, trying to find the most rapid method of obtaining metal capillary tubes to repeat Golay's work, taking advantage of McWilliams hydrogen flame detector. A few days later we had obtained several hundred feet of thick-walled stainless steel, 10 thou capillary, from stocks held by an instrument company for their pressure recorders.

Fig. 4 . 3 . First crude flame ionization detector and thick-walled stainless steel capillary column (1958).

40

We coated 250 feet with squalane using the dynamic method described by Dijkstra at Amsterdam ( 9 ) , constructed a crude version of McWilliams' detector in a cocoa tin and the assembly was completed by the first splitter injection system put together by Whyman from a few surplus pipe connectors ( 2 0 ) . Even with our knowledge of Condon's results our first chromatogram was a complete surprise. Our crude first capillary column, which was strewn all over the floor in coils, with the cocoa tin detector at one end and the splitter at the other, produced 100,000 theoretical plates and the injection of a microlitre of light petroleum distillate'groduced peak after peak of well resolved individual hydrocarbon isomers. The excitement was intense and we all ran around the lab finding interesting materials to examine with this fascinating tool of amazing power. Within a few weeks Whyman had, with great enthusiasm, designed and constructed a complete apparatus for operation at temperatures as high as 250oC ( 1 1 ) . The ability to separate in this equipment much higher molecular weight hydrocarbons once again led us into another spectacular ad hoe exploratory examination of all sorts of

Fig. 4.4.Capillary column gas chromatography apparatus for operation up to 25OoC (1958).

41 samples. We were particularly impressed by the separation of a normal paraffin concentrate obtained from petroleum wax by urea adduction. Someone suggested sticking in a few cubic centimetres of tobacco smoke and we were all amazed by the vast number of peaks which kept rolling out. The next two or three years represented a period of activity with capillary columns which, almost every day, produced new surprising results. There was never enough time in any one day to complete the work which had emerged from the previous efforts. By 1961 we had produced long columns with 1 million theretical plates and chromatograms went on and on for 12 hours or more ( 1 2 , 2 3 ) . Earlier in this text I mentioned the work of API Research Project under Rossini, which had struggled to separate the lighter components of a typical petroleum, prior to the advent of gas chromatography. One chromatogram on such a capillary column not only repeated their 25-year effort in a few hours, but in addition produced resolved components which in total tripled the number separated by Rossini's group. At the other extreme Goldup, who was with me around that time, had constructed extremely narrow-bore columns which could resolve 15 low molecular weight hydrocarbons in a rush of peaks on an oscillograph over 2 seconds.

Fig. 4.5. The first glass capillary drawing machine (1959).

42

Insofar as this account is concerned primarily with the story of my involvement in gas chromatography in the early days, there seems no point in continuing into the consolidation period which occurred during the sixties and seventies. I must, however, refer to the emergence of glass capillaries in 1959. Whyman, in his characteristic way, once again so effectively met our needs for a cheap self-produced capillary column and his first machine produced hundreds of feet of beautiful glass helical coils in a few hours ( 1 4 ) . Rudolf Kaiser, amongst many others, was responsible for the wide-spread adoption of these glass capillaries a decade later and has organised in Hindelang, a beautiful mountain village in Bavaria, a series of meetings wholly devoted to the application of these elegant columns. It is difficult to summarise in a few words the enthusiastic dedication which that splendid 10 first years of gas chromatography produced in a select body of pioneers. I remember this decade with very warm and fond nostalgia. It seems unlikely that another such period will occur in modern chemistry for a very long time. REFERENCES 1 D . H . Desty and B.H. Whyman, Anal. Chem. 29 (1957) 320. D.H. Desty, ed., Vapour Phase Chromatography ( 1 9 5 6 London Symposiwn), Butterworths, London, 1957. 3 A.J.P. Martin, in Gas Chromatography (1957 Lansing Symposium), V.J. Coates, H.J. Noebels and I.S. Fagerson, eds., Academic Press, 1958, pp. 237-247. 4 A.T. James, in Gas Chromatography (1959 Lansing Symposium), H.J. Noebels, R.F. Wall and N. Brenner, eds., Academic Press, New York, 1961, pp. 247-254. 5 R.P.W. Scott, in Gas Chromatography 1958 (Amsterdam Symposim), D.H. Desty, ed., Butterworths, London, 1958, pp. 189-199. 6 I.G. McWilliam and R.A. Dewar, in Gas Chromatography 1958 (Amsterdam Symposium), D . H . Desty, ed., Butterworths, London, 1958, pp. 142-152. 7 J. Harley, W. Nel and V. Pretorius, Nature (London) 181 (1958) 177. 8 M.J.E. Golay, in Gas Chromatography 1958 (Amsterdam Symposium), D.H. Desty, ed., Butterworths, London, 1958, pp. 36-55. 9 G . Dijkstra and J. de Goey, in Gas Chromatography 1958 (Amsterdam Symposim), D.H. Desty, ed., Butterworths, London, 1958, pp. 56-68. 10 D.H. Desty, in Gas-Chromatographie 1 9 5 8 , H.P. AngelB, ed., Akademie Verlag, Berlin, 1959, pp, 176-184. 11 D.H. Desty, A. Goldup and B.H.F. Whyman, J . I n s t . Petrol. 45 (1959) 287. 12 D.H. Desty and A. Coldup, in Gas Chromatography 1960 (Edinburgh Syntposiwn), R.P.W. Scott, ed., Butterworths, London, 1960, pp. 162-183. 13 D.H. Desty, A. Goldup and W.T. Swanton, in Gas Chromatography (1961 Lansing Symposium), N. Brenner, J.E. Callen and M.D. Weiss, eds., Academic Press, New York, 1962, pp. 105-138. 14 D.H. Desty, J.N. Hareanape and B.H.F. Whyman, Anal. Chem. 32 (1960) 302. 2

43

GREULT DIJKSTRA

GREULT DIJKSTRA was born in 1923, in Lemmer, Friesland, the Netherlands. He started his chemistry studies at Amsterdam University in 1941 and graduated in 1951, a few years being spent in prodigious efforts to escape deportation by the occupation forces. After graduation he joined the Unilever Research Laboratory at Vlaardingen as a spectroscopist. He received his doctorate at Amsterdam University in 1957. Since 1962 he has been professor of analytical chemistry at Utrecht University. Dr. Dijkstra is the author of a number of publications. His main fields of interest have been infrared and mass spectrometry and gas chromatography. He stimulated the application of instrumental methods in various fields, from agricultural research to the investigation of art objects and is currently director of the "Government Service for Culture Conservation", dealing with the investigation and restoration of art objects. In 1976, Dr. Dijkstra served as the president of the Royal Netherlands Chemical Society. Dr. Dijkstra's involvement in gas chromatography started in 1953 with the development of a high-temperature apparatus. He also contributed to the introduction of open tubular (capillary) columns, in 1958.

44

The Time, t h e Place and t h e Atmosphere When il d i s t i n g u i s h e d b u t e Zderly s c i e n t i s t s t a t e s t h a t something is p o s s i b l e he is almost c e r t a i n l y r i g h t . When he s t a t e s t h a t something is impossible he i s very probably wrong. A.C. Clarke ("Profile") Scientists, in those post-war years, had a great confidence in their abilities to solve all problems, given a lot of money and a little time. So had everybody, as a matter of fact. Most of it was due to the realization that science-based technology can win wars and had indeed decisively influenced the last one. And if it did so much for war, why not for peacetime society? So down we went into the atomic nucleus, down into the living cell, down into the Earth's crust and up into ozone layer and beyond. Some of the products of a technology that would never get off the ground in the present mood towards science are still racing for the outer planets. Budgets for the luxury products of our science-based culture were justified by the promise of spin-off, a motive which was just as true and just as insincere as a justification of the creation of the Parthenon as a long-term investment in tourist trade would have been to the ancient Athenians. One just wonders whether we have witnessed another fifty-years miracle from 1920 to 1970 like Athens from 470-420 BC or Florence from 1420-1470. O r will science regain its full momentum by showing its problem-solving abilities for all to see? However it will be hereafter, theclimate was excellent for the development of new techniques like gas chromatography in the post-war years. So it was in the Netherlands. Originality was perhaps not at a high pitch after the wartime near-closure of the universities by the deportation o r going into hiding of all but a few collaborating students and the counterproductive atmosphere of the occupation in the industrial research laboratories. What was at a high pitch was the urge for creative development in that reconstruction period and mainly thanks to the presence of several large research laboratories such as those of Shell, Unilever, Dutch State Mines most of the new instrumental methods of organic analysis like infrared spectroscopy, mass spectrometry, gas chromatography and perhaps NMR knew an initial period when there were more instruments working in the Netherlands than in the rest of continental Europe put together. Interest in chromatography was keen, with Martin and Synge's papers of 1941 being well read. Paper chromatography and the aluminium oxide column were widely applied, and Boldingh at Unilever Research had developed reversed-phase chromatography of fatty acids on rubber columns. Not unnaturally he advised me when I was going to attend the 1952 Analytical Chemistry Conference at Oxford, to see what Martin was up to with the gas-liquid chromatography which he had predicted to be more effective than liquid-liquid chromatography in 1941 and which he now apparently was demonstrating there. Martin was there, grumpy because, as he explained to me, ten years after having clearly described the advantages of gas chromato-

45

graphy ( I ) he had had to go out of his biochemical way to work it out himself when he needed a good fatty acid separation. He also jabbed a finger at me: "Can you suggest a method for detecting traces of organic compounds in a gas stream?" I lamely suggested heat conductivity, which he of course knew about, and infrared nondispersive detection. His comment, firm but friendly, his eyes gazing towards the distant aims he always sets himself: "I want something much more sensitive; we shall need detection of fractions of a microgram. I'

EarZy GLC i n the NetherZands You knm t h i s appZied science i s j u s t as i n t e r e s t i n g as pure science and what's more, i t i s a darn sight more d i f f i c u Z t . W.B. Hardy At this time in the Netherlands, Shell were most actively interested. To the Shell Research Laboratory in Amsterdam mass spectrometric analysis of mixtures of up to twenty hydrocarbons assisted by one of the countryls first computers was a going concern. Although this served company needs, it could be anticipated that customers would ask for specifications based on gas chromatograms and Keulemans and others were convinced of the proplise of the method. After the unavoidable gestation period, in which management has to decide whether proposals for an exciting new research could be of value to the firm's operations, the nod was given late in 1952 for the involvement of a considerable number of scientists and engineers in the development of analysis by gas chromatography on a large scale. Keulemans acted as coordinator, travelled widely, contacting Martin, the Nobel division and the Billingham group of I.C.I. and of course the Shell laboratories at Houston (Texas) and Emeryville (California) in the United States and Thornton in the United Kingdom. These efforts had a number of consequences. The first was the development of the Van Deemter equation in which the Shell experience in diffusion of vapours in packed sand-beds proved fruiftul. After the original expos6 (2, 3) the equation was presented by Keulemans and Kwantes at the first internation symposium of the Gas Chromatography Discussion,Groupheld in 1956, in London ( 4 ) . This was very important both because the operating parameters, such as the gas speed, to be chosen in any gas chromatographic experiment were so clearly linked to the column properties like packing density and stationary phase loading and because it had been presented to a dedicated audience in an easily accessible form. In this case it is easy to verify for any amateur scientific sociologist how important the latter factor is. The majority of authors of later papers referring to the Van Deemter equation use the form given in the 1956 Symposium proceedings ( 4 ) with a numerical factor in the second term missing, although many give the original articles as references. The second main consequence of the Shell effort was the rapid spread of news of new developments; the best components like

46

syringes, flowmeters, detectors to be used. Although information from Shell itself was restricted, the unrestricted technological small talk from many places spread through Keulemans and helped people to overcome frustrating holdups in the development of their equipment. I think everybody, including Shell, profited. The third consequence was the rapid spread of the katharometer, the mainstay of detection. And lastly, Shell went into an agreement with the firm of Becker, Delft, for the production and sale of their standard instrument. The instrument, although falling short of giving a new start to the Dutch scientific instrument industry that collapsed in Napoleonic times, was robust, reliable and versatile and one of the early Becker models holds its own to the present day in the company of a collection of more modern ones in my laboratory. It certainly helped to establish the technique in the Netherlands. Back in 1953 all instruments had to be home-constructed. The story of the construction of the first gas chromatograph with working temperatures up to 200 OC is worth relating because it must have been characteristic of many situations (Keulemans is on record with a similar experience), particularly for the passionate way in which problems were tackled. Gas chromatography was of evident use to a laboratory working on flavours and off-flavours like the Unilever Research Lab in Vlaardingen and I had advocated its development on those general grounds which seldom appeal to management and probably rightly so, because too many cases. for research projects can be made out that way. When however the identification of hardening flavours of fats ran aground on the problem of separation of microgram amounts of a close mixture of unsaturated aldehydes in the C6 to C 1 2 range, another offhand suggestion that GLC would do the job elicited a baffling response from Boldingh, our research director. I was given seven days off from my spectroscopic work provided by the end of that period a GC apparatus would be in working order. The specification included the analysis of fatty acid mixtures up to C 2 0 . Anything required to achieve this end would have absolute priority during that period. I accepted the challenge, but did not meet it. It took a fortnight. The snag was the temperature range of up to 220 OC considered necessary on the basis of distillation data for fatty acids at a time when 120 was the recorded maximum operating temperature for columns. Above that, equipment used to disintegrate in various places due to the approaching melting point of solder at 150 OC. A trivial problem until one realized that the alternative of hard solder was impossible because some essential materials would not stand more than 280 OC, far below the softening point of hard solder. The problem was one of going through every part of the process rigorously, from stabilizing the gas stream through sample vaporization, finding a low vapour-pressure stationary phase, avoiding dead volumes, cold spots, adsorptive materials and contact resistances. First of course the pilgrimage (barefoot) to the people who were six months ahead (Shell) or more (Martin). The Shell workers were quite understandably not allowed to divulge technical details but did all

47

they could do to indicate possible obsta,cles and give the names of component suppliers like Negretti and Zambra for pressure stabilizers and of the man who had tuned heat conductivity measurement to a fine pitch: my namesake (Dr. J. Dijkstra) at "Staatsmijnen", the Dutch State Mines in the South of the Netherlands. He had developed a robust instrument for checking coal mine atmospheres for the presence of methane and was delighted to give it the name katharometer purity meter his Protestant tongue in check in a Roman Catholic area where Protestants still were considered "ketters", locally more or less synonymous with devil's offspring, but, as Dijkstra was delighted to point out, a word derived from Greek "katharosT1= pure, meaning somebody who wants to purify the Church from its sins. He also pointed out that the taut platinum wires would sag because the strings would lose elasticity at 220 OC. That week was further taken up by a trip to Martin, whose friendly acceptance and free advice to any who turned to him with their problems, had a great influence on the rapid spread of GLC. There were also visits to various factories making pieces of equipment or components, like the producers of glass-to-metal seals, who on hearing of my quantitatively modest requirements and my predicament regarding delivery times just stuffed a handful of the things in my pocket, or the vacuum grease importers and phosphor bronze spring manufacturers who provided similar services. The apparatus worked in afortnight. It even worked very well three months later and brought a cavalcade of visitors when it had been described ( 5 , 6 ) . Like the poet says: A caprious goddess i s fame

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She e i t h e r neglects you o r coolly s e l e c t s you for things wide apart from your aim ( 7 ) . One anecdote is illustrative of the non-availability of instruments at the time. As no commercial instruments seemed to be forthcoming and an urgent need for high temperature gas chromatographs became apparent in Unilever laboratories it was decided reluctantly to start the construction of thirty-five instruments in batches of about eight. A production prototype was constructed, but in the meantime fatty acid ester mixtures were sent to every manufacturer announcing a new instrument. None met our specifications until one week before the 1958 conference in Amsterdam we received a perfect chromatogram of our test sample from Pye, Cambridge. On the first day of the Amsterdam conference a small but select Unilever committee tested the instrument on exhibition, and on the second day Boldingh in person ordered seventeen instruments on the spot, with strict delivery times. No wonder Pye was flabbergasted and doubled initial production plans for an instrument with such obvious customer appeal. I think a final remark on the episode of the development of high-temperature apparatus is in order. Its success was undoubtedly due partly to the play-safe policy of using the best available components, even when experts gave their considered opinion to the contrary. Potentiometers and switches made t o the seemingly needless specifications of the Royal Navy for resistance to salt spray, gave

48

Fig. 5.1. The early high-temperature apparatus was far from compact. A triple-column instrument in the Unilever laboratory in 1955. us years of operation, free from corrosion troubles, as we found out to our detriment later when building a cheaper version. The main lesson from the episode is that the free flow of information and a cooperative atmosphere stimulates the development of a new technique immeasurably.

Some Phases i n Capillary C o l m Development Let us work without t h e o r i z i n g , said Martin, it is t h e only way t o make l i f e endurable. Voltaire, Candide 1758 XXX. To many people thinking about the nature of the separation in GC columns it must have been clear that near-ideal conditions for

separation were to be found in straight capillaries compared with the maze of widening and narrowing channels in a packed column. How much better would a separation in a capillary be? Plain common sense with a bit of speculation gives the answer: consider a straight capillary column. The wider it is, the longer diffusion from the

49

Fig. 5.2. The first glass capillary, a remarkable bit of glass blowing considering the capillary channel had to remain open when joining the bends to the straight stretches.

centre of the column to the wall will take. During that time there will be an equal amount of longitudinal diffusion raising the HETP, the column length required for one exchange with the stationary phase, by the equivalent of the column radius. It will be larger of course, because of flow profile and diffusion in the stationary phase. The flow should be kept to the low limit where it offsets the longitudinal counter diffusion by a factor of, say ten, and the liquid layer thickness should be Dliq/Dgas * r if the D's are the respective diffusion coefficients and r is the column radius; this makes the contributions to diffusion in the stationary and mobile phase equal. The number of theoretical plates then is f/r in which f is one of the "fudge factors" abhorred so much by Golay, indicating that we know we have neglected a few effects so we know the result will be out by a factor of f. The surprising result (at a time when I first made the "calculation" in 1954 three hundered plates were quite reputable) was f times 100.000 plates for 50 m of a 0.5 mm radius capillary, (thinner columns would give pressure drop difficulties) with f probably between 0.1 and 0.5. So the hunt was on, but had to be called off almost immediately for practical reasons. No detector would give a signal for the microgram loads which a 0.5 Um liquid layer would take without overloading. Thicker columns destroy the advantages, so let us wait for more sensitive detectors. In 1957 we took up the thread again. Detectors had improved, we thought of low pressure detectors to obviate dead volume problems (later we used a glow discharge type devized by Pitkethly with considerable success) and we decided to have a try for the 1958 Symposium in Amsterdam (8). Glass capillaries were constructed, one 50 ft. long, 1 mm diameter, folded into 5-ft.lengths, and one 70 ft. long and 0.5 mm diameter. The glass material allowed us to follow the column loading procedures and to develop the method still in use of

50 sending lengths of solution of the coating material through the column leaving a film on the wall of the capillary. It also made the behaviour of the coating at various gas speeds visible, helping to define conditions for stable coatings. The decrease of separating efficiency due to the coating being driven towards the end of the column was eliminated by reversing flow direction after every few

runs. Results were below expectation, although with a 400-ft.copper capillary we reached 6000 plates. The cause was stupid impatience. Contrary to our own calculations we used far too high speeds and reported as much at the conference, where Golay not only showed how a rigid calculation of capillary column parameters (9) can be made but also pointed out in the discussion that according to him gas speeds were too high. Two days after the conference we jumped to 20,000 plates by lifting the foot from the accelerator pedal. From that time on development and exploitation of the capillaries was in the hands of Perkin-Elmer with Golay as the intellectual force indicating the best parameters. This was partly due to the patent taken by this firm and this had a few consequences, not all of them pleasant. An amusing incident occurred at the 1958 conference, where in the combined discussion after Golay's and our paper he dropped a clanger by stating that the capillary was very interesting in gaining insight into the column processes but that he personally frankly did not expect any practical use for this type of column. He added that his Perkin-Elmer friends had a quite different opinion. This caused a commotion that went unnoticed by the audience. As the discussions were taken down verbatim the remark could be exploited against the patent and immediately after the session the stenographers room and the session chairmen were besieged with the result that the remark was edited out. I personally did think at the time and still do that this was the right thing to do as the significance of the remark was out of proportion to the possible consequences.

The Historical Lesson

I t is not t u i c e , but times without number t h a t the same ideas make t h e i r appearance. Aristotle (Wn the Heavens") I have attempted to describe a few parts of the microcosm of science rather than memoirs, although superficially it may look like the latter. This is because I am convinced that historical cause and effect act only by letting "cause" create a preference for certain developments in a hundred incidents, such as brainwaves, opportunities seen when circumstances require action, goals that are made to appear desirable, ideas that become f'gelaufig'l. So the "effect" surfaces from one of many events whence it might have originated. Many developments took place in many laboratories, and we now recognize the pattern of the sprouting and blossoming of a branch of science. But rather than considering this process as a

51 series of unique single strands of thought and hard work I see it as a coherent texture of progress with a few recognizable main colouring agents. I think the remarkedly quick development and exploitation of gas chromatography was strongly influenced by (a) Martin's combination of practical sense, theoretical insight and willingness to let everyone share them; (b) the climate of confidence in science; and (c) the fact that to none of the organisations primarily involved was the method of gas chromatography of primary interest. Information flowed freely and no one bothered about patents, almost. REFERENCES 1 A.J.P. Martin and R.L.M. Synge, Biochem. J . 35 (1941) 358. 2 A. Klinkenberg and F. Sjenitzer, Chem. Eng. S c i . 5 (1956) 258. 3 J.J. van Deemter, F.J. Zuiderweg and A. Klinkenberg, Chem. Eng. S c i . 5 (1956) 271. 4 A.I.M. Keulemans and A. Kwantes, in Vapour Phase Chromatography (1956 London Symposium), D.H. Desty, ed., Butterworths, London, 1957, pp. 15-34. 5 G. Dijkstra, J.G. Keppler and J.A. Schols, 'Rec?. Trav. Chim. 7 4 (1955) 805. 6 J.G. Keppler, G. Dijkstra and J.A. Schols, in VapoUr Phase Chromatography (1956 London Symposium), D.H. Desty, ed. , Butterworths, London, 1957, pp. 222-234. 7 W.S. Baring-Gould, The Lure of t h e Limerick, 1968. 8 G. Dijkstra and J. de Goey, in Gas Chromatography 1958 (Amsterdam Symposium), D.H. Desty, ed., Butterworths, London, 1958, pp. 56-68. 9 M. J. E. Golay, in Gas Chromatography 1958 (Amsterdam Symposium) , D.H. Desty, ed., Butterworths, London, 1958, pp. 36-55.

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53

LESLIE S. ETTRE

LESLIE STEPHEN ETTRE was born in 1922, in Szombathely, Hungary. He graduated in chemical engineering at the Technical University) Budapest, in 1945, and later obtained a technical doctorate from the same school. Prior to 1957, he was active in Hungary, in the chemical industry, industrial research, research management and academic teaching. In 1957-1958, he was a chemist at the laboratories of the LURGI companies in Frankfurt/Main, German Federal Republic. After coming to the United States, in the fall of 1958, he joined the Perkin-Elmer Corporation, in Norwalk, Connecticut, and served as applications chemist, product specialist and chief applications chemist in gas chromatography. Between 1968 and 1972, he took over the executive editorship of the EncycZopedia of indztstriaZ ChemicaZ Analysis, a 20volume series published by J. Wiley & Sons. In 1972, he returned to Perkin-Elmer as a senior staff scientist, his present position. In the 1977/78 school year, he also served as a research associate at the Department of Engineering and Applied Sciences of Yale University) New Haven, Connecticut and since September 1, 1978, he is also connected to the College of Natural Sciences and Mathematics of the University of Houston, Houston, Texas, as an adjunct professor. Dr. Ettre is the author and coauthor of close to 100 scientific and technical papers and a number of books which his Open TubuZar CoZwrms i n Gas Chromatography (1965) , Practice of Gas Chromatography (1965; with A. Zlatkis) AnciZlary Teehniques of Gas Chromatography (1969; with W.H. McFadden) and @en !7'ubuZar Cohnns - An Introduction (1973) are best known. The book on ancillary techniques was also published in 1972 in Russian edition while the last introductory book on open tubular columns in 1976 appeared in German translation. Dr. Ettre has lectured widely in the world and has been active in the organization of several international symposia in chromatography. He served as the chairman of the Anniversary Symposium on Chromatography (164th National American Chemical Society Meeting, New York, Fall 1972) and the Symposium on Selective Chromatography Detectors (172 National ACS Meeting, San Francisco, California, Fall 1976) and co-chairman of the Summer Symposium in Analytical Chemistry of the ACS

54

Analytical Division in 1973. He is one of the editors of Chromatographia and serves on the editorial advisory board of the Journal of Chromatographic Science. In 1978, he received the M.S. Tswett Chromatography Medal. Dr. Ettre's involvement in gas chromatography started in 1957, at LURGI, in Frankfurt/Main and has continued ever since. His activities covered a wide variety of fields including trace analysis, studies on detector response, the retention index system and particularly open tubular (capillary) columns. In recent years, Dr. Ettre's interest focussed more-and-more on the history of chromatography, particularly on the early evolution of the various techniques, looking at them in the proper historical and political context and investigating their interaction with other scientific disciplines.

55 My involvement in gas chromatography started in 1957, as the result of a misunderstanding. Christmas 1957 found me as a pennyless refugee in Frankfurt am Main, in West Germany, and I immediately applied for an immigration visa to the U . S . A . However, soon I was told that it would take at least a year to get it; therefore, I should settle there, even if only for a temporary period. So, I started to look for a job. Between 1949 and 1953 I was associated in Hungary with the Research Institute for the Heavy Chemicals Industries and engaged in investigations of coal tar and related products. In connection with these I happened to know the name of the technical representative of the LURGI Companies for Eastern Europe and thus, I sent him my resume. LURGI is one of the largest European companies engaged in developing chemical processes and building factories for these processes; they are located in Frankfurt. I was soon called for an interview and there, I was told that they did not have an opening in process development (after all, my background up to then was in applied chemical engineering); however, there is another possibility. They have a number of analytical instruments in the laboratory among them a "new wonderful machine" - which are being used by everybody. However, since nobody is in charge of them, they never work when needed. Therefore, they decided to hire a chemist and give him this responsibility. "You have a few publications in analytical chemistry" - they said - "would you take this job"? Actually, I did not have an3 analytical publications: those they referred to dealt with pilot plant separation of various tar components, but they misinterpreted their titles. In fact, except for College, I never performed any chemical analysis (and even then, very poorly). However, when you are on the street, the only answer you give is "aye, aye, Sir:", and this is what I said. Of course, the "wonderful new machine" was a gas chromatograph: the PerkinElmer Model 154B Vapor Fractometer. Within a couple of months, everything went.wel1. I purchased a second Model 154, constructed myself a Jan4k-type gas chromatograph* and was able to build up a nice group. It is worthwhile to stop here for a moment and reflect on an interesting question: how can a former chemical engineer who never worked in an analytical lahoratory, jump from one day to the other head on into analytical chemistry? I believe that-the credit for this is not mine but belongs to my former azma mater, the Technical University Budapest. The strength of this school was to give an

*

Today, only a few "old timers'' know what a JanBk-type gas chromatograph was. Here, carbon dioxide was used as the carrier gas and the column effluent was conducted to a nitrometer with sodium hydroxide solution; C02 was absorbed and the separated sample components measured volumetrically. Thus, if the sample volume was known (I used a calibrated Perkin-Elmer gas sampling valve), the concentrations could be calculated directly in vol-%, without the need of any detector response factors.

56

Fig. 6.1. The author in the LURGI laboratory, in 1958, with a home-built Janhk-type gas chromatograph. excellent background on which almost any activity can be built up; after all Hungary is a small country and, particularly in the pre-1950 period, one had difficulty to plan ahead in which type of industry would he be employed after finishing school. It is interesting to note that all three contributors to this volume who are from Hungary (Cs. Horviith, E. s z . Kovhts and myself) studied in the same school, in the Faculty of Chemical Engineering. Also, in general, it is interesting to note the relatively high percentage of scientists with chemical engineering background who are represented in this volume. In this respect, it is proper to quote Dr. C.D. Scott (Oak Ridge National Laboratory) who, in a Chemical & Engineering News interview emphasized how much his chemical engineering schooling helped him in his present, apparently unrelated field. Going back to my activities at LURGI, it was by no means a kind of "routine" analytical laboratory: we had to handle a great variety of samples practically all coming from pilot plant type operations. At the beginning, the bulk consisted of different cracking gases representing the usual problem of separating the Cz-Cq hydrocarbons. For everybody, the separation of butene-1 and isobutylene represented a problem and I learned an excellent method from Ruhrchemie AG how to resolve this: by reaction-gas chromatography. After preseparation on a tetraisobutylene or dimethyl sulfolane column where all C q ' s except these two were separated the stream (using hydrogen as the carrier gas) entered a catalyst column containing finely dispersed platinum on chromatographic support. Here, these two were hydrogenated

57

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Fig. 6.2. Chromatogram of a C4 hydrocarbon mixture obtained by reaction-gas chromatography, by hydrogenation of the olefins after preseparation followed by an additional separation column. (1958; from the author's notes). to n-butane and isobutane. A short separation column followed the catalyst column; there, these two were also separated. I later found out that this method was originally developed at Farbenfabriken Bayer AG. I should add here that years later, at Perkin-Elmer, we again picked up this technique in connection with pyrolysis-gas chromatography, in investigating the thermal degradation products of polyethylenes (I). Another interesting problem was the routine determination of p-xylene in a mixture of aromatics. I could easily separate ?n- and p-xylene from the rest but, of course, could not separate them from each other. Therefore, we separately determined the m/p ratio by IR spectrophotometry; knowing this, it was a simple calculation to establish the composition of the original sample. (A few months ago, I learned that about the same time, Ted Adlard in England had the same problem and solved it in exactly the same way). Probably the most difficult problem I had was the routine determination of isopropyl ether in water, in concentrations of 0.010.05% using a thermistor detector. I solved it by preconcentration, followed by GC analysis on a Carbowax column, using an internal standard. Speaking about my activities at LURGI, I must mention one scientist there: Dr. Karl Bratzler. He was a student of Eucken in the 1930s and spent practically his whole professional life in various studies related to the field on adsorption. In fact, he was one of the first German scientists who was involved in its industrial applications, a field in which LURGI pioneered, and he was also one of the first who wrote a book about the technique (2). Dr. Bratzler was not my boss in the organization structure: he was rather a kind

of senior stateman having his office next to my laboratory and serving as my unofficial mentor. He was teaching me the (quite different) ways how things are done in the West, helped me in solving a variety of scientific, technical and administrative problems, and encouraged my interest in chromatography and adsorption. Without his help, I would never have achieved the knowledge I did. While at LURGI, I participated at my first two symposia. The subject of the first was "Analysis of Gaseous and Liquid Hydrocarbons and their Derivatives Using Physical Methods" and was organized by the German Chemical Society on January 22-24, 1958, in Essen; four months later, I had the good fortune to attend the famous International GC Symposium held in May, in Amsterdam. I still have a copy of my official report on the papers and exhibit; when speaking about Dr. Golay's paper on capillary columns, my only remark was that "his paper consisted of complicated mathematical treatments of the various columns which were difficult to understand" (which really meant that I didn't understand them at all - you don't say this in a report to your boss). I didn't know then that within five months, 1 would be working with him ... I finally immigrated to the U.S.A. at the end of August, 1958 and in a few weeks joined Perkin-Elmer, the company with which I have been associated (not counting a four-year hiatus between 1968 and 1972) ever since. I can thank this relationship to a thunderstorm. During the last week at LURGI, I had an emergency, and urgently needed a part for one of our gas chromatographs. The easiest approach was to go to Perkin-Elmer's office in Frankfurt and pick it up personally. I had a good working relationship with Joe Wolff, then Perkin-Elmer's sales manager for Germany and thus, I also wanted to use this opportunity to say good-bye to him. The moment I opened his door, a very heavy thunderstorm started and I had no other possibility than to wait it out. When chatting with him, he asked me what I planned to do in the United States and I told him that I would stay for a couple of weeks in New York City, until I could find a suitable job. "Would you like to work for Perkin-Elmer?" - was his question and my answer was that certainly, I would be happy to consider this. Then, he sat down at his typewriter, wrote a letter without saying a word, put it in an envelope addressed to Mr. Vincent J. Coates, manager of Applications Engineering in Norwalk, Conn., placed a stamp on the envelope and handed it over to me to put it in the mail box outside the building. Naturally, I did s o . Much later, I found out the content of Joe Wolff's letter. He told Vinnie Coates that "we had so much trouble here with this guy, as our customer, that you better hire him so that he won't be a customer anymore.'' At Perkin-Elmer I was fortunate to be able to join an excellent group at just about the right time: this was the period when gas chromatography probably underwent the most rapid evolution and every day brought something exciting and new. My first job was to do trace analysis with the thermistor detector and I was able to solve it with preconcentration. This work - which was presented at the Spring American Chemical Society Meeting in Boston

by my boss and friend Nate Brenner ( I did not know enough English to present a paper) (3) - had the dubious distinction of becoming obsolete almost at the moment it was done because, obviously, the flame ionization detector introduced commercially in March 1959, at the Pittsburgh Conference, was a much better tool for organic trace analysis. However, for inorganic sample components present in trace concentrations the technique is still valid; and, in fact, present-day more sophisticated preconcentration methods could be traced back to this work. Another early work in which I was involved concerned the use of molecular sieves as subtractors. Nate Brenner had a paper scheduled for the 1959 Pittsburgh Conference and wanted one additional slide. Since I planned to fly to Pittsburgh only on Wednesday (at that time, the GC papers were on Thursday and Friday), he asked me to quickly analyze on Monday a mixture consisting of a small amount of acetone in propionaldehyde which otherwise could not be separated on the column used - to show that now, by removing the matrix, one could analyze the small amount of acetone, have a slide made of the run and bring it with me. However, it didn't work as expected: neither compounds emerged from the molecular sieve column. Subsequent investigations ( 4 , s ) showed that we had a secondary effect here, a reaction between the adsorbed substance (in this case, the aldehyde) and the other component (in this case, the ketone); similar secondary reactions could also be proven for other component pairs. At the Spring 1959 ACS Meeting, in Boston, I heard a lecture by Professor Emmett of John Hopkins University on instrumental techniques used in studying the mechanism of catalysts and in it, he described the combination of a "microreactor" with a gas chromatograph, developed at Gulf Research and the Mellon Institute in Pittsburgh, Pa. I immediately connected this lecture with my past experience: I remembered the difficulties we had back in Hungary and particularly at LURGI in collecting representative gas samples from laboratory and pilot plant reactors and realized the advantages such a system would have as a kind of miniature pilot plant with built-in analyzer; also, I realized that the Perkin-Elmer gas sampling valve could provide a very easy way to build such a system. The system was built in a short time and a number of investigations made. I reported on them first at the Fall 1959 Meeting of the Gulf Coast Spectroscopic Group (this was my first presentation in English!), in Houston, Texas, and then, in more detail at the Spring 1960 ACS Meeting in Cleveland (6). I had the good fortune that Chemical & Engineering News picked up this paper as one of the highlights of the meeting. I have already mentioned that the flame ionization detector of Perkin-Elmer was introduced at the Pittsburgh Conference, in March 1959. About the same time my colleagues at Perkin-Elmer, particularly H.N. Claudy, also developed the so-called hydrocarbon detector essentially a flame ionization detector without a column - to analyze the total contents of hydrocarbons and other organic substances in gases and the atmosphere. A particularly important question related to this instrument was the relative response of organic compounds on

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60

Fig. 6.3. At the First GC Symposium organized by the Chemical Institute of Canada, in Toronto, Ontario, February 1, 1960. Left to right: A. Zlatkis (University of Houston), L.S.Ettre, J.L. Monkman (Canadian National Health and Welfare), S. Sandler (University of Toronto) H. Felton (Du Pont), B.W. Taylor (Fisher Scientific), and D.A.M. Mackay (Evans Research & Development Co.). the flame ionization detector and I became involved in these studies which have been reported at various places, the first being the Symposium on Gas Chromatography held at Toronto, Canada, on February 1, 1960 (7). In 1958, Nelsen and Eggertsen of Shell Development Co. (Emeryville, California) reported on a new (“gas chromatographic”) method for the determination of the surface area of solids ( 8 ) , and, at the Spring 1959 ACS Meeting in Boston, Lee and Stross from the same laboratory described the prototype of an instrument developed for this measurement. This was the so-called Sorptometer and Perkin-Elmer was licenced by Shell to further develop, build and market this instrument. Starting in the Spring of 1959, a major part of my activities for the next two years was related to this instrument and investigations on surface area measurements. These activities also permitted me to build up a closer contact with a number of scientists outside Perkin-Elmer such as Professor Emmett of John Hopkins University, in Baltimore, Maryland, researchers at Shell‘s laboratory in Emeryville, California, and Professor Erika Cremer in Innsbruck, Austria.

61 When I joined Perkin-Elmer, I became part of their Applications Engineering Group and, in the next 10 years, I was associated with this group in various capacities. Particularly in the early development of GC, it was the place where most of the activities centered: every day, visitors came to us, just to say hello or bring new types of samples for analysis by GC. These samples ranged from simple to impossible, and each represented a new challenge. One could tell stories about some odd cases such as the analysis of volatiles in potato chip bags when (after asking for ''a small sample") we received 144 bags (the smallest "samplet1they could ship), or the used basket ball sneekers to be analyzed for bad odor... A favorite sample was represented by the various alcoholic beverages where the remaining part of the samples could always be conveniently consumed. A particular study to remember was related to the direct analysis of such samples with a flame ionization detector in which we concentrated on the peaks emerging before ethanol; these substances are said to be mostly responsible for the well-known headache and hangover. We had a paper on our investigations at the Spring 1962 ACS Meeting in Washington, D.C., and it was given by Frank Kabot, one of my collaborators ( 9 ) - I was in bed with high fever and a bad virus flu during that week. The publicity group of the American Chemical Society picked out this paper and included it in the usual news release; as a conclusion, Frank was surrounded by newspapermen after his lecture, asking the craziest questions. f still remember one of the newspaper headlines which resulted from this interview:"Chemists Claims to Eliminate Hangover." Speaking about newspaper interviews, I will never forget my trip to Winnipeg, Canada, in 1964. I was invited to participate at a symposium together with Dr. W.E. Harris of the University of Alberta. It was the second week of May, and we already had temperatures in the high 8 0 s at home while, upon arriving, the headlines in the newspaper greeting me were: "the ice starts to me1 t..." After the symposium, we had an interview with a newspaper reporter and he tried to get a simple explanation what gas chromatography really does. We presented a number of examples: the planned use in space, the success of the Royal Canadian Mounted Police in cracking a multimillion dollar narcotics ring, the analysis of the alcohol content of blood in case of drunken drivers, etc. However, the reporter still continued to press on and, finally, Dr. Harris gave the ultimate answer: one can determine from the odor of perspiration whether somebody is a schizophrenic. This is all duly printed in the May 11, 1964 issue of the Winnipeg Free Press, and you can imagine the scores of letters we received after this article ... A special field in which I became involved was the utilization of the retention index system developed originally by Dr. Ervin Kovlts at the Eidgenossische Technische Hochschule, in Zurich, in 1958 ( 1 0 ) . Ervin and I both graduated at the Chemical Engineering Faculty of the Technical University Budapest and I have met him many times in Switzerland. His original publication was in German and in a Swiss chemical journal usually not read by analytical chemists.

62

Fig. 6.4. At the 1962 International Symposium in Hamburg. In the middle: R.D. Condon (PerkinElmer), right: L.S. Ettre, left in the background: K.I. Sakodynskii (Moscow). Thus, for some years, I was probably the only chromatographer in the U.S.A. who really read it. More-and-more people started to ask me about this work and, after the 1964 "Zlatkis Meeting", I was the logical choice of Dr. Hallett, then editor of A n a l y t i c a l Chemistry to write a Report for Analysts, based on a couple of papers presented at that meeting ( 1 1 ) . My interest continued in this, most logical system ever since. I have carried out - together with my collaborator Ken Billeb - a number of investigations and I tried to be instrumental in helping the understanding and use of this system in the United States. When, in the fall of 1958, I joined Perkin-Elmer, everybody was excited about the superb chromatograms obtained by Dick Condon on capillary columns invented by Marcel Golay just a little over a year earlier. Slowly, I also became involved in various questions associated with their theory, preparation, utilization and the corresponding instrumentation. In fact, since the early 1960s, this was probably my major field of activities. Major credit should go to Dr. W. Averill who, for a long time, w a s the indisputable master of

63

Fig. 6.5. After the 1963 "Zlatkis Symposium" in Houston, Texas: Professor A.I.M. Keulemans (left) visiting Perkin-Elmer. column preparation and application, and naturally, I learned a lot from Dr. Golay with whom I had the good fortune to work together for many years. It is very interesting to survey the special fields in which we were active (apart of routine application-type work where we had to demonstrate the applicability of the column for certain sample types) because these also show the general concerns which gas chromatographers had. Probably the first such question was related to the split type sample introduction systems where their "linearity" was questioned mainly because of unsatisfactory experiences with the early, crude systems. We thus, had to carry out some basic work to prove their "linearity" (i.e., non-discrimination) and describe ways how this can be tested ( 1 2 ) . We also had to demonstrate a number of times the possibility of doing quantitative analysis with GC systems employing capillary columns ( 1 3 ) . Another interesting question was to investigate the possibility of using larger diameter tubing for open tubular columns and to relate their performance to the fundamental relationships of such columns (which are independent of diameter) ( 1 4 ) . In the very first papers on open tubular columns Dr. Golay already proposed to try to add (or form) a porous layer on the inside wall of column tubing. Many ways were tried to accomplish this but the first really reproducible method was developed by Csaba Horvlth in his Ph.D. Thesis work at the University of Frankfurt/Main, in Professor Hallsz' laboratory. The first paper on these columns was presented at the first "Zlatkis Meeting", the International Symposium on Advances in Gas Chromatography held in January 1963 ( 1 5 ) . I knew both of them for a long time: in fact, Dr. Horviith w a s a classmate

64

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Fig. 6.6. Chromatogram obtained on a 50-ft. SCOT column coated with squalane liquid phase. Phase ratio: 67. Column temperature: 75OC; carrier gas (He) average velocity: 21.5 cm/sec. The HETP values for peak 22 (b1.31) and 36 (k=3.07) were 0.42 mm and 0.47 mm, representing a 83% and 81% utilization of the theoretical efficiency, respectively. Peaks: 13, n-pentane; 17, cyclopentane; 22, methylcyclopentane; 26, benzene; 28, cyclohexane; 36, n-heptane and 39, methylcyclohexane. From ( I ? ) . of my wife at the Technical University Budapest; thus it is natural that I learned about his work very early. It was fortunate that Perkin-Elmer also became interested in these columns representing a logical extension of Golay's original work and thus, in the next five years the main activity of my group was related to the further development of the support-coated open tubular (SCOT) columns. The principal credit for our achievements is due to John Purcell, my collaborator and, in 1968, my successor as the head of the GC Applications Group. Our investigations concerned many aspects: theory, preparation, optimum conditions, development of various types of columns with different phase ratios, etc., and, last but not least, technical marketing to make these columns and their characteristics known to everybody. Our first report was published in 1965 ( 1 6 ) , and it was followed by a number of publications, data sheets, reports and lectures.

65

Fig. 6.7. With Dr. Boer (Koninklijke/Shell, Amsterdam) in 1974 during the International Chromatography Symposium, in Barcelona, Spain, in the palace where Queen Isabella received Columbus after his triumphal return from the New World. A personal recollection of the events of the past 20 years would not be complete without mentioning two very special features of chromatography development: the organization of periodic national and international symposia and the publication of specialized journals. As already mentioned, I started in 1958 with the German nation-wide symposium soon followed by the 1958 Amsterdam Symposium of the Gas Chromatography Discussion Group and participated at most international symposia ever since, from Las Vegas, Nevada (U.S.A.) to Samarkand, Uzbekistan (U.S.S.R.). In 1963 Dr. Zlatkis of the University of Houston asked me to help in the organization of the first International Symposium on Advances in (Gas) Chromatography and from then on, I had the good fortune to be able to cooperate with him in the organization of these "Zlatkis Meetings". International Symposia represent a very important forum for the free exchange of ideas; they bring scientists from various countries together; and it is safe to say that without them, chromatography could not have developed as rapidly as it did. The other special feature of chromatography is the existence of specialized journals devoted solely to this discipline. The first, of course, is the Journal of Chromatography, founded by M. Lederer in 1958. The two others are the Journal of Chromatographic Science (originally the Journal of Gas Chromatography) and Chromatographia and I have been involved in both of them. The Journal of Gas Chromatography was founded by Seaton T. Preston Jr., himself a pioneer in gas chromatography at Podbielniak Co., an old friend of mine. I remember well our discussion during the Fall 1962 National American Chemical Society Meeting in Atlantic City (my wife presented a paper there on the first modern pyrolysis-GC unit) when he told me about his plans to start this journal in January 1963 and asked me to participate in the Advisory Board. For certain

66

reasons, I was not listed as such in the first couple of years but this did not restrict me to closely cooperate with Seaton every since. The other journal I am closely associated with is Chromatogrqhia which originally was the brainchild of Rudolf Kaiser, then with BASF in West Germany. In September 1970, I passed through Frankfurt/Main and he came to see me in the hotel to persuade me to accept the editorship for the Americas. I have been involved in editing this journal since then. I believe that the three journals now existing for a number of years serve a very important purpose and that, by having somewhat different profiles and editorial philosophies, they really do not compete with each other. Recently new journals started to mushroom in the field of chromatography. Time will tell whether this overspecialization serves a purpose or simply splits the available talents and funds. Twenty years represent a long time in an individual's life and particularly in the period of his professional activities. Looking back to my 20 years in this field, my most important conclusion is that I had the good fortune of working together with many scientists, chemists and engineers in the field of instrumental analytical chemistry in general, and at Perkin-Elmer in particular. I would like to use this opportunity to thank all of them for their friendship, cooperation and help. REFERENCES 1 B. Kolb, G . Kemner, K.H. Kaiser, E.W. Cieplinski and L.S. Ettre, 2. Anal. Chem. 209 (1965) 302-312. 2 K. Bratzler, A d s o q t i o n von Gases und Diimpfen i n Laboratorium und Teehnik, Steinkopff Verlag, Dresden und Leipzig, 1944. 3 N. Brenner and L.S. Ettre, Anal. Chem. 31 (1959) 1815-1818. 4 N. Brenner, E. Cieplinski, L.S. Ettre and V.J. Coates, J . Chromatogr. 3 (1960) 230-234. 5 L.S. Ettre and N. Brenner, J . Chromatogr. 3 (1960) 235-238. 6 L . S . Ettre and N. Brenner, J . Chromatogr. 3 (1960) 524-530. 7 L . S . Ettre and H.N. Claudy, Chem. Canada 12 (9) (1960) 34-36. 8 F.M. Nelsen and F.T. Eggertsen, AnaZ. Chem. 30 (1958) 1387-1390. 9 F.J. Kabot and L.S. Ettre, I n s t m e n t flews 1 3 (4) (1962) 1-9; B r i t i s h Food J . 65 (1963) 72-74. 10 E. KovAts, HeZv. Chim. Aeta 41 (1958) 1915-1932. 11 L.S. Ettre, Anal. Chem. 36 (8) (1964) 31A-47A. 12 L . S . Ettre and W. Averill, Anal. Chem. 33 (1961) 680-684. 13 L.S. Ettre, E.W. Cieplinski and N. Brenner, IsA Transactions 2 (1963) 134-140. 14 L.S. Ettre, E.W. Cieplinski and W. Averill, J . Gas Chromatogr. 1 (2) (1963) 7-16. 15 I . Hallsz and Cs. HorvPth, Anal. Chem. 35 (1963) 499-505. 16 L.S. Ettre, J.E. Purcell and S.D. Norem, J . Gas Chromatogr. 3 (1965) 181-185. 17 L.S. Ettre, in Gas Chromatography 1966 (Rome Syrnposiwn), A . B . Littlewood, ed., Institute of Petroleum, London, 1967, pp.115118.

67

PER FLODIN

PER FLODIN was born in 1924 in Ljushult, Sweden and studied at the University of Uppsala where he received his Ph.D. in 1962. Between 1950 and 1953, he worked with Professor Tiselius at the Department of Biochemistry and obtained a licentiate degree in 1953. Between 1954 and 1962, he was a research scientist at the pharmaceutical company Pharmacia AB, in Uppsala. In 1962, he was appointed as the research director of Perstorp AB, a chemicals and plastics manufacturing company. In 1972, he was appointed docent (associate professor) at the Royal Institute of Technology in Stockholm and in 1977, professor of polymer technology at Chalmers University of Technology, in Goteborg. Dr. Flodin is the author and coauthor of a number of scientific publications and patents. In 1962, his book on Dextran Gels and Their Application t o Gel F i l t r a t i o n was published by Pharmacia. He is vice president of the Swedish Chemical Society and a committee member of the Swedish Board for Technical Development. He was actively engaged in the foundation of the Swedish Plastics and Rubber Institute and is now a member of its Board. He is a recipient of the Arrhenius medal of the Swedish Chemical Society (1963) and of the gold medal of the Swedish Academy of Engineering Sciences (1968). Dr. Flodin’s major activities in chromatography started in 1956, when jointly with Dr. J . Porath, they made the fundamental observations leading to the gel filtration method. He developed the Sephadex range of dextran gels, the Sephadex ion exchangers, and various other derivatives of dextran gels, e.g. for use in gel chromatography in nonaqueous media. In methodological studies, he developed experimental techniques and gel types for”variousapplications, such as the separation of small molecules from macromolecules, fractionation of watersoluble oligomers and polymers, and preparative separation of proteins. A qualitative theory of gel filtration and the development of a method for industrial gel filtration are among his achievements. He also had major contributions to biochemistry arid polymer technology.

68 In 1950 chemistry in Uppsala was dominated by two Nobel prize winners, The Svedberg and Arne Tiselius. Svedberg was still very active as director of the Gustaf Werner Institute for Nuclear Research. He had retired a few years earlier from the chair in physical chemistry but still his spiritual influence was strongly felt by those working in the laboratories. The Department of biochemistry under Arne Tiselius had laboratories in the physical chemistry building. Since he received the Nobel Prize in 1948 his department was soaring with activity. A new laboratory was being built, money for equipment was available and a stream of scientists visited the lab and some stayed for extended periods. At times almost half of the staff was non-Swedish. Visitors came from well-known laboratories all over the world and the department certainly was one of the centers of international development in the field. This year I finished my undergraduate studies and was admitted to the Tiselius laboratory as a graduate student. Since the lab was crowded, I was squeezed into his private lab in which at times up to ten people worked. Tiselius had received the Nobel prize for his contributions to electrophoresis and chromatography. His personal interest at that time was mainly chromatography and in particular displacement chromatography on activated carbon. The heart of the equipment at disposal was two interferometers used to analyze the effluents from columns. The group was quite international with some people working on methodology and others on applications to biochemical problems. Being surrounded by skilled scientists working on a variety of separation tasks, it is no wonder that I became deeply involved in chromatography in spite of the fact that I did not work directly with it. Among the applied problems tackled, one was the separation of hormones. It was inspired by C.H. Li and the organic chemist Jerker Porath was engaged to work with the problems. He and I became close friends and had almost daily discussions over a cup of coffee. The first result of our "brain storming" was a short communication in Nature on a new method to detect amino acids on paper chromatograms. When Henry Kunkel of the Rockefeller Institute left Uppsala I continued his work on paper electrophoresis of serum proteins. My task was to find out how to diminish zone spreading in the type of arrangement they used, i.e. a paper sandwiched between glass plates. Though much effort was put into the work only marginal improvements were obtained. It was now 1951 and we moved into the new building of the Department of Biochemistry. The space available was so much larger that I more or less lost contact with chromatography. Electrophoresis gradually became more important to me. I shall briefly describe it here since it was of decisive importance for my later chromatographic work, Tiselius' work on electrophoresis had mainly concerned boundary electrophoresis. This is an analytical method and not suitable for preparation since the proteins were only partially separated. Great efforts were made to find preparative procedures by Harry Svensson

69 (now Rilbe), Ingrid Brattsten, Henry Kunkel and others. In one approach continous electrophoresis in a trough packed with glass beads or in a paper curtain was used. In another batchwise separation in columns packed with glass beads was used (Svensson and Haglund). However due to adsorption effects relatively large beads had to be used which in turn caused operational difficulties, particularly when eluting the electrophoretically separated proteins. Porath and I decided to try starch granules as a support in zone electrophoresis in columns. We were quite successful and used the method for many types of preparative separations ( I ) . Later Kupke and I got still better results using a form of partially hydrolyzed cellulose similar to mycocrystalline cellulose (2). In this work it was necessary to keep zone spreading as low as possible, A lot of time was therefore devoted to the study of techniques for packing, application of samples and elution. We also observed that proteins and small molecules behaved differently on starch compared to filter paper and cellulose columns. In the first place only about half of the water in the column was available to proteins. To low molecular weight substances almost all the water was available. Such a difference was not,observed for paper and cellulose. We concluded that the effect could be used to separate large and small substances but did not pursue the subject further. Later, Lindqvist and Storgards used the phenomenon to separate peptides (3) and Lathe and Ruthwen studied the elution behaviour of a number of substances in starch column ( 4 ) .

Pharmacia Late in 1953 I obtained a licentiate degree and was immediately offered a job at the pharmaceutical company AB Pharmacia at Uppsala. The manager of the research laboratory, Bjorn Ingelman, was one of the inventors of Yacrodex, a plasma extender based on the polysaccharide dextran. It was growing rapidly and was one of the most profitable products of the company. However, management wanted a new product based on dextran and I was to help Bjorn Ingelman in this applied research. Thus I believed I had left chromatography and electrophoresis for good. On of the reactions I studied was the one between dextran and epichlorohydrin. Ingelman had earlier studied the reaction and had obtained gels. I confirmed his results and also found that swollen gels were brittle and could easily be disintegrated while dry ones were extremely tough. Jerker Porath and I kept on meeting to discuss scientific methods in spite of our seemingly diverting interests. Among other things he was busy looking for more inert supports for column electrophoresis. The structure of dextran was well known from the work of Kirsti Granath and others and therefore we considered it worth while to test crosslinked dextran as support. It took me some time to mdke so much material of the narrow particle size distribution required for electrophoresis. He found it to be an excellent support with very little adsorption of proteins. Among other things he noted that it behaved like starch and not like cellulose when large and small molecules were passed through the column.

70 Gel filtration The significance of these results were only gradually apparent to us. For chromatography in the traditional manner the separation volumes were too small. Thus great care had to be taken to avoid zone spreading and only small samples, compared to the bed volume, could be separated. For me the decisive moment was when I realized that the dextran gels could be used to replace dialysis. Since I was employed by a commercial enterprise I had to be able to indicate a potential market to get authorization to continue. The dialysis concept made this possible since I could indicate a market for lab use and also as a unit operation for industrial separations. According to lab records, all this took place between October 1956 and March 1957. In May that year I made a formal proposal to the management of Pharmacia. In it I suggested that the company engaged in the development of dextran gels for four applications: (1) as a support in electrophoresis in columns; ( 2 ) as a backbone for ion exchangers; ( 3 ) as molecular weight selective membranes for electrophoresis of blood serum; and ( 4 ) for separation by "restricted diffusion" in gels; in particular to replace dialysis. Much due to the wholehearted support by Bjorn Ingelman, the project was authorized and more people became involved. Among these was the late Bertil Gelotte, who later on was to be managing director of Pharmacia Fine Chemicals. Now things began to develop rapidly. In one year gel materials were developed, produced in development quantities, the limits of the separation methods investigated, material for patent applications developed and so on. In a PM dated March 1958 Gelotte and I reported much of the basic facts of gel filtration. Since we cooperated closely with Porath at the Department of Biochemistry we were also able to report his work on peptide separation on dextran gels and on ion exchangers based on dextran gels. Later in 1958 a project group was constituted with responsibility for all aspects of the project. The members were Sven Boode, chairman (now executive vice president of the Fortia Group to which Pharmacia Fine Chemicals belongs), Bertil Gelotte and I. It was decided to introduce the first products in the summer of 1959. Since we had a very limited budget, we had to start with only a single market. The U . S . market was chosen due to the large number of potential users there and also because we felt there was a real need for such a method. The products were given the name Sephadex (Separation, Pharmacia, dextran) and the separation method was called gel filtration as suggested by Arne Tiselius. He also arranged that I got an invitation to a Gordon Conference on Proteins in New Hampton, N.H., from the chairman, Professor Stanford Moore of the Rockefeller University. All activities were from now on (Jan. 1959) directed towards introduction in connection with my lecture at the Gordon Conference. Porath and I wrote a communication to Nature, information material was prepared for distribution to biochemists and Sephadex was produced and shipped to the U.S.

71 At the Gordon Conference which was attended by about one hundred protein chemists, I presented the results of a large number of separation experiments made by Porath, Gelotte and me. The report created quite a sensation and most of those present believed in the results. A few doubted them and there was a hot debate. During the meeting Stanford Moore told us that he had just obtained the issue of Nature where our paper was published ( 5 ) . The timing had been perfect. News about the method evidently spread very rapidly over the U.S. since when I returned to New York two weeks later all Sephadex in stock had been sold. In fact throughout 1959 production had difficulties to keep pace with demand. First electrophoresis in filter paper and in columns had been the means. Now together with. Johan Killander I was fortunate enough to be able to introduce a new dimension, i.e., size into plasma protein separation (6). An early development was the ion exchangers made by etherification of hydroxyl groups of Sephadex. Tertiary amine, carboxyl and sulfonic acid containing varieties were introduced in 1960. Like the cellulose ion exchange developed by Sober and Peterson they were useful for protein separations. However, the particle volume was very sensitive to the ion environment which made the exchange somewhat difficult to handle in chromatographic experiments. In spite of this and a competitive market they have been relatively successful. It was obvious to us that gel filtration in organic solvents should be a logical extension of the field of application of the method. Since a policy decision was made to give the water systems (i.e., biochemical applications) top priority relatively little effort was made to develop gels for other solvents. However, some were made at an early stage, notably acetate esters and hydroxypropyl ethers of Sephadex. It was left to others to explore the field. Thus Vaughan used lightly crosslinked rubber to separate a number of substances in hexane. The great leap forward came, however, in 1964, when J.C. Moore of the Dow Chemical Company in Freeport, Texas introduced macroporous styrene-divinylbenzene gels for separation over a wide range of molecular weight.

The Second Phase of Development The instant success with Sephadex convinced us that we had hit upon something important, not only from the scientific but also from the commercial point of view. The market introduction had been made with a minimum number of gel types and with only gel filtration applications in mind. However, we had a number of improvements and new products available for development in our labs as well as numerous ideas of applications. Of great help in the further development was the very fine feedback we had from the customers. At first the gels were made in blocks by performing the crosslinking reaction of dextran with epichlorhydrin in troughs. After reaction the blocks were ground, dried and sieved. The particles obtained were irregular in form and had a broad size distribution. A major improvement was the development of a method to make spherical beads. It facilitated production and made more uniform quality possible. However, it was on the application side the greatest advan-

72

Interferometer

reading

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Fig. 7.1. Separation of oligosaccharides from cellulose in a column packed with Sephadex G-25. Peaks: 1, glucose; 2 , cellubiose; 3, cellutriose; 4 , cellutetraose; 5 , cellupentaose and 6 , celluhexaose. From ref. 5. tages were gained. Firstly the spherical form and the narrow particle size distribution gave low hydraulic resistance in the chromatographic columns. Thus smaller particles could be used with improved resolution as a consequence. Secondly, more open gel structures could be used allowing separation in higher molecular weight ranges. This also was a consequence of the lower hydraulic resistance of the spherical beads which made possible the use of gels with lower compression modulus and thus a more open structure. Thus the G-200 type of Sephadex came into existence. It made possible the separation of moderately large proteins according to size. Since my first contact with biochemical research ten years earlier, I had a desire to improve separation of the proteins of blood plasma.

Farewell t o Sephadex Late in 1961 I was offered a leading position in another company. I then decided to write a thesis to get a doctor's degree before leaving Pharmacia. The problem was that I had so much material to present and only six months at my disposal. The result was a monograph with the title Dextran Gels and t h e i r AppZications i n Gel F i l t r a t i o n ( 8 ) . In the monograph I had to treat the preparation of gels and derivatives without much detail being added to the information given in our patents. Three parts of the monograph concerns work which has not been mentioned above but has been important to me. In the first place, dextran was not only used as a raw material for Sephadex, but also as a test material for the performance of columns. Thanks to the work of Dr. Kirsti Granath at Pharmacia, it was one of the best characterized polymers and we had numerous fractions with known molecular weight distributions at our disposal. It was natural for us to try to substitute the tedious

73

A* 500

700

ELUTION

Fig. 7 . 2 . Elution patterns of bovine plasma proteins in columns packed with three types of Sephadex. Upper diagram: G-75; center diagram: G-100, the right-hand peak contains albumin; lower diagram; G-200, the right-hand peak contains albumin, the center peak y-globulin and the left-hand peak high molecular weight globulins. From (8). 900

1100 ML

VOLUME

precipitation fractionation methods by gel filtration. Consequently we reported results on the determination of distributions already in 1959 ( 9 ) and a more comprehensive report later ( 1 0 ) . The second point I should like to mention is the efforts to make gel filtration a unit separation for production use. One of the strongest arguments for the company management to authorize the gel filtration project in 1957 was the possibility to use the process for technical scale production. An automatic process operating in cycles was developed to meet this requirement. It was designed to separate macromolecules from low-molecular-weight material, i.e., for applications similar to what could be achieved by dialysis. Much due to the efforts of Mr. Arne Emnbus this work was successful. In my thesis we reported an experiment comprising 396 cycles in 33 days without interruption. However, the industrial use of gel filtration developed much slower than we had anticipated but it was more than compensated for by the rapidly increasing lab use. The question of which mechanism governed the gel filtration process bothered us a lot. To be able to work in a constructive way it was necessary for me to have a simple concept to start from. A "molecular sieve" mechanism in which the diffusion process in and out of the gel particles were fast in comparison to the rate of transport along the column, i.e., near-equilibrium conditions, was an intellectually satisfactory concept. I made experimental investigations to test it and found strong support for the hypothesis. Accordingly I tried to formulate a more detailed mechanism which was presented in

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Fig. 7.3. Arrangement used for automatic gel filtration to remove salts and other low molecular weight solutes from macromolecules. From (8). A, column; B , distributor; C1 and C2, pumps; D1, two-way solenoid valve; D2 and D3, three-way solenoid valves; E, timer; F1 - F3, cam pairs.

my thesis. It has later been confirmed by several authors that my basic thinking was correct. Only two weeks after the dissertation I left Pharmacia for another company and thus had to say goodbye to Sephadex and gel filtration. Now, sixteen years later when I look back at the "chromatography period" these years stand out as some the best of my life. REFERENCES 1 P. Flodin and J. Porath, Biochim. Biophys. Acta 13 (1954) 175. 2 P. Flodin and D.W. Kupke, Biochim. Biophys. Acta 21 (1956) 368. 3 B. Lindquist and T. Storgards, Nature 175 (1955) 511. 4 G.H. Kathe and C.R.J. Ruthven, Biochem. J . 62 (1956) 665. 5 J. Porath and P. Flodin, Nature 183 (1959) 1657. 6 P. Flodin and J. Killander, Biochim. Biophys. Acta 6 3 (1962) 403. 7 P. Flodin and K. Aspberg, in Biological Structure and Function, J. Goodwin and B . Lendberg, eds., Vol. 1, Academic Press, New York, 1960, pp. 345-349. 8 P. Flodin, Dextran Gels and T h e i r Application to Gel F i l t r a t i o n , Pharmacia, Uppsala, 1962. 9 P. Flodin and K. Granath, Symposiwn iiber MakromoZekuZe, Wiesbaden, W. Kern, ed., Verlag Chemie, Weinheim, 1959-1960, lecture 11-C-6. 10 K. Granath and P. Flodin, Makromol. Chemie 4 8 (1961) 160.

75

CHARLES W. GEHRKE

CHARLES WILLIAM GEHRKE was born in 1917 in New York City. He studied at the Ohio State University receiving a B.A. degree in 1939, a B . S . degree in education in 1941 and a M.S. degree in 1941. From 1941 to 1945 he was professor and chairman of the Department of Chemistry at Missouri Valley College. In 1946, he returned to Ohio State University as an instructor in agricultural biochemistry and received his Ph.D. degree in 1947. In 1948 he joined the College of Agriculture of the University in Missouri, Columbia, where at present he is professor of biochemistry and manager of the Experiment Station Chemical Laboratories. His duties also include those of State Chemist for the Missouri Fertilizer and Limestone Control Laws. Dr. Gehrke is the author of over 175 scientific publications in analytical and biochemistry. His research interests included the automation of analytical methods for nitrogen, phosphorus and potassium in fertilizer and for other biologically important molecules, e.g. spectrophotometric methods for lysine, methionine and cystine, the development of quantitative gas and liquid chromatographic methods for fatty acids, amino acids, purines, pyrimidies, nucleosides and biological markers in cancer detection, and the characterization and interaction of proteins. Dr. Gehrke has been an invited scientist on GLC analysis of amino acids at many universities and institutes in the United States, Europe and Japan. As an invited teacher under the sponsorship of five Central American governments, he taught chromatographic analysis of amino acids at the Central American Research Institute for Industry, in Guatemala. He participated in the analysis of lunar samples brought back by the Apollo 11-17 missions for amino acids and extractable organic compounds with Professor Cyril Ponnamperuma (University of Maryland) and a consortium of scientists with the National Aeronautics and Space Administration. In 1974 he was invited by the Soviet Academy of Science to make the summary presentation on organic substances in lunar fines at an international symposium held in Moscow. Dr. Gehrke is the recipient of the Harvey W. Wiley Award in Analytical Chemistry of the Association of Official Analytical Chemists,

76 the Senior Faculty Member Award, College of Agriculture of the University of Missouri and the Faculty Alumni Gold Medal from the University of Missouri Alumni Association. Professor Gehrke's involvement in chromatography started in the early 1960's first with investigations on improved methods for fatty acid analysis. He is most widely known, however, for developing a reliable gas chromatographic method for the gas chromatographic analysis of amino acids. This method was applied to the analysis of lunar samples when he was a co-investigator with NASA during this period. In the 1970's Professor Gehrke's major interest slowly shifted towards the development of quantitative HPLC methods for the analysis of various important substances in biological samples especially the modified nucleosides as biomarkers in cancer research.

77 I am deeply honored to participate in this treatise commemorating the 75th anniversary of the first reported research on chromatograph by M.S. Tswett. Analytical chemistry and biochemistry,are changing disciplines and, since 1951, revolutions have occurred that will have a dramatic impact through the coming years. These changes have been brought about to a large extent by chromatography. We are now in a period of chromatographies interfaced with high?> and low-resolution mass spectrometry and computers for data reduction. Some of our most important environmental problems are being solved with this array of instrumentation combined with sensitive and selected analytical and chromatographic methods. At Missouri, in the Experiment Station Chemical Laboratories, our major goals since 1962 have been the development of analytical and chromatographic methods as tools useful in research in biochemistry, agriculture, space sciences, and the medical sciences. Automation of these methods has also been of importance. A concerted effort has been placed on the development of quantitative GLC methods for amino acids from the macro to nanogram levels; non-protein amino acids (about 50 molecules); the search for amino acids in the Apollo 11-17 lunar sample fines. As we found, the analysis of lunar samples for indigenous amino acids is a search for the nonexistent. A considerable effort has also been made to develop methods for major and modified nucleic acid bases as markers for the diseased state. Our latest research efforts (1976-78) have been directed toward the development of quantitative high performance liquid chromatography methods for nucleosides in biological materials (plasma, tissue, urine), and hydrolysates of RNA and DNA with the measurement of more than 30 major and modified nucleosides. Also, simple, sensitive, quantitative, high performance liquid chromatographic methods have been developed at the nanogram level for measuring neurotransmitters as histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin, and tyramine in plasma, tissue, or other biological fluids. The world of analytical chemistry has not been the same since 1951. The analytical laboratory of today is far different from that in which we were trained, and the diverse types of chromatographies available to us have greatly modified our approaches to the problems of analysis and constitute important methods in every discipline of the biological sciences. Some of our earliest work was on the gas chromatography of the volatile fatty acids in rumen fluids and, in 1977, we again published a definitive report on a Rapid Microdetermination of Fatty Acids i n Biological Materials by Gas-Liquid Chromatography ( I ) using a an internal standard method and 'on-column' methylation with trimethyl (a,a,a-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH). I will now discuss in more detail the four most important areas of our activities involving different chromatographic techniques in research on amino acids, lunar sample studies, biologic markers in cancer, and HPLC of biogenic mines.

78 Amino a c i d s Our work in this field started in 1962 with the gas chromatographic analysis of protein amino acids. At that time amino acids were analyzed by bacteriological, paper chromatographic and manual ion-exchange methods. A method was needed to rapidly and accurately determine the amino acids in agricultural and other biological samples. Our investigations covered a number of questions. The results can be summarized in the following points: (a) Direct esterification of the amino acids; (b) development of special columns and analytical conditions; (c) investigation on the hydrolysis of proteins as a function of time and temperature; (d) the successful routine analysis of amino acids in biological substances (blood plasma, corn and soybean grain hydrolysates, urine); and (e) the analysis of nanogram amounts of amino acids using a "solvent venting" system. GLC ANALYSIS OF A FINGERPRINT AMINO ACID N-TFA g-BUWL ESTERS FINAL SAMPLE VOLUME 6081 INJECTED: 4 5 ~ 1 VENT TIME: 45 SEC. INITIAL TEMh: 55% PROGRAM RATE: 6'1MIN. TO 230 COLUMN 0.65 w/w X EGA ON uUl00 MESH CHROMOSORB W, WITH SOLVENT VENTING SYSTEM

j

AMINO ACID STANDARD

1

VJVq#i

TY ,o/&c R

IIILJU 80 0

5

100

120 10

140

15

160

180 20

200'w220230-lsotC 25

30

35MIN

Fig 8.1. GLC analysis of a fingerprint and a standard mixture containing 20 ng of each amino acid. Conditions and peak identification are indicated in the figure. "Vented solvent" refers to the use of the device developed by us to prevent interference from TFA. From the number of publications I would like to quote only a few from the early period dealing with fundamentals (2-6) and then a later one discussing never developments in the GLC analysis of protein amino acids ( 7 ) . The use of gas-liquid chromatography (GLC) has become an important method for the analysis of amino acids. The classical ion-exchange methods of Nobel Laureates Stein and Moore developed in the

79 ;

1

NOTHYDROLYZED

h

\

E/

2 -o_ 55

coulyk Om%ro1ON W/l/1oD YBI A.W. aydKMu W. 1.5 rn x 4 r m 1.D. aulu

6 91

127

163

----

24 33 MIN 199 2040 Is0 -204 oc

Fig. 8.2. GLC Analysis of the water extract of Apollo 17 fines. Note three peaks: they are not amino acids and disappear on hydrolysis. early 1950's are now complemented and supplemented by these excellent GLC methods. Several types of derivatives have been used but the most reliable and common are the N-trifluoroacetyl (N-TFA) n-butyl esters developed by us. We have also developed a special device ("Sol-Vent") to the injection port which allows injection of large amounts of samples ( 8 - 9 ) . This results in greater sensitivity, accuracy, and precision, especially for very small samples. These GLC methods opened doors to researchers because of their rapidity, sensitivity, simplicity, accuracy, and economics and are now being adopted widely throughout the world. In our research, attention was directed to sample preparation methods as these are as vital in amino acid analysis as the methods of measurement. We conducted experiments to obtain a rapid, accurate, and precise procedure for protein hydrolysis and sample cleanup with subsequent gas-liquid chromatographic analysis. The use of ultrasonication and reduced pressure to remove dissolved air from the sample solution prior to hydrolysis assured a good recovery for methionine and cystine. These techniques combined with a 4 hour hydrolysis at 145O using 6 N HC1 gave results in good agreement with the hydrolysis conditions of 18-24 hours at llOo. We have prepared the physiological fluids for free amino acid analysis by precipitating the protein with saturated picric acid followed by cation-exchange clean-up, The techniques for sample preparation and chromatographic analysis of amino acids developed by us provide the chemist with valuable tools for the analysis of biological samples by gas-liquid chromatography. Over 5,000 requests for reprints have been made for our papers on the GLC analysis of amino acids; Norway set up a central laboratory

80

x PROCEDURAL BLANK, HYDROLYZED

APOLLO 17. 72501.62, UNHYDROLYZED

E APOLLO 17.72501.62. HYDROLYZED

--v-(

::

long AMINO ACID STANDARD

ASP ;THRnSER ,

GLY fl +LA

I

F i g . 8 3 c 1 s s i c a l ion-exchange (CIE) a n a l y s e s o f Apollo 17 Lunar fines

.

APOLLO 17.70011.37. UNHYDROLYZED

APOLLO 17,70011.37, HYDROLYZED

2ng AMINO ACID STANDARD

F i g . 8 . 4 . C.W. Gehrke ( l e f t ) and R . Zumwalt i n t h e Lunar S c i e n c e Clean Room a t NASA's Ames Research Center, i n 1 9 7 2 .

81 at its Agricultural Research Station in Aas for this determination and at least 5 0 scientists each year, in the early 1970's, from many laboratories in England, on the continent, Sweden, South Africa, Japan, Central and South America, and others have come to our laboratories in Columbia, Missouri, to learn directly of these methods.

Lunar science In recognition of our work, I was selected as a co-investigator in 1969, with Professor Cyril Ponnamperuma of the National Aeronautics and Space Administration for the analysis of amino acids and selected organic molecules in the lunar samples returned by Apollo missions 11-17. The search for extraterrestrial life or evidence of chemical evolution has been one of the main driving forces in space exploration. The theory of chemical evolution postulates that when this planet was much younger, a sequence of chemical events took place leading eventually to the origin of life. The chemical events started with interaction of the simple elements, C, H, N, and 0, and led to the molecular monomers of amino acids and the genetic bases. We were privileged and honored to be included in the select group of scientists who were first fo analyze the soils of the moon which had been unreachable since its origin 4-5 billion years past. In 1969 our methods of GLC analysis of amino acids had a sensitivity factor of at least lOOX over then current commercial models of classical ion-exchange chromatography (CIE). We could detect with certainty any amino acids which might have been present in lunar fines at a level of 2 ng/g ( 2 ppb). Later, in 1973, we also used the sophisticated and equally sesitive Durrum amino acid analyzer and GLC in our search for indigenous amino acids in the lunar samples ( 1 0 ) . These were exciting years for me and my staff as we conducted our investigations in organic clean rooms across the United States, travelling to the NASA Ames Research Center near San Francisco, to Houston and the L.B. Johnson Manned Spacecraft Center, the University of California at Berkeley, and the Laboratory for Chemical Evolution at the University of Maryland. I take this oppotunity to recognize members of my staff and graduate students who contributed a part to history and worked untiringly and with dedication over the years in the search for a trace of amino acids in lunar fines: R. Zumwalt (assistant professor at College of Veterinary Medicine, University of Missouri, Columbia, Missouri) and K.C. Kuo (senior research chemist at the University of Missouri Experiment Station Chemical Laboratories, Columbia, Missouri) for their brilliance and innovative ideas throughout the total program of studies, D. Stalling (chief chemist, National Fish Pesticide Research Laboratories, Department of the Interior, Columbia, Missouri), W. Aue (professor at Dalhousie University, Halifax, Nova Scotia, Canada), J. Rash (chief chemist at Pfizer Pharmaceutical Co., Groton, Connecticut) and D. Roach (chairman, Department of Chemistry, Miami Dade Community College, Miami, Florida). It was most stimulating to work with such noted scientists as Cyril Ponnamperuma, Project Leader and Principal Investigator, Keith Kvenvolden, Lunar Laboratory Manager, as well as with Elso Barghorn,

Sherwood Chang, Chao-Nang Cheng, Paul Hamilton, K. Harada, P . E . Hare, Jim Lawless, Bart Nagy, Glenn Pollock, Carl Sagan and Akira Shimoyama. A high point in our investigations was in 1972 at the Space Sciences Laboratories in Berkeley, when the "Missouri group" was assigned to check out the "cleanness" of the new Berkeley clean room. The Apollo 14 lunar sample returned by Admiral Shepard was not opened at Houston, but at Berkeley, but not until it was known that the Berkeley laboratory was clean to the satisfaction of the searchers for amino acids; we found it so. The lunar samples from Apollo flights 11-17 provided a unique opportunity for the study of chemical evolution via the examination of extraterrestrial materials for evidence of prebiological organic chemical processes. The characterization of carbon compounds indigenous to the lunar surface was of particular interest as these investigations could result in findings which would advance our knowledge of the processes of chemical evolution.

Fig. 8.5. C.W. Gehrke as the U.S.A. representative at the International Seminar on "The Origin of Life" organized in August 1974 at Lomonosov State University, Moscow by the Academy of Sciences of the Soviet Union on the occasion of the 50th anniversary of the publication of Oparin's book The O r i g i n of L i f e . Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ion-exchange chromatography and gas chromatography combined with mass spectrometry were

83 used for the analyses. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigations of 1 to 2 ng/g. In Apollos 11, 12, and 14, wide publicity was given to the announcement that some amino acids were found in lunar fines by other investigators. As the refined techniques of GLC and C I E with the Durrum analyzer were brought to bear, it was convincingly shown that the level of amino acids in all samples of lunar fines was not above the background level of 1-2 ppb. Glycine was found by GLC and CIE at 19 ppb in a special Apollo 17 sample. This sample was known to be contaminated from rocket exhaust and the glycine was synthesized from rocket exhaust and deposited on the moon. Earthly contamination was excluded as the samples and blanks did not show an array of common amino acids. In our lunar investigations, a venting device was developed ( 8 , 9 ) which allows one to inject 50-100 p1 of sample into a gas chromatograph permitting much greater sensitivity and led to new approaches in GLC research not previously published. It was convincingly shown that the most likely source for any trace of amino acids in returned lunar samples is contamination, either in acquisition or return of samples, or during preparation and analysis in laboratories on earth. Sagan clearly showed, under simulated lunar conditions of proton flux, that the presence and survivial of amino acids in the environment of the moon was highly unlike1y .

Biologic markers Fifteen years ago at Columbia University, Professor Ernest Borek discovered the highly specific methyltransferase enzymes which modify the primary structure of tRNA macromolecules, and he reported increased activity of these enzymes in tumor cells. With Professor T. Phillip Waalkes of Johns Hopkins University and Dr. Ernest Borek at the University of Colorado Medical Center, we have undertaken joint investigations on biologic markers and their place in the management of the cancer patient (11-14). Modified nucleosides are found in the urine of normal and cancerous animals and humans. Since there seems to be no mechanism for reincorporation of these post polymer-modified nucleosides into tRNA, their levels in urine reflects the extent of modification as well as a measure of the turn-over rate of tRNA. Therefore, quantitation of modified nucleosides in urine could indicate changes in the tRNA profile during differentiation or tumor induction. Advantage has been taken of these excretion products to search for biologic markers of cancer. Such markers would either be indicative of the presence of cancer or it would parallel changes in tumor mass and be useful in following therapy. Development of methods for the analysis of nucleic acid components has been a major thrust in our laboratory since 1967 with the early work utilizing gas-liquid chromatography (GLC). The GLC methods we developed have been used to monitor the levels of pseudouridine, N2,N2-dimethylguanosine and 1-methylinosine in urine. Further , reports

84

Fig. 8.6. Reversed-phase HPLC isocratic separation of nucleosides. by Waalkes e t al. have indeed demonstrated that elevated levels for these markers do occur in urine of cancer patients with Burkitt's lymphoma, colon, and other types of cancer. A comprehensive and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine ($, m'A, m'I, m2G, A , m$G) ( 1 4 ) . An initial preliminary group isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic aicd was used to improve selectivity and sensitivity. The high resolution of the reversed-phase column allowed the complete separation of 30 nucleosides from other unidentified UV-absorbing components at the 1-ng level. Supporting experimental data have been presented on the complete method, and this technique has been applied to the analysis of urine of patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration of nucleosides in urine from patients with various types of cancer and in therapy response studies.

jicgenic m i n e s In 1977-78 we have developed a high-performance liquid chromatographic method with fluorescence detection for the biogenic amines in plasma, urine, and tissues ( 1 5 ) . This method, using pre-column treatment with 0-phthalaldehyde for the derivatization and separation of the biogenic amines on a reversed-phase phenyl column, provides a rapid, highly sensitive, simple and quantitative method for the

85

I

...................

SAYPLE. 10 pl STDS. 10 nu .a. COLUMN .................. IgONMPAM PHENVL. 4 rnm I jOOmm BUFFER.. 0.05 M N.Hm+ p#i 5.10

1

...............

om VIV cnan

A.

o.

45% V

cnm

~ V

................................. 1.6mlRIlN ......... SCHOEWEL FSWO. 0.1s AFS,

FLOW

DETECTOR

EX. Y O nin EM. 480 nm

TEMP.

0

10

-BUFFER

20

......................................

30

A

40

50

0

MINUTES +BUFFER

!

70

80

B-4

Fig. 8.7. Reversed-phase HPLC two-step isocratic separation of biogenic amines. Code of peak identification: HI=histamine; NE=norepinephrine; OCT= octopamine; NMS=normetanephrine; DA=dopamine; 5-HT=serotofifri. TYM=tyramine. simultaneous analysis of many biogenic amines at the nanogram level. This method provides a powerful research and clinical tool for studying various diseased states in both man and animals. In closing, the "chromatographies" are a major "bridge" or "common denominator" as analytical methods in biological sciences research. The importance of research and chemical analyses to the advancement of our society is increasing to the point where our society depends upon new knowledge from every source for its continued growth. Problems in nutrition, pollution, cancer, drugs, and other areas of medical science and space studies are now being solved by chromatography in days and weeks, where, formerly, months and years of study were involved. The genius of Tswett has had a profound impact to this point in history, and promises to open even greater vistas, through science, to mankind. SEFERENCES 1 K.O. Gerhardt and C.W. Gehrke, J . Chromatogr. 143 (1977) 335. 2 C.W. Gehrke, W.M. Lamkin, D.L. Stalling and F. Shahrokhi, Biochem. Biophys. Res. Corn. 19 (1965) 328. 3 C.W. Gehrke and F. Shahrokhi, Anal. Biochem. 15 (1966) 97 4 M. Lamkin and C.W. Gehrke, Anal. Chem. 37 (1965) 383.

86 5 D.L. Stalling and C.W. Gehrke, Biochem. Biophys. Res. Commn. 2 2 (1966) 329. 6 C.W. Gehrke and D.L. Stalling, in Separation Techniques i n

Chemistry and Biochemistry (19th ACS Summer Symposium on Anal. Chem., Edmonton, Alberta, June 1 9 6 6 ) , R.A. Keller, ed., M. Dekker, Inc., New York, 1967, pp. 21-58. 7 E. Kaiser, C.W. Gehrke, R.M. Zumwalt and K.C. Kuo, J. Chromatogr. 94 (1974) 113. 8 C.W. Gehrke, R.W. Zumwalt and K.C. Kuo, J . A g r . Food Chem. 19 (1971) 605. 9 C.W. Gehrke, K.C. Kuo and R.W. Zumwalt, J . Chromatogr. 57 (1971) 209. 10 C.W. Gehrke, R.W. Zumwalt, K.C. Kuo, C. Ponnamperuma and A . Shimoyama, Origin of L i f e 6 (1975) 541.

11 S.Y. Chang, D.B. Lakings, R.W. Zumwalt, C.W. Gehrke and T.P. Waalkes, J . L a b . CZin. Med. 8 3 (1974) 816. 12 T.P. Waalkes, C.W. Gehrke, R.W. Zumwalt, S.Y. Chang, D.B. Lakings, D.C. Tormey, D.L. Ahman and C.G. Moertel, Cancer 36 (1975) 380. 13 E. Borek, B.S. Baliga, C.W. Gehrke, K.C. Kuo, S. Belman, W. Troll and T.P. Waalkes, Cancer Res. 37 (1977) 398. 14 C.W. Gehrke, K.C. Kuo, G.E. Davis, R.D. Suits, T.P. Waalkes and E. Borek, J . Chromatogr. 150 (1978) 455.

15 T.P. Davis, C.W. Gehrke, C.W. Gehrke Jr., T.D. Cunningham, K.C. Kuo, K.O. Gerhardt, H.D. Johnson and C.H. Williams, CZin. Chem. 24 (1978) 1317.

87

J. CALVIN GIDDINGS

JOHN CALVIN GIDDINGS was born in 1930, in American Fork, Utah. He received a B.S. degree from Brigham Young University in 1952 and a Ph.D. from the University of Utah in 1954. His graduate advisor was Henry Eyring. In 1957, following postdoctoral work at Utah and the University of Wisconsin, he joined the faculty of the University of Utah as assistant professor of chemistry. He became associate professor in 1959, research professor in 1962 and professor in 1966. Dr. Giddings is the author of more than 180 scientific papers. His research interest is wide ranging. His early studies on flame theory, quantum mechanics and chemical kinetics soon yielded to his interests in chromatography and related methods. However, he also published papers on snow and avalanche physics, steady-state kinestics, prediction of diffusion coefficients and probability factors in nuclear holocust. In addition to his book, Dynm-ics of Chromatography (M. Dekker, 1965) he also wrote a textbook on Chemistry, Man and Environmental Change (Canfield Press , 1973) which reflects his longstanding interest in the environment. He is also the co-editor of 16 volumes of Advances i n Chromatography. His participation in outdoor activities and exploration, which has been the source of five articles, culminated in 1975 when he organized an expedition for the first successful navigation of Peru's Apurimac River, source of the Amazon. This exploration, through what the Encyclopedia BKtannica calls "one of the deepest canyons of the hemisphere", is the subject of his forthcoming book. Dr. Giddings is the recipient of the American Chemical Society Award in Chromatography and Electrophoresis, the Utah Award of the local section of the Society, and the ROMCOE Award for Outstanding Environmental Achievement in Education. In 1974, he received a Fulbright Grant for work in Peru, and he has received lectureship awards from Nebraska, North Carolina and the State University of New York at Buffalo. In 1978, he was awarded the M.S. Tswett Chromatography Medal.

88 Dr. Gidding's interest in chromatography dates back to his graduate years. His work in this area has shed light on nearly every chromatographic process, including nonequilibrium, diffusion, eddy diffusion, pressure changes, flow in paper and thin-layer chromatography, preparative-scale and programmed-temperature GC, exclusion chromatography, electrophoresis and the generation of nongaussian zones. He has worked extensively with the optimization of chromatography and has developed new high-pressure chromatographic systems and the one-phase system called field-flow fractionation.

89 It was a day in late autumn, 1953. Footsteps and creaking boards telegraphed that someone was coming down the hall 09 the old barracks. The moaning of wood and the firm, almost hurried thumping of soles on worn linoleum penetrated the thin walls, establishing a pattern of sound as characteristic as a 50-peak chromatogram. I raised by head from my work, not because hearing steps in the hall was unusual, but as a Pavlovian response to those particular steps. With those steps, you could always count on a wild confrontation with the unknown: a burst of imagination on flame dynamics, an animated discussion of energy flow in vibrating molecules or a new concept of the liquid state, any one of which might be punctuated with the question of why were you up skiing yesterday when there is so much science left to be done. I wondered what was in the air this time. Then Henry Eyring burst through the door and before I had time to greet him, he posed a fateful question: “Would you like to know all about chromatography?” The question, not the answer, was fateful because the answer was foregone: a graduate student never answered no to Henry. ”Sure,” I said, trying to act enthusiastic, knowing I was in for a siege,,andwondering when I would get back to my work. The barracks was an old, war surplus annex to the Physical Science Building, home of the Chemistry Department. Henry Eyring shuttled back and forth between here and the Park Building, where he served as Dean of the University of Utah Graduate School. In the barracks he had an office at the far north end of the hall on the right, and it was his custom when he passed through the door to pivot right again, which brought him in front of a big blackboard. It was his favourite place. Henry Eyring has never been afraid to tackle anything. With profound intuition, deep physical insight, and razor-sharp mathematics, he could break any process down into essential parts, and then synthesize a physical-mathematical picture. Why not chromatography? I think someone else joined us but I don‘t remember who. But I remember like crystal the exposition: it was classic Henry Eyring. Enthusiastic, whirlwind fast, he talked about molecules as if they were his friends. He had them dancing off adsorptive sites in average time , then gyrating through the mobile phase and coming back to S stick again in time T Clearly, he pointed out, R (or RF ) was the m simple ratio T / ( T These times could be written in terms of rate m m ‘s) constants, and suddenly Henry had developed a kinetic picture of chromatographic migration. We were left breathless. Henry wanted me to explore it further. It seemed like he had just solved the whole thing - which was quite new to me - and I couldn’t for life of me imagine what else there was to do. But that soon came. Serendipitously, I was at that time taking a graduate class, Principles of Physical S t a t i s t i c s , from Walter Elasser, a noted theoretical physicist. We had recently covered Poisson’s distribution, and it suddenly occurred to me that the passages of a molecule from the mobile to the stationary phase constituted a Poisson process. The distribution in the number of such passages was therefore given by

.

+

.

TABLE 9 . 1 . T i m e d i s t r i b u t i o n of my research a c t i v i t i e s i n d i f f e r e n t chromatographic a r e a s s i n c e i n c e p t i o n of work. A s t e r i s k s correspond t o i n d i v i d u a l p u b l i c a t i o n s , and t h e r e f o r e l a g about one year behind a c t u a l r e s e a r c h . Categories are a r b i t r a r y , and i n v o l v e considerable o v e r l a p ; publicat i o n s bridging s e v e r a l c a t e g o r i e s are a r b i t r a r i l y a s s i g n e d t o j u s t o n e .

Year

-+

1.

Statistical theory

2.

Nonequilibrium theory

3.

Plate height & column parameters in GC

4.

Basic paper chromatography ( & TLC

5.

Eddv diffusion

6.

Programmed temperature GC

7.

Optimization and separation

&

h955 I 5 6 I57 I58

models

8. Physicochem. constants from chrom.

9. Non-Gaussian zones, kinetics I

0.

Electrophoresis

1. Misc. general

& theoretical chromatography

2.

Preparative scale GC

3,

Theory of high efficiency LC

4.

High pressure & supercritical GC

5.

Steric exclusion chromatography

6.

Field-flow fractionation (1-phase chromatography

7. Non-chromatographic ( 1 9 5 4 *)

l

l

(13

0

91 Poisson's equation. Molecules coming o u t of t h e s t a t i o n a r y phase d i s t r i b u t e d themselves i n t i m e according t o another Poisson equation. Combining t h e two equations, I obtained a Bessel f u n c t i o n , and i n t h i s form (and a s i m p l i f i e d expansion) I i r r i v e d a t t h e d i s t r i b u t i o n of e l u t i o n times. I t was quick and simple. Now w e had both t h e mean and t h e d i s t r i b u t i o n i n terms of k i n e t i c parameters. I was happy, a l l a t once e n t h u s i a s t i c about chromatography. Now Henry wanted m e t o do some experiments, so I set up a g l a s s column packed with Magnesol and C e l i t e , made s t r i k i n g l y c o l o u r f u l l i k e raspberry marble ice cream with bands of B r i l l i a n t S c a r l e t 3R washed down with water. I t w a s i n c r e d i b l y i n e f f i c i e n t , But I found t h a t t h e dye molecules took an average of 43.7 adsorption s t e p s i n t h e run. In t h i s way I got r e v e r s i b l e rate parameters o u t of chromatographic experiments, and I became i r r e v e r s i b l y adsorbed t o chromatography. I completed t h e p r o j e c t and received my Ph.D. i n t h e summer of 1954 ( 1 ) . In t h e next few y e a r s I was engaged i n p o s t d o c t o r a l work on a v a r i e t y of t h e o r e t i c a l p r o j e c t s : flame k i n e t i c s , d e t o n a t i o n s , nucleat i o n k i n e t i c s and s t r a i n electrometry. Chromatography became a s p a r e time a c t i v i t y , but I succeeded i n expanding t h e s t a t i s t i c a l treatment ( 2 ) , and applying i t t o e l e c t r o p h o r e s i s ( 3 ) . Most of t h e flame work was done i n 1955-56 under t h e t u t e l a g e of Joseph 0. H i r s h f e l d e r , an eminent t h e o r e t i c a l chemist a t Wisconsin. One q u e s t i o n t h a t nagged m e a good deal a t t h a t t i m e was how c l o s e an atom o r r a d i c a l could come t o i t s s t e a d y - s t a t e l e v e l i n a flame f r o n t i n which r e a c t a n t concentrations were changing l i k e w i l d f i r e . I subsequently developed a general r e l a x a t i o n model t o p r e d i c t such steadystate departures ( 4 ) . From flames I went on t o o t h e r problems, and i n 1957 I accepted a p o s i t i o n as A s s i s t a n t Professor of Chemistry a t Utah. I w a s now f r e e t o go my own way, and I got quickly back t o chromatography. My research w a s boosted by two g r a n t s , one from N I H t h a t is s t i l l ongoing today, and b y 1958-59 I had begun s t u d i e s i n about t e n chromatographic a r e a s , The t a b l e shows each a r e a , roughly when I started i t , and t h e e v o l u t i o n of my work from t h a t time. These a r e i n d i c a t e d v i a my publ i c a t i o n s divided up by s u b j e c t and year of appearance. Normally, t h e p u b l i c a t i o n lagged about one year behind t h e a c t u a l work. The f i r s t t h i n g I d i d was t o s i m p l i f y t h e s t a t i s t i c a l treatment i n t o a random walk model of chromatography (5). I l a t e r used t h i s w i d e l y i n my book (6) because i t o f f e r s a d i r e c t and meaningful r o u t e f o r s t u d e n t s l e a r n i n g chromatography. A t t h e same t i m e I began t o f e e l d i s s a t i s f i e d w i t h t h e s t a t i s t i c a l approach because i t w a s so hard t o apply without guesswork t o r e a l i s t i c adsorption and p a r t i t i o n processes. I wondered what t h e p l a t e model r e a l l y o f f e r e d , and how i t related t o nonequilibrium, and I s t a r t e d t h i n k i n g again about t h e r e l a x a t i o n model f o r flames. I brought a l l of these t o g e t h e r i n a 1959 paper ( 7 ) , and a t l e a s t f o r me, everything began t o f i t i n p l a c e . The r e l a x a t i o n model w a s t h e key t o f u r t h e r progress, and l e d t o t h e nonequilibrium theory, a powerful t o o l f o r d e s c r i b i n g band broadening i n terms of t h e complex k i n e t i c and d i f f u s i o n processes of r e a l columns. The r e s u l t s were both simple and a c c u r a t e , a very n i c e s i t u a t i o n f o r a phenomenon as complex as chromatography.

92

Nonrquili brium disploccmr nt,

Fig.9.1. Shift or displacement in mobile phase (solid line) from its equilibrium concentration. The magnitude of the shift determined band broadenIn ref. 7 , I showed that the plate height ing (7). is exactly twice the nonequilibrium displacement distance, vtr ( 1 - R ) , where v = flow velocity, t, relaxation time and R = retention ratio.

I had returned to the relaxation model for flames because, kinetically, a chromatographic zone resembles a flame. A zone washing through a bed is like a flame eating through combustibles: species strive constantly for equilibrium, but the requirements of equilibrium keep changing. It is like trying to hit a moving target: with a fast gun (rapid kinetics) you can come close, and knowing the speed of the bullet you cqn calculate the miss. In a chromatographic column, the moving target is the solute profile, which by its migration constantly changes the concentration and thus the equilibrium condition at any point (see Fig. 9.1). The rate processes strive to keep up with equilibrium only to find the target shifting with each new increment of migration. This is crucial because band spreading increases with the shift in equilibrium (6,7). Relaxation theory gives the lag, and therefore, the band spreading. It is a simple, effective theoretical tool. This nonequilibrium theory was applied over a period of ten years to obtain plate height terms for a host of chromatographic processes: one-site, two-site, and general n-site adsorption; adsorption with partition; adsorption of large molecules by segments; chemical change; gas-solid chromatography; mass transfer in preparative, coiled and capillary columns; diffusion in uniform layers, rods, spheres, and glass bead interstices; diffusion in nonuniform pores and collections of unequal pores; field-flow fractionation; and most comprehensively, diffusion in the presence of complex flow (8). All except the most recent results can be found in my book (6). For me this work was like laying the bricks of a satisfying edifice, tying diverse chromatographic results to dynamical roots, and leading to predictions of efficiency (3).

93

Fig.9.2. Special cases of stationary liquid configuration and the corresponding plate height equations from generalized nonequilibrium theory

(9). While the dynamics of peak broadening long occupied me, I had become increasingly fascinated with chromatography as a whole physicochemical system, within which separation was influenced by complex flows accompanied by pressure changes, surfaces that adsorbed molecules and deflected streams of matter, pores that accommodated and complicated diffusion, scattered pools of liquid stationary phase that soaked up solute, capillary forces that shaped the pools and sometimes drove the mobile phase over them, and transport processes that were complicated by every accident of geometry and twist of current conceivable. I grew determined to cut to the core of this interesting system. In 1957-58, I began two broad studies of whole systems. One was on paper chromatography (PC), and the other on gas chromatography (GC). Both entailed a mix of experimental and theoretical work, and important collaboration. I will describe here the PC work, which is least known, shortest, and earlier of the two projects, but still quite relevant to modern TLC.

94

5hr\

J

i

i

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J 4 6 Reduced Ddrtoncr. y =

e

I

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Fig.9.3. Figures from a 1959-paper (lo) showing (top) individual solvent concentration profiles in a paper strip, and (bottom) the common profile they superimpose to when plotted in terms of the reduced distance to the front. Our work on PC started with the premise that to understand the chromatography, we first had to understand solvent flow. Flow in PC is more complex than that in GC or LC, where a pump or tank provides a steady input and the main concerns are pressure and velocity gradients. In PC (as in TLC) flow is born in the chromatographic bed, through capillarity. As a result of this unique mechanism, both the content and velocity of solvent in the bed vary with time and distance. Because chromatographic zones migrate according to the velocity and amount of solvent engulfing them, these solvent parameters govern chromatographic behaviour. It had been known since the 1950's that a liquid front in paper advances with (time)i. What clues did this offer? It suggested diffusion, but clearly did not offer a diffusion mechanism. Would a diffusion analogy work - diffusion equations minus the appropriate mechanism? Art Ruoff, George Stewart, and I set up experimental tests. The results were affirmative (10). Suddenly, out of this complicated mess of capillarity and viscous drag in a complex pore space, we had a simple rule: the solvent profile on a paper strip (or TLC plate) always retains the same shape, but the whole thing expands outward with (time)* Theory in this case provided simplicity (always a goal), but it also provided the power of prediction, control, correlation, and calculation. Given a solvent profile in a rectangular stip, profiles in other geometries could be predicted ( 1 0 1 , and controlled ( 1 1 ) . More importantly, given a solvent profile, we could correlate zone migration rates with solute partition coefficients ( 1 2 ) . Here we found something unexpected - the solvent behind the front moves as much as 40% slower than the

95

Fig.9.4. A Summer 1964 photograph of John H. Knox, Roy A. Keller, Stephen J. Hawkes and J.C. Giddings at Utah. John Knox spent most of 1964 in Utah, working primarily on the mechanism of eddy diffusion. Stephen Hawkes, now at Oregon State University, was doing postdoctoral work at the time. Roy Keller, now at the College of Fredonia of the State University of New York, a former graduate student of Eyring, was visiting. front itself. This alters migration rates, and requires revision in the old relationships between RF and partition coefficients. Another goal of theory is the elucidation of mechanism. Pursuing this goal we found that an interconnected capillary model satisfied the physical requirements of flow, and the mathematics of diffusion as well (13). George Stewart, who did part of the research for his Ph.D., later carried the work over to TLC (14). With flow characterized, we could get on with zone (spot) studies. This aspect of PC was pursued with my good friend, Roy Keller. Roy also did his Ph.D. work with Henry Eyring, studying PC systems. His work had raised questions on spot distribution, shape, size, identity, and resolution. Roy had joined the faculty of Arizona in 1956, but in the summers of 1958 and 1959 he came back to Utah where we attacked these questions. The first summer we showed that the normal chromatographic process tends to generate elliptical spots; we confirmed the empirical quantitation rule that spot area is proportional to the logarithm of

96

I

I

-0.4 -0.2

I

0

I

I

1

I

0.2 0.4 0.6 08 X

I

I

J

1 . 0 1.2 1.4

Fig.9.5. Spot outlines at different cutoff values for an asymmetrical case. The range of phenomena that can be found is partially illustrated here. All the spots illustrated will, in practice, have a diffuse rather than a sharp boundary (16). sample size, and showed as incorrect a similar rule for spot length (15). The following summer we addressed a class of abnormal chromatographic processes - slow kinetic steps that convert single species into the confusion of multiple spots. We surveyed the literature, and showed that many anomalies could be traced to such kinetics (16). The productive relationship with Roy has continued over the years with no end in sight, spawning research and special projects, such as the Advances in Chromatography series. Space does not allow me to describe my work with eddy diffusion, column parameters in GC, preparative scale, programmed temperature GC, exclusion chromatography, and other categories. Instead in the lines remaining, I will touch on some of my efforts to improve chromatography. In the early years, my central interest was in understanding chromatography and in finding ways to describe it with physical and mathematical models. However, in the first paper I had begun to wonder about optimization, and had discussed optimum velocity as a compromise between diffusion and rate processes ( I ) . My interest in optimization grew, and soon turned into an effort to design new kinds of columns, then new systems. I worked out a continuous, rotating column in 1961 ( 1 7 ) , only to find that A.J.P. Martin had beaten me to it. Soon afterwards I became excited about high pressure chromatographic systems. John Knox had shown theoretically that GC analysis speed could be pushed up indefinitely with increasing drop ( 1 8 ) . My own theoretical work confirmed and extended this conclusion for both GC and LC. I became interested in optimum LC, not as a system different from GC, but as the same system with different parameters. I saw no need for new theory, only new numbers and operating conditions. I analysed this in a paper published in 1963 (19), and concluded that it pressure drops in LC and GC were equal, "then for the fastest analysis the particle

97

Fig.9.6. S q u a l a n e , r e a d i l y d i s s o l v e d i n dense C02 a t 4OoC, condenses i n t o a c l o u d o f l i q u i d d r o p l e t s a s i t emerges from a h i g h - p r e s s u r e column t o t h e atmosphere. S p e c i e s up t o s e v e r a l hundred thousand m o l e c u l a r weight can b e s o l u b i l i z e d i n dense gas chromatographic s y s t e m s . (Photo by A l e x i s K e l n e r , 1 9 6 8 ) . d i a m e t e r f o r LC w i l l be about 1/30 of t h a t f o r t h e analogous GC system." On t h e b a s i s o f p a r t i c l e s i z e s t h e n employed, t h i s g i v e s dp 2-20 u m , which s p a n s t h e range o f modern p r a c t i c e . A t t h e same t i m e , I s u g g e s t e d t h a t h i g h e r p r e s s u r e s might be used i n LC. While my c o n c l u s i o n s a r e n o t always p r o p h e t i c , I b e l i e v e t h a t t h e p a p e r i l l u s t r a t e s t h e power of t h e o r y , when v e r y , v e r y c a r e f u l l y a p p l i e d , t o g u i d e t h e development and optimization of s e p a r a t i o n systems. I n 1964 John Knox came t o Utah on a S e n i o r F o r e i g n F e l l o w s h i p from NSF, and p r o v i d e d f u r t h e r i n s p i r a t i o n f o r o u r h i g h p r e s s u r e work. I n 1964-66 Marcus Myers and I developed s e v e r a l h i g h p r e s s u r e ( 150 atm) GC s y s t e m s . A t one extreme w e c o n s t r u c t e d a 4000-foot column t o r e a c h a m i l l i o n t h e o r e t i c a l p l a t e s , a r e c o r d f o r packed columns ( 2 0 ) . A t a n o t h e r extreme w e developed a high-speed t u r b u l e n t GC system (21). A t a t h i r d extreme, w e b u i l t microcolumns u t i l i z i n g p a r t i c l e s down t o a f e w U m ( < 1 u m i n one c a s e ) t o a c h i e v e f a s t s e p a r a t i o n s and t h e l o w e s t GC p l a t e h e i g h t s known - 0.082 mm. ( 2 2 ) . These were good columns f o r GC, b u t more b r o a d l y t h e y were s u p e r b p r o t o t y p e s f o r h i g h p r e s s u r e LC columns i n t h a t t h e y c o n s t i t u t e d a v e r i f i c a t i o n of t h e b a s i c p r i n c i p l e s ( a p p l i c a b l e t o a l l chromatography) t h a t u l t i m a t e l y l e d J a c k K i r k l a n d and o t h e r s t o modern LC s y s t e m s . The apex o f o u r h i g h p r e s s u r e program w a s o u r d e n s e ( s u p e r c r i t i c a l ) gas work. Here w e used p r e s s u r e s t o 2000 atmospheres t o compress g a s e s t o l i q u i d - l i k e d e n s i t i e s , w h e r e t h e y a c q u i r e l i q u i d - l i k e s o l v e n t power.

-

98 The physical chemistry of t h i s s y s t e m w a s engrossing, but t h e concept of using a c o n t r o l l a b l e mechanical parameter, p r e s s u r e , t o run s o l u b i l i t i e s up and down as d e s i r e d seemed (and s t i l l seems) an i n c r e d i b l y usef u l concept f o r chromatography ( 2 3 ) . In 1965 I s t a r t e d o f f i n a new d i r e c t i o n : I developed t h e concept of a chromatographic-like system i n which r e t e n t i o n i s e s t a b l i s h e d and c o n t r o l l e d by an external f i e l d r a t h e r than by t h e s t a t i o n a r y phase ( 2 4 ) . This s y s t e m , which w e c a l l field-flow f r a c t i o n a t i o n (FFF), extends t h e range of chromatography upwards t o i n c l u d e macromolecules and p a r t i c l e s of almost every type and s i z e , from 0.001 t o 10 u m and beyond ( 2 5 ) . The experimental work w a s a t f i r s t f r a u g h t with d i f f i c u l t i e s , but thanks t o t h e s k i l l s of Marcus Myers and o t h e r c o l l e a g u e s , i t has y i e l d e d g r a t i f y i n g r e s u l t s i n r e c e n t y e a r s . Field-flow-fractionation has occupied most of my a t t e n t i o n i n t h e 1970's. I w i l l add no more, f o r t h e s t o r y of FFF belongs more in t h e f u t u r e than i n t h e p a s t . REFERENCES

1 J .C. Giddings, Topics i n Chemical Kinetics and Chromatography, Ph.D. t h e s i s , University of Utah, J u l y 1954; J . C . Giddings and H. Eyring, J. Phys. Chem. 59 (1955) 416. J.C. Giddings, J. &ern. P h p . 26 (1957) 169. J . C . Giddings, J. &em. Phys. 26 (1957) 1755. J . C . Giddings, J. fiem. Phys. 26 (1957) 1210. J . C . Giddings, J . Chem. Ed. 35 (1958) 588. J.C. Giddings, D y n m k s of Chromatopqhy, M. Dekker I n c . , New York, 1965 7 J . C . Giddings, J. Chromatogr. 2 (1959) 44. 8 J.C. Giddings and P.D. S c h e t t l e r , J . Phzjs. Chem. 7 3 (1969) 2577. 9 J . C . Giddings, Ber. Bunsenges. 69 (1965) 773. 10 A.L. Ruoff, D.L. P r i n c e , J . C . Giddings and G . H . Stewart, KoZZoid Z. 166 (1959) 144. 11 A.L. Ruoff and J . C . Giddings, J. Chromatogr. 3 (1960) 438. 12 J . C . Giddings, G.H. Stewart and A.L. Ruoff, J . Chromatogr. 3 (1960) 239. 1 3 A.L. Ruoff, G . H . Stewart, H.K. Shin and J . C . Giddings, KoZZoid 2. 173 (1960) 14. 14 G.H. Stewart, Sep. S e i . 1 (1966) 747. 15 J . C . Giddings and R.A. Keller, J . Chromatogr. 2 (1959) 626. 16 R.A. Keller and J . C . Giddings, J . Chromatogr. 3 (1960) 205. 17 J . C . Giddings, Anal. &em. 34 (1962) 37. 18 J . H . Knox, J. Chem. Soc. (1961) 433. 19 J . C . Giddings, AnaZ. Chem. 35 (1963) 2215. 20 M.N. Myers and J . C . Giddings, AnaZ. Chem. 37 (1965) 1453. 21 J . C . Giddings, W.A. Manwaring and M.N. Myers, Science 154 (1966) 146. 22 M.N. Myers and J . C . Giddings, A n a l . Chem. 38 (1966) 294, 23 J . C . Giddings, M.N. Myers, L. McLaren and R.A. Keller, Science 162 (1968) 67. 24 J . C . Giddings, Sep. s&. 1 (1966) 123. 25 J . C . Giddings, J . Chromatogr. 125 (1976) 3.

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99

EUGEN GLUECKAUF

EUGEN GLUECKAUF was born in 1906, in Eisenach, Germany. He first studied at the University of Freiburg (Breisgau) and at the University of Berlin, but then changed to the Technical University of Berlin where he received the doctorate (Dr.-Ing.) in chemistry in 1932. He left Germany in 1933 in anticipation of the racial persecution by the Nazis and continued research in England in collaboration with Professor F.A. Paneth, first at the Imperial College, London, and later at the University of Durham, largely on the analysis of atmospheric gases and of helium produced by radioactive events. In 1942-1944, he was a MacKinnon Research Student of the Royal Society, and in 1944-1947 a Research Fellow at Durham, working for the D.S. I.R. on the separation of isotopes by chromatography. He then joined the United Kingdom Atomic Energy Research Establishment at Harwell. In 1951, he received the D.Sc. degree from the University of London and became a Deputy Chief Scientist (personal merits) and head of the Physical- and Radio-Chemistry Branch. Dr. Glueckauf is the author and co-author of over 100 scientific publications, about a third of which are associated with gas and ionexchange chromatography, mostly from the period between 1945 and 1960. His other scientific interests include the analysis of atmospheric gases, solution chemistry, treatment for atomic waste and water desalination. He was elected a Fellow of the Royal Society in 1969. Dr. Glueckauf pioneered in the modern theory of gas (and ionexchange) chromatography and in the application of the technique to the analysis of inorganic gases and isotopes.

100 My recollections concerning my involvement in chromatography can be divided into two fields: experimental chromatographic separations, and the evolution of the theory of the chromatographic separation process.

ExperimentaZ Chromatographic Separations My first contact with the chromatographic procedure took place around 1937-1938 at which time I was collaborating with Prof. F.A. Paneth at the Imperial College, London in studies involving the determination of helium, both in the atmosphere and also produced by artificial radioactive decompositions involving the formation of a-particles. The accurate determination of helium in air involved a fractional separation of helium and neon by low-temperature absorption on charcoal and this was carried out best by a stepwise adsorption and desorption involving several hundred adsorption/ desorption steps, and as many as 12 adsorption units ( I ) . This work anticipated the Craig multiple extraction process (2) by many years, though Craig's work was published two years earlier. Though a stepwise separation process is not quite identical with a chromatographic column operation - which operates continuously both in space and time - there are considerable similarities in the two processes. Thus when my results were finally published (after the war) in 1946 ( I ) , the discussion of the theory of fractionation contained a paragraph on the "comparison with the process of chromatographic separation". Of course, helium and neon, being the least adsorbed gases, could never be separated quantitatively by elution gas chromatography, as there is no (less adsorbed) gas that could act as carrier gas. But the technique of displacement chromatography was later used to separate helium and neon qualitatively for the purpose of leak-testing large vacuum plants (3). I have, however, used a quantitative gas chromatographic separation by the elution technique for the separation of krypton and xenon fission products ( 4 ) in a low temperature charcoal column, using hydrogen as the carrier gas, and this technique had in fact been evolved much earlier (1948-1949) for the quantitative determination of the krypton and xenon contents of atmospheric air. Unfortunately this work too could not be published until 1956 (5). Our earliest gas chromatographic work (in 1948-1949) was concerned with the partial separation of the isotopes of neon (6) by 0 adsorption on charcoal at -196 C . The technique used the break-through method. In view of its historical importance - it was the first gas chromatographic isotope separation - I shall describe this work with some detail. The charcoal tube was originally filled with nitrogen and the rear-end was connected to a neon supply. The charcoal tube is then slowly immersed in liquid nitrogen and progressively cooled from the rear. The small amount of nitrogen in the tube is strongly adsorbed and this results in a constant inflow of neon which passes through the low temperature region. A si nificant depletion of 22Ne, which is more strongly adsorbed than 2'Ne, takes place at the advancing

101

BREAKERSEAL GAS SAMPLE TUBE

Fig. 10.1. Apparatus for gas chromatographic separation of neon isotopes

front (i.e. at the level of the liquid nitrogen bath), as shown by the mass-spectrometric analysis of small samples ( - 0.5 ml NTP). Theoretical analysis of the results gave a single-stage separation factor of 1.0020 only, and a theoretical plate height of 0.02 cm, so that the column used comprised about 2500 theoretical plates. Considerably earlier (around 1945) than this gas chromatographic work, I had done some work at Durham on the separation of the Li-isotopes by ion-exchange chromatography (6). This separation had been attempted earlier by Taylor and Urey in 1938, but in the absence of theoretical guidance, their columns of up to 30 m length did not have an adequate number of theoretical plates to achieve significant separations. In those experiments seven years later, I worked close to the kinetic optimum conditions, using a linear flow rate of about 0.006 cm/sec, and particle diameters of 0.0013 cm, thereby achieving about 70.000 theoretical plates in a 90 cm column. In order to obtain the best result, tilting of the front boundary had to be avoided, and this was done by passing a solution of Li-acetate into an acid exchanger column. The conversion of H-exchanger to Li-exchanger produced a slight swelling of the particles, which caused a self-adjustment of the advancing (self-sharpening) front of Li-acetate. Thus in one experiment a perfectly straight boundary was produced, and the first drops of Li-solution arriving at the bottom of the column did contain practically pure 7Li (which is less adsorbed than 6Li). Needless to say, that the very low separation factor of 1.0025 excluded any possible technological application. We could achieve a much more successful separation with the isotopes of hydrogen in 1956-1957 (6). We have used a displacement

102

Fig. 10.2. Observed depletion of 6Li at the front boundary. technique, where a mixture of H2 + D2 + HD was separated on a column of Pd-asbestos, by using (the more strongly adsorbed) pure H2 as a displacing gas. It was possible to purify much of the deuterium to a purity of up to 99.5%. From the chromatographic point of view this isotopic separation has no problems as the single stage separation factor for H-D at room temperature has a value of around 1.75. As the adsorption of hydrogen on palladium takes place in "atomic" and not in "molecular" form, mixed species like HD do not cause complications. This separation technique (which lends itself to continuous operation, by moving the Pd-adsorbent countercurrent to the gas stream) may have some technical importance for recovering tritium and deuterium from HDT mixtures arising during future thermonuclear power generation. A discussion of the whole field of chromatographic isotope separation was given by me in an article (6) for a textbook on the Separation of Isotopes edited by H. London.

Theory of Chromatography When I took up work on chromatography (in 1944), the theoretical understanding of the process was still in its infancy, being confined to the concepts of Wilson ( 7 ) on "linear" "ideal" chromatography, and of de Vault (8) who dealt with %on-linear" "ideal" chromatography of a single solute. There was also some understanding of linear nonideal chromatography ( 9 ) . "Linear" and 'Inon-linear" refers here to the adsorption isotherms, while "ideal" and %on-ideal" refers to the absence or presence in the column of discontinuities (theoretical plates) or other non-ideal behaviour arising from diffusion processes. Being myself concerned with the separation of isotopes, preferably in bulk, the most important combination.at that time was non-linear chromatography of more than one solute; because working at relatively high concentrations always leads to non-linear isotherms in which the concentration of one species affects the adsorption equilibrium

103 r BUFFER GAS

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Fig. 10.3. Gas chromatographic separation of an H-D mixture containing 40% deuterium, on palladium black. Column was originally filled with helium. of the other. This was a problem which had not previously been properly investigated. If we write the amounts q adsorbed of two substances to be separated as q 1 = fl(clc.2) and 42 = f2(clc2) then the simultaneous presence of both substances in the chromatographic column involves the condition d fl(c1c2)

-

d f2(c1c2)

c2

This differential equation cannot be generally solved, except in the case of adsorption following a Langmuir isotherm e.g. fl = alcl/ (1 + blcl + b2e2) and v i c e versa for fa, which accounts for the mutual displacement of the two solutes on the adsorbent. This study lead us to a number of publications in 1945 and 1946, and a complete analysis of the process of a separation of two solutes following a Langmuir isotherm was given in a paper in 1946 ( 1 0 ) . This theoretical work was supported by experimental studies on the separation of two solutes, and by theoretical studies on the phenomena arising at self-sharpening frontal boundaries under non-ideal nonlinear conditions. The results of these studies were summarized in 1947 in four papers (11-14) while a paper published in 1949 ( 1 5 ) dealt with the general theory (non-linear and ideal) for two or more solutes. I gave a theoretical discussion of mono-layer adsorption of two species on non-uniform surfaces, i.e., of conditions that for single species would produce Freundlich isotherms ( 1 6 ) while in a subsequent paper, I presented a study of diffusion into spheres as applying to chromatographic separations (27). One of the important papers which has been largely neglected by gas chromatographers, perhaps on account of the title, dealt with the principles of operation of ion exchange columns (ref. 19 in (6)) though the equations apply really to all other forms of chromatography

104 as well. It summed up in detail that the Effective Height of One Theoretical Plate is not only a function of the column alone (particle size), but is affected by the equilibrium between adsorbent and mobile phase, to which in the case of gas chromatography has to be added longitudinal diffusion in the gas phase. In particular the paper gives a mathematical description of the non-ideality effects on the shape of the frontal boundaries. These frontal boundary effects are particularly important in the case of isotopic separations of low separating power, where the selfsharpening effect of boundaries between two isotopes is negligible, and where the quality of the separation is almost entirely dependent on the non-idealities of the operating procedure, i.e., H.E.T.P. Thus the usual picture of a displacement process giving a clear separation of the species changes to partial enrichment or depletion only. The equations for the gradient of the boundary, the detailed effects of the various non-idealities, and the attainable enrichment are discussed in the Appendix of my 1956 paper coauthored with G.P. Kitt ( 5 ) . One of my best known theoretical papers dealt with the "theoretical plate" concept in column separations (18). This showed clearly the importance of having to treat the chromatographic process as a continuous flow operation, rather than with a discontinuous model as used by Mayer and Tompkins for the prediction of the separation power of columns in 1947 (which would have been appropriate to a Craig extraction process). The paper deals with the shape of break-through curves, with the elution of bands of one or more solutes of linear isotherm, and with the prediction of the number of theoretical plates needed to obtain a given purity of products for the separation of species having a given separation factor (i.e. a given ratio of their adsorption coefficients on the stationary phase). The treatment was followed in considerable detail in the textbook on gas chromatography by A.I.M. Keulemans ( 1 9 ) who also summarised the results in a diagram. The application of this theory leads to a very simple graphical assessment of isotopic (or other very small) separation factors from data obtained by elution chromatography of an isotopic mixture (20) where due to the separating action the local ratio of any two isotopes changes with the distance from the common peak of the eluted band. Thus, if we plot the log of the local separation factor [(cl/c2)/ (e$/ep)] as ordinate against the fraction of the eluted mixture on a probability scale abscissa, we obtain a straight line plot the gradient of which has the value of 6 / ( N ) f where N is the number of theoretical plates in the column and (1+6) is the single stage separation factor of the two isotopes. This treatment when applied to the data obtained by Betts, Harris and Stevenson (ref. 11 in (6)) with 22Na and 24Na mixtures on an ion exchanger column of about 9400 theoretical plates gave values of 6 of 1.38 x at 2 4 . 8 O and 1.78 x at 5.5OC. These are probably the lowest separation factors of any two substances which have been accurately established by chromatography. Of less general interest is my paper on the chromatography of highly radioactive gases ( 2 1 ) . Here I quantitatively discussed the

105 n

n

Fig, 10.4, Relationship between the number of plates ( n ) (ordinate), the separation factor (a), and the fractional band impurity (n) (abscissa). Keulemans' diagram ( 1 9 ) , based on my treatment. effect of a temperature rise which occurs in the adsorbent column during the passage of a highly radioactive band of adsorbate. As the result of this temperature rise, the rear end of the band moves in a temperature zone which is higher than that at the front, and as higher temperature generally reduces the adsorption coefficient of gases, this results in a marked band contraction. The band eventually attains a width which is independent of the width of the feed band

106

F i g . 10.5. Maximum f r a c t i o n a l l o a d i n g of column No/N ( a b s c i s s a ) for g i v e n s e p a r a t i o n f a c t o r k and column l e n g t h ( e x p r e s s e d a s t h e p l a t e number N i n t h e o r d i n a t e ) , comp a t i b l e w i t h a p r o d u c t p u r i t y o f 99.9%. and t h e l e n g t h of t h e column. T h i s phenomenon t h u s a f f o r d s a s i m p l e means o f c o n c e n t r a t i n g d i l u t e r a d i o a c t i v e g a s e s i n t o s m a l l volumes w i t h o u t t h e need f o r r e f r i g e r a t i o n - p r o v i d e d t h a t t h e t o t a l r a d i o a c t i v e power i s i n t h e w a t t r a n g e . My f i n a l p a p e r ( 2 2 ) e x t e n d e d t h e t h e o r y ( g i v e n i n (18)) t o t h e b e h a v i o u r of "wide bands" i n c h r o m a t o g r a p h i c columns. I n p a r t i c u l a r , t h e r e e x i s t s t h e problem o f how to assess t h e t h e o r e t i c a l p l a t e h e i g h t and number (N) e x p e r i m e n t a l d a t a o b t a i n e d w i t h wide f e e d bands. For narrow f e e d bands (No<(N/lO)*) t h e t h e o r y ( 2 8 ) l e a d s t o 2

= ~(D/f3)

N'

where 3 i s t h e e l u t i o n volume o f t h e peak maximum and @ t h e bandwidth

at the concentration C

e

= c /e max Y

For bands w i d e r t h a n t h a t , t h i s g i v e s an u n d e r e s t i m a t e f o r N and a c o r r e c t i o n f a c t o r h a s t o be a p p l i e d which i s g i v e n i n t h e q u o t e d paper ( 2 2 ) .

107 But t h e most i m p o r t a n t q u e s t i o n r e a l l y i s how t h e width of t h e f e e d band a f f e c t s t h e p u r i t y of t h e s e p a r a t e d p r o d u c t , because i n t h e case case of p r e p a r a t i v e work, t h e economy of p r o d u c t i o n g e n e r a l l y demands t h e u s e of "wide bands". T h i s q u e s t i o n c a n be t r e a t e d i n nomograms p l o t t i n g a f a m i l y of c u r v e s f o r t h e s e p a r a t i o n f a c t o r of two subs t a n c e s f o r columns of given t h e o r e t i c a l p l a t e numbers, depending on t h e d e s i r e d product p u r i t y . From t h e s e p l o t s t h e f r a c t i o n a l l o a d i n g can be d i r e c t l y o b t a i n e d . One f a m i l y of c u r v e s w i l l r e f e r t o a p r e s e t p r o d u c t p u r i t y ( e . g . 99.9%); f o r o t h e r p r o d u c t p u r i t i e s , similar c u r v e s can t h e n be c o n s t r u c t e d , u t i l i z i n g t h e e q u a t i o n s g i v e n i n r e f . 22. REFERENCES 1 E . Glueckauf, Proc. Roy. SOC. A185 (1946) 98. 2 L.C. C r a i g , J. Biol. Chem. 155 (1944) 519. 3 E . Glueckauf and G.P. K i t t , J . sci. I n s t r . 35 (1958) 220. 4 E . Glueckauf and G.P. K i t t , Analyst 7 7 (1952) 903. 5 E . Glueckauf and G.P. K i t t , Proc. Roy. Soc. A234 (1956) 557. 6 E . Glueckauf, Isotope Separation by Chromatographic Methods. I n Separation o f Isotopes, H. London, e d . , George Newnes L t d . , London, 1961, pp.209-248. 7 T.N. Wilson, J. h e r . Chem. Soc. 6 2 (1940) 1583. 8 D. de V a u l t , J. Amer. Chem. Soc. 65 (1943) 532. 9 A.J.P. M a r t i n and R.L.M. Synge, Biochem. J . 35 (1941) 1358. 1 0 E . Glueckauf, Proc. Roy. Soc. A186 (1946) 35. 11 E . Glueckauf J. Chem. Soc. (1947) 1302. 12 J . I . Coates and E. Glueckauf , J . Chem. Soc. (1947) 1308. 13 E . Glueckauf and J . I . Coates , J . Chem. Soc. (1947) 1315. 1 4 E . Glueckauf, J. Chem. Soc. (1947) 1321. 1 5 E . Glueckauf, Disc. Faraday SOC. 7 (1949) 12. 16 E. Glueckauf , Trans. Faraday SOC. 49 (1953) 1066. 1 7 E . Glueckauf, Trans. Faraday Soc. 51 (1955) 1540. 18 E . Glueckauf, Trans. Faraday Soc. 51 (1955) 3 4 , 1 9 A . I . M . Keulemans, Gas Chromatography, Reinhold, New York, 1957. 20 E. Glueckauf, Trans. Faraday SOC. 54 (1958) 1203. 2 1 E . Glueckauf, i n Gas Chromatography 1958 (Amsterdam Symposium), D . H . Desty e d . , B u t t e r w o r t h s , London, 1958, pp.69-89. 22 E. Glueckauf, Trans. Faraday Soc. 60 (1964) 729. ~

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109

MARCEL J.E. GOLAY

MARCEL JULES EDOUARD GOLAY was born in Neuchltel, Switzerland, in 1902. He studied at the Eidgenossische Technische Hochschule, in Zurich, receiving his licentiate in Electrical Engineering in 1924. He then joined the staff of Bell Telephone laboratories in the United States where he worked until 1928 when he entered the University of Chicago to pursue graduate studies. He received his Ph.D. in 1931 with a thesis in atomic physics. On graduation, Dr. Golay joined the U.S. Signal Corps Engineering Laboratories at Fort Monmouth, N.J., where he remained until 1955. His last position was that of chief scientist of the Components Division. Between 1955 and 1961 Dr. Golay served as a consultant, t o the Philco Corporation, Philadelphia, Pa., and to the Perkin-Elmer Corporation, Norwalk, Conn. In 1961-1962 he was a visiting professor at the Technische Hogeschool (Technological University) in Eindhoven, The Netherlands, and since 1963, he has been a senior scientist at Perkin-Elmer. Dr. Golay has received numerous awards during his professional career, among them the Harry Diamond Award of the Institute of Electric and Electronic Engineers, the American Chemical Society Award in Chemical Instrumentation, the Distinguished Achievement Award of the Instrument Society of America and the M.S.Tswett Chromatography Medal. He has a honorary docteur ds sciences degree from the Ecole Polytechnique F6dBrale of Lausanne, Switzerland. Dr. Golay has made significant contributions in various scientific disciplines. His activities in gas chromatography started in 1955 with theoretical studies leading to the development of open tubular columns. Other activities in this field include the development of the theory of preparative chromatography and the utilization of mixed washers; investigations on the effect of various parameters on the efficiency of sample introduction systems; and various theoretical considerations aimed at the improvement of the separation ability of gas chromatographic columns.

110 My earliest brush with chromatography came without me even knowing the name. It was, I believe, during the War and I was working with long copper tubes through which I was trying to pipe the small pressure changes picked up by infrared pneumatic detectors at the end of airplane wings to the cockpit for observation. The experiments came to naught, as they deserved to, because I should not have expected to pipe successfully the tiny pressure changes involved against the background of noise present in such a system. Still, in the course of working with and studying these not very clean copper tubes, the thought came to me that if one were to pass in them a mixture of vapors with various affinities for the walls, there would be a separation of the individual components. I did nothing with the idea - there were too many army officers breathing down my neck for results more pertinent to the problems at hand - but when, in 1955, Perkin-Elmer decided to put its best foot forward in the budding field of chromatography, and asked me to prepare a theoretical treatment of the subject for presentation at the National American Chemical Society Meeting in Dallas, Texas, in April 1956, I was somewhat primed by my earliest musings, and found little difficulty in applying communication concepts to the molecular separation problem. After my presentation ( I ) I was shown in the Perkin-Elmer suite in the hotel the Model 154 Vapor Fractometer which had been developed by Harry H. Hausdorff and designed by Emmett Watson and was impressed as I am always impressed by sophisticated hardware. About this time, I was asked to make sense out of the chromatographic columns then in use, which were of the packed type. Jim Friend and Emmett Watson raised the question whether one could not define a measure of the goodness of columns. This led me to the development of the performance index (P.I.). When I applied this concept to the packed columns existing at that time, I found an enormous discrepancy between actual and ideal values: the packed columns were horrifyingly poor, the actual P.I. values were about 10,000-times larger than theoretically expected. As expressed in my paper submitted in May 1956 to Nature ( Z ) , "this very large ratio between the actual and ideal performance indexes constitutes a measure of how much room for improvement there is in basic column design." In the subsequent months, I continued my studies to understand the reason for this discrepancy and tried to assimilate a packed column to a bunch of parallel identical tubes. Returning to my wartime idea of long, small diameter tubes with a retentive layer, I decided to try a rather large bore tube - a 1-meter long pyrex tubing with 0.8 mm internal diameter the walls of which I had coated with polyethylene glycol. I was disappointed by the frightfully asymmetric peaks obtained but my colleagues at Perkin-Elmer marvelled encouragingly about the fact that a separaOion had been obtained with such an unorthodox means; thus, I continued the experiments with longer tubes made of copper, having a smaller diameter. My overriding worry at that time was the poor shape of the peaks, which I had expected to be gaussian, but which were not. I remember making an experiment with an uncoated Tygon tubing, 10 meter in length with 3 mm internal diameter which gave an air peak having the right

111

Fig. 11. 1. One of the early chromatograms on open tubular columns. This chromatogram was obtained by using a specially built micro thermal conductivity detector and oscilloscope readout. The column was 16 ft. x 0.25 mm I.D., coated with Carbowax 1540. Peaks: l=air; 2= acetone; 3=carbon disulfide; 4=chloroform; 5=methylene chloride. Retention time bf the last peak is 15 seconds. order-of-magnitude width and a more nearly gaussian shape. I was then using a small, experimental thermal conductivity detector having a thermistor bead and a volume of only a few 1-11 instead of the close to 1-ml volume of the standard detectors used at that time with packed columns, in order to be sure that the peaks' long trailing edges could not be ascribed to the detector volume. In this way, I could eventually obtain somewhat better peaks. My first report on the preliminary work was presented at the 1957 Lansing Symposium (3). In the subsequent months I had cleaned up the theory of open tubular columns and presented a more detailed paper at the 1958 Amsterdam Symposium ( 4 ) . In this paper, I also presented the theory for the case of columns with rectangular cross-section with a high width-to-thickness ratio. This was alright as a mathematical exercise, but subsequently, when I decided to investigate what the effect of the corners of such rectangular tubes was, I discovered that their effect was to increase the HETP of such tubes by a factor of around six - as I recall - and that this effect was independent of the width-to-thickness ratio: with a higher ratio, the fractional effect was less but that effect had to be diffused over a greater distance so that the overall effect was the same HETP increase as for lower width-to-thickness ratio. The only way to avoid it was to have rectangular cross-section tubes without corners, i.e., the tube was to be bent around and have a ring-like cross-section of extremely uniform width, and with minimal support of the inner rod or wire. I dropped the idea completely.

112

EOllr0 GlOin ca?lary I% &decyl phthdldte

201bln’ Temp LO’C Flow-0 %mllmn

Star1

t

Fig. 11. 2 . A chromatogram shown at the 1958 Amsterdam Symposium ( 4 ) . The columns was 150 ft. x 0.25 mm I.D., coated with didecyl phthalate; column temperature was 4OoC and the carrier gas (He) flow rate 0.96 ml/min. A specially built micro thermal conductivity detector was used as the detector. The sample consisted of a c6 hydrocarbon mixture. Going back to the small diameter open tubular columns of circular cross-section, one of the early observations was the slowness of the molecular diffusion in the retentive liquid layer when compared to the much higher speed of molecular diffusion in the gas phase. This consideration led me to suggest that the way out of this impasse was to have the walls of the capillary tubes coated with a porous layer of much greater area than the bare walls, so that the retentive layer could be dispersed in a much thinner layer or in a quantity of tiny droplets. This suggestion was briefly mentioned in both my Lansing and Amsterdam papers and then further elaborated in a plenary lecture presented at the 1960 Edinburgh Symposium ( 5 ) . I was happy to see that my suggestion was taken up by Drs. HorvHth and Halhsz who succeeded in the preparation of such columns utilizing the coating technique used by me in the early stage of capillary column development and by Leslie Ettre and his group at Perkin-Elmer who continued this work developing the so-called SCOT (support-coated open tubular) columns which are widely used in analytical laboratories. In conjunction with this work, I reexamined the theory of open tubular columns and adjus€ed it to take the effect of the porous layer into consideration ( 6 , 7 ) . Later on, I took a look at liquid chromatography and decided that here, the theoretical discrepancy between capillaries and packed columns was seven orders of magnitude. I subsequently discovered that I was quite wrong but had to accept the impossibility of liquid chromatography with capillaries for which a diameter of the order of one micrometer was calculated. This is the reason for my present interest in liquid chromatography with packed columns.

113

Fig. 11. 3. 1965 Meeting of the G.A.M.S. and the Greek Chemical Society, in Athens. M.J.E. Golay (right) and L.S. Ettre. One of the things I enjoyed in connection with my gas chromatography activity was to study how gas chromatography presented itself from a thermodynamic viewpoint. This led to a somewhat fraudulent paper I gave at the 1964 Brighton Symposium (a), in which I showed that the chromatographic separation property could lead to a lowering of the enthalpy of a system. I was not quite sure where the fallacy was, albeit I had some vague idea about it, but I thought I would challange my audience with the idea and see if someone would pick up the gauntlet. I am not aware that this has been done. REFERENCES 1 M.J.E. Golay, AnaZ. Chem. 29 (1957) 928. 2 M.J.E. Golay, Nature (London) 180 (1957) 435. 3 M.J.E. Golay, in Gas Chromatography (1957 Lansing Symposium), V.J. Coates, H . J . Noebels and I . S . Fagerson, eds., Academic Press, New York, 1958, pp.1-13. 4 M.J.E. Golay, in G a s Chromatography 2958 (Amsterdam Symposim), D.H. Desty, ed., Butterworths, London, 1958, pp.139-143. 5 M.J.E. Golay, in Gas Chromatography 1960 (Edinburgh Symposium), R.P.W. Scott, ed., Butterworths, London, 1960, pp.139-143.

114 6 M . J. E. Golay, Nature (London) 199 (1963) 370. 7 M.J.E. Golay, Anal. Chem. 40 (1968) 382. 8 M . J . E . Golay, in Gas Chromatography 1964 (Brighton Symposiwn), A . Goldup, ed., Institute of Petroleum, London, 1965, pp.143-153.

115

DAVfD W. GRANT

DAVID WALTER GRANT was born in 1929, in London, England. Between 1946 and 1949 he studied at Medway Technical College, Kent, England.and in 1949 obtained from London University an external B.Sc. in chemistry with honors. In 1973 he was awarded the D.Sc. degree by London University. After a two-year army service, he joined the Distillers Company as an analytical chemist. In 1953 he moved to the Coal Tar Research Association and since then, he has been active in the development of gas chromatography and its application to coal tar. At present he is a principal scientific officer with the British Carbonization Research Association leading a team responsible for the use of chromatographic techniques in a study of environmental problems within the carbonization industry. Dr. Grant has authored and coauthored about 50 papers on various aspects of chromatography; his book on Gas-Liquid Chromatography was published in 1971 by Van Nostrand Reinhold Company. He holds several patents related to sampling devices and porous-layer open tubular columns. He was one of the early members of the (Gas) Chromatography Discussion Group, member of its Executive Committee in 1967 and served as its Honorary Secretary from 1968 to 1971. Dr. Grant's involvement in gas chromatography started in 1953 and continued ever since. Besides investigations related to the investigation of coal tars and associated products his achievements cover various questions related to sample introduction, automation and column development particularly concerning porous-layer open tubular columns.

116 When I was asked to provide these reminiscences on my early days in chromatography and to reflect on the circumstances of my introduction to this fascinating subject, it was with something of a shock to realise that I had to cast my mind back a quarter of a century. In those days I was responsible for the development of new methods for the analysis of coal tar distillation fractions. This involved searching for chemical techniques that would specifically determine relatively inert compounds such as aromatic hydrocarbons, as well as the more reactive phenols, bases, and heterocyclics. My director at that time was Dr. D. McNeil who had previously been a member of the Billingham staff of I.C.I. and it was he who first drew my attention to the new technique of gas-liquid chromatography which had already been employed with success at I . C . I . for the analysis of volatile samples. I was in fact given the task of investigating the possibilities of the technique in relation to our own industry. It is interesting to reflect that at this time one of my colleagues, Don White, was working on applications of 'liquid-liquid partition chromatography' and he succeeded in producing some excellent separations of phenolic isomers (I). My own first printed words on the subject were contained in a 1953 internal report describing various methods that would be worth pursuing for this type of analysis, and I have reproduced this below:

" ( b l Gas Liquid Chromatography The most important advance i n chromatographic a n a l y s i s since t h e deve Zopmcnt of p a r t i t i o n chromatography has been t h e development by James and Martin of what i s called gas-liquid chromatography. I n t h i s a n a r r m tube i s packed u i t h an i n e r t f i l l e r s l i g h t l y wetted with a high-boiling l i q u i d , f o r example l i q u i d p a r a f f i n , glycerol or JinonyI phthalate, and the whole i s heated by enclosure i n a vapour or e l e c t r i c a l l y heated j a c k e t . A few milligram of the sample is introduced i n t o one end of the tube i n t o which a l s o a slow stream of nitrogen i s f e d . The sample i s carried by t h e gas through the tube where t h e separation of i t s components takes place. The separated components i s s u i n g from t h e other end of t h e tube are passed through ct s u i t a b l e d e t e c t o r , sueh as an automatic t i t r a t o r f o r a c i d i c or basic materials, or for neutral substances a thermal conductivity ce I 1 incorporated i n an e l e c t r i c a l bridge system, which compares the conductivity of t h e entrance and e x i t gases." My first practical task was to survey the available literature. This was comparatively easy since, up to the end of 1953, there had been no more than a handful of publications in GC, including the historic papers of James and Martin. I suppose one of the major events to fire my own enthusiasm was an early meeting with these two remarkable men. The contrast between the two stands out sharply in my memory in spite of the intervening years - Tony James, talkative, gregarious, disseminating new ideas with almost every sentence; Archer Martin, quiet and thoughtful, unassuming and brilliant. I was priviledged to see their well-known gas chromatograph which was equipped with the ingenious automatic titration detector. In retrospect

117 I'm sure this had a profound effect on my attitudes to experimental work and the realization that one can accomplish a great deal with simple homemade apparatus. The modern generation of chemists would perhaps do well to learn that it is not always essential to purchase the most expensive apparatus to achieve the best results. In 19535 4 of course there was no commercial equipment for GC and so we had to build a chromatograph for ourselves from very basic materials. That is we used home-made glass columns encased in cylindrical glass outer jackets which we wound with resistance wire for heating purposes. The detector was a primitive katharometer fabricated from glass tubing and fitted with lengths of 0.001 inch diameter platinum wire as sensors. An exasperating routine job was the fitting of these

Time in minutes, from s t a r t SEPARATION OF MIXTURE

OF MONOCYCLIC AFIOMATICS BY GAS-LIQUID CHROMATOGRAPHY

Fig. 12.1. Early gas chromatogram obtained by plotting the peaks from spot galvanometer readings. sensors when they burnt-out, as they frequently did. The circuit consisted of an elementary Wheatstone bridge fed by wet acid accumulators, which of course, required regular recharging. The bridge output was monitored by a rather ungainly commercial recorder with a five-second response and a frustrating propensity to 'go dead' at

118 critical times (as for instance when a peak was due!). Nevertheless our efforts produced dividends in the shape of our first-ever chromatograms, which engendered that indescribable fascination of watching a new chromatogram being generated, a fascination that has hardly faded in the intervening years. To be strictly accurate, our homemade apparatus was completed some weeks before the delivery of the recorder and so we ran our initial chromatograms by plotting them from spot galvanometer readings. However, even s o , the results were sufficiently coherent to reproduce it in an internal report. Another milestone for me during these early years was enrolling as a member of the Institute of Petroleum Gas Chromatography Discussion Group, which provided a meeting ground for so many Pioneers. I well remember attending meetings at which the entire membership sat round a single table and where I met many well-known personalities. R.P.W. Scott was an early associate, largely because of our common affiliation to the coal carbonization industry. A gifted experimentalist, Ray never fails to impress me with his enthusiasm and seemingly unflagging energy. The 1956 London Symposium was the first international meeting organised by the Group and was the occasion at which I presented my first paper, written in collaboration with my colleague, Mr. G.A. Vaughan (2) and -1 have been lucky enough to have attended all of this series of meetings up to the 1976 Birmingham Symposium. The London meeting clarified a number of fallacies and misconceptions. Who would believe today that reduced column exit pressure was often advocated because "this is what one does in fractional distillation". I remember Dr. A.J.P. Martin standing up after an extended argument over this very point and politely reminding us of a few facts of elementary physical chemistry. My paper, written in collaboration with Mr. G.A. Vaughan, was concerned with the gas chromatography of coal tar naphthas, but in my introduction I described a very simple new detector which I called the 'emissivity detector'. I described this in more detail at the next symposium in Amsterdam (3) and I believe this was the first detector to be based on a photometric principle - a fact not often recognised in subsequent publications. Perhaps I might modestly claim that it was also the first detector to claim to be selective, and to be used in a selective sense with regard to its application. If asked to select the most memorable of this series of symposia I would, without hesitation, nominate the 1958 Amsterdam meeting. This was particularly notable as the first major occasion at which Golay described the theory of capillary columns. During his introduction he showed the two slides which had such an immense impact on everyone present, showing in one case the separation of six C6 hydrocarbon isomers in under nine minutes and in the other, the separation of isomeric xylenes. Although the Amsterdam meeting was the second formal symposium to be organised by the Discussion Group I would regard it as the first truly international meeting in chromatography. For many of us it was the first real opportunity to meet prominent chromatographers from other countries who, until then,

119

Fig. 12.2. At the 1958 Amsterdam Symposium. Leftto-right: D . H . Desty, D.W. Grant and s. Dal Nogare

.

had merely been names in various journals. The late Stephen Dal Nogare was one of these personalities and one of my most treasured photographs includes Stephen taken at the informal reception before the meeting started. ~ laboratory was equipped with a fairly By the early 1 9 6 0 ' ~my large number of gas chromatographs. Although all were home-made apparatus, they included a great variety of detectors such as electron capture, micro argon ionization,.khermal conductivity and emissivity detectors. We also built a preparative scale gas chromatograph. The glass capillary columns were also made by ourselves, using the machine originally designed by Desty's group at British Petroleum. We were using these columns successfully at that time for the analysis of complex phenols; our results were reported at the 1962 Hamburg Symposium ( 4 ) . One must not, however, remember only one's triumphs. In the late 1950's I borrowed Dr. Martin's famous perspex model of the gas density balance and set about attempting to fabricate a working version in our own workshops. We never succeeded in getting this device to work and eventually abandoned it as beyond our instrumental capability. Thus I learned my next most important lesson - "know your limitations". However, in fairness to our workshops who undertook many difficult tasks for me, the fabrication of a Martin gas density balance was no ordinary feat, as I'm sure many others have since had cause to realise. In fact I'm not at all sure that it wasn't necessary to have the Martin personal spell placed on the device before one could get it to work!

120

Fig. 12.3. My laboratory in the early 1960's. Twelve home-made chromatographs are visible in this photo. Glass capillary columns can be seen hanging from the ceiling in tQe left. Naturally, making ones own equipment was not without its share of hazards. On one occasion I was testing a newly built flame ionization chromatograph which I had designed myself and of which I was overtly proud. I ceremoniously pressed the button to ignite the hydrogen flame and immediately the entire column unit exploded. Fortunately, although I was standing over the unit, I only suffered singed hair and eyebrows - but of course my pride was fatally injured! Another rather comical event that used to occur regularly with our very early columns was the result of using rather flimsy septa for sample injection. These devices were made primarily for medical purposes since in those days custom-made septa for GC were not yet available commercially. Often, as a sample was being injected, the septum would be forced off the column by the sudden increase in inlet pressure and the entire column packing would then vacate the column as a picturesque white fountain, a large proportion of which would be deposited upon ones head. Another memorable mishap occurred when we tried to chromatograph corrosive materials on a preparative column. Puzzled as to why nothing seemed to be reaching the detector we found on closer examination that the material had

121 preferred to find a different way out - through the column wall : There is no doubt that the advent o f capillary columns initiated a remarkable breakthrough in chromatographic resolution. When Golay announced this development at the Amsterdam meeting most of us were impatient to return to our own laboratories to try out this new technique. In my case the only material of capillary dimensions that I could find relatively quickly was copper tubing manufactured by a local company. I purchased several hundred feet of this material, charged for by the pound weight, and this was coated with squalane and operated with one of our home-made micro-argon detectors. By modern standards the column was not very efficient, possibly because of the substantial layer of copper oxide in the bore, but it nevertheless gave a rather better performance than we had previously obtained using packed columns. These early experiences with capillary columns emphasized the importance of the capacity (partition) ratio in resolution and hence not to attach too much significance to plate values alone. Nevertheless there was a considerable spirit of competition between chromatographers to achieve very high plate efficiencies, and I must admit to as being as guilty as others in participating in this race. Although the connection between these efforts and my professional duties may have been rather hard to detect at times, I believe that the modern science of capillary chromatography owes a considerable debt to the enthusiasm which prevailed, as it soon became clear that many of our difficult separation problems would be much easier to solve on this type of column than on the more conventional packed columns. Golay, -in his early papers, had hinted at the possibility of even better columns if the inner wall of the capillary were to be roughened or coated with finely divided support material. In considering possible ways to accomplish this I thought it may be feasible to sinter diatomaceous support into the wall ofha glass column by drawing it down to capillary dimensions. In fact we made many such columns successfully in our laboratory over a period covering some ten years ( 5 ) . Unfortunately the idea did not seem to be commercially attractive although successful patent cover was completed in several countries including the U.S.A. and Great Britain. Thus we had a unique situation in which all of our important analyses were based on the use of columns that could not be purchased. Of course, support-coated open tubular columns of rather different character are now available commercially but it remains a fact that Golay's predictions with regard to the theoretical improvements afforded by their use have never been fully realised in practice. This is a pity considering the time and attention presently devoted to finding better ways of treating wall-coated capillary columns. Surely this subject could still be an attractive field of study for enterprising young research workers? Another major event in my professional life was my first visit to the United States, where I met many of the American school of chromatographers. This first visit occurred in 1964 when I attended the Second International Symposium on Advances in Chromatography in Houston, Texas; an opportunity given to me through the generosity

122

Fig. 12.4. At the 1972 Montreux Symposium. Leftto-right: M . J . E . Golay, H. Boer, D.W. Grant and R.P.W. Scott. of Professor A1 Zlatkis, who is known to everyone in the chromatography world as the leading character behind that immensely successful series of meetings. I have been fortunate to have attended several of these symposia in the intervening years and have never failed to be impressed by the variety of papers and the personalities that these meetings attract. I have earlier mentioned the Gas Chromatography Discussion Group which for a long time performed its activities under the aegis of the Institute of Petroleum but now operates independently. This organization is well-known for its series of formal and informal symposia and for the dissemination of chromatographic information through the publication of Abstracts. I personally have had many an occasion to bless the existance of the Abstracts but none more so than during the preparation of my book Gas-Liquid Chromatography (6). Of course much of the original motivating force of the Group was created by the enthusiasm of D.H. Desty, a fact that has been recognised in recent years by awarding him the Honorary Life Membership in the Group. In reminiscing over past events in the development of chromatography, and my personal involvement in it, is easy to become nostalgic. However I feel that the state of the subject even today offers many

123 e x c i t i n g p r o s p e c t s f o r t h e f u t u r e , p a r t i c u l a r l y i n t h e realms o f HPLC and HPTLC. For m e t h e s u b j e c t h a s n o t l o s t i t s g l i t t e r and I am c e r t a i n t h a t i t s t i l l i n f e c t s t h e new e n t r a n t s t o i t s r a n k s j u s t as c o m p l e t e l y as i t i n f e c t e d m e t w e n t y - f i v e y e a r s ago. I f e e l p r i v i l e d g e d t o have been so c l o s e l y a s s o c i a t e d w i t h t h e e a r l y development o f g a s chromatography, a s i t u a t i o n c r e a t e d by my p r e s e n c e i n t h e r i g h t p l a c e a t t h e r i g h t t i m e and w i t h t h e r i g h t c o l l e a g u e s , r a t h e r t h a n by any o f my own good management. I n o t h e r words, by s h e e r good l u c k I became a chromatographer, and n e v e r f o r one moment have I regretted it. REFERENCES 1 D. White and G.A. Vaughan, Anal. Chim. Acta 16 (1957) 439. Grant and G . A . Vaughan, i n Vapour Phase Chromatography (1956 London Symposiwn), D . H . D e s t y , e d . , B u t t e r w o r t h s , London, 1957, pp. 413-421. 3 D.w. G r a n t , i n Gas Chromatography 1958 (Amsterdam Symposiwn), D.H. D e s t y , e d . , B u t t e r w o r t h s , London, 1958, pp. 153-164. 4 D.W. Grant and G . A . Vaughan, i n Gas Chromatography 1962 (Hamburg Symposium), M. Van Swaay, e d . , B u t t e r w o r t h s , London, 1962, pp. 305-313. 5 D.W. G r a n t , J. Gas Chromatogr. 6 (1968) 18. 6 D.w. G r a n t , Gas-Liquid Chromatography; R.A. Chal’mers, e d . , Van Nostrand Reinhold Co., London, 1971. 2 D.W.

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125

ERICH HEFTMANN

ERICH HEFTMANN was born in 1918, in Vienna, Austria, where he studied medicine from 1936 to 1938. When he emigrated to the United States in 1939, he was awarded a scholarship, which enabled him to attend New York University. After obtaining a B.A. degree in chemistry from N.Y.U. in 1942, he became an Instructor in Chemistry at the University of Maryland, where he took graduate courses in organic chemistry. In 1943 he accepted a Graduate Assistantship in the Department of Biochemistry and Pharmacology of the University of Rochester, and in 1947 he fulfilled the requirements for a Ph.D. degree in biochemistry. Since that time, Dr. Heftmann has been in government service, first in the Diabetes Branch of the U . S . Public Health Service and then at the National Institutes of Health from 1948 to 1963. At N.I.H. he worked in the Endocrinology Branch of the National Cancer Institute until the establishment of the National Institute of Arthritis and Metabolic Diseases in 1950, when he became a member and ultimately the Acting Chief of its Steroid Section. During that time, Dr. Heftmann taught a course in chromatography at the U.S. Department of Agriculture Graduate School and at Georgetown University. In 1959 he joined the Division of Biology at the California Institute of Technology, first as a Research Fellow, and from 1964 to 1969 as a Rsearch Associate. He also was a Visiting Associate Professor of Medicine at the University of Southern California from 1966 to 1969. While at Caltech, Dr. Heftmann transferred to the U.S. Department of Agriculture, and in 1969 he moved to its Western Regional Research Center in Berkeley, California, where he organized a Plant Biochemistry Research Unit in 1976. Dr. Heftmann's main scientific contributions have been both in steroid biochemistry and in the field of chromatography. He is an author of over 130 publications, including two books on Steroid Biochemistry and one on the Chromatography of Steroids and he is the editor of a book on Chromatography (now in its third editiov) and one on Modern Methods of Steroid Analysis. Dr. Heftmann serves on the Editorial Board of Phytochemistry, Lloydia, Journal of Chromatography

126 and JoiuvlaZ of L i q u i d Chromatography and he has served on the Advisory Board of the Advances i n Chromatography and Chromatography Science series (both M. Dekker Inc.), as well as on the Subcommittee on Lipids of the National Research Council. In 1975 he was awarded the Alexander von Humboldt Prize of the German Federal Republic. Dr. Heftmann is one of the pioneers in the utilization of liquid chromatography for the analysis of steroids and other biological substances. A whole generation of American biochemists and chromatographers learned the details of the technique from him.

127 If my Major Professor, the famous lipid chemist Walter R. Bloor, Professor of Biochemistry at the University of Rochester, had been the man to interfere with somebody's research, my thesis work would have been much easier. But his favorite expression was "It's your funeral," and so I struggled along with the isolation of vitamin E from carrot oil, using molecular distillation, which at that time had its home in Rochester, N.Y., at the Eastman-Kodak subsidiary, Distillation Products, instead of using chromatography. Of course, I had read H.H. Strain's classic, Chromatographic Adsorption AnaZysis but chromatography did not impress me until I met C.E. Dent. Not of prepossessing appearance, but endowed with a brilliant mind, Dent had come from A.J.P. Martin's laboratory to spread the word about paper chromatography and soon had us graduate students involved in excited discussions. Unfortunately, my dissertation was almost finished by then, but one of my younger colleagues, Alejandro Zaffaroni, was just starting his own. In order to apply the aqueous partition systems then used for amino acids to the separation of ketosteroids, he decided to convert them to the water-soluble Girard's derivatives. This work created quite a bit of interest, but Alex was not content to rest on his laurels and proceeded to devise for the adrenocortical hormones the nonaqueous partition systems, which are still referred to as Zaffaroni systems. I was quite impressed with his work, but I had already begun to work on my first job, which involved the detection of diabetes, and all I could do in chromatography was to practice the identification of sugars in urine by paper chromatography. It was not until I transferred to the National Cancer Institute in the National Institutes of Health (N.I.H.) that I could start the work on the chromatography of steroids, which was to become my chief scientific interest. My first publication on the paper chromatography of estrogens ( I ) was influenced by Zaffaroni's work. To facilitate the detection of the estrogens, I chromatographed them in the form of azo dyes with solvent-systems composed of petroleum ether, toluene, ethanol, and water. My work came to the attention of Erich Mosettig, who was just organizing a Steroid Section for the newly established National Institute of Arthritis and Metabolic Diseases, and he asked me to join his group. My assignment was to help the U.S. Department of Agriculture expeditions, which were in Africa collecting plants containing certain steroids, suitable as starting materials for the partial synthesis of cortisone. With the help of Alma L. Hayden, I developed some screening methods for plant steroids, based on paper chromatography. In time, my horizon widened, and I became interested in every aspect of chromatography. I must have read most articles on chromatography and electrophoresis that had been published until then, and when the U.S.D.A. Graduate School invited me to organize one of the first courses on chromatography ever taught, I accepted enthusiastically. After struggling through seven years of teaching without a textbook, I decided to produce my own text through a collaborative effort. This book has now gone through three editions (2) and, although it has not made me wealthy, it has enriched me by bringing me

128

Fig. 13.1. (Left-to-right): Erich Heftmann, Alma L. Hayden and David F. Johnson, "looking busy" for the N.I.H. photograph, in 1956. in contact with many outstanding scientists in the field of chromatography. For six years I have also assumed the immense task of reviewing the field of chromatography for A n a l y t i c a l Chemistry. It has earned me nothing but friendly twits about my spiritual descent from Beilstein. Thus, L b z l 6 Zechmeister once told me what Beilstein said when somebody asked him how he keeps up his encyclopedia. "Das ist sehr einfach," he said, "Ich lese alles und schreibe alles auf am richtigen Platz.I r i My study of the chromatographic literature as well as the steroid literature convinced me that individual steroids in biolog-

+ "This is very simple. I read everything and note everything the right place."

-

in

129 ical extracts can only be analyzed after chromatographic separation. In nature, steroids invariably occur in association with a large number of closely related precursors and metabolites, and they give virtually no specific color reaction. The problem is compounded by the fact that there may be large differences between the relative amounts of the various analogs and homologs in the mixtures. Because the load capacity of most chromatographic systems is quite small, minor components may he present in the sample in amounts smaller than the limit of detection and, thus, they remain unaccounted for. The reason why the original Tswett columns are still used in steroid laboratories is simply that they have the load capacity required, especially, for crude extracts (3). With this in mind, I started work in 1954 on an analyzer for adrenocortical hormones, based on liquid column chromatography ( 4 ) . In this work I was greatly aided by my only graduate student, David F. Johnson, and members of the Instrument Engineering and Development Branch of N.I.H. The Steroid Analyzer, which was finally completed in 1961 ( 5 ) , featured a gradient elution system, a UV analyzer, a colorimetric analyzer, a fraction collector, and a recorder. It is now obsolete, of course, but some of its details have survived in other instrument designs. With the advent of thin-layer chromatography, my research group, particularly Raymond D. Bennett, has pioneered in applications of TLC to various classes of steroids and has also forayed into other fields, such as the gibberellins and purines. We have developed these methods as we needed them for our research in plant biochemistry and phytoendocrinology and had only rare occasions to advance the science of chromatography. One of these was my rediscovery of the Chromatofuge (6). I was unaware of the fact that this extremely useful device for preparative chromatography had already been invented 25 years earlier, until Michael Lederer pointed this out to me. Like reinventing the wheel, this will make any inventor feel both proud and stupid. Regardless of my personal role, the Chromatofuge needs to be popularized, manufactured, and improved. It is easy to pack, load, and use; it is fast, efficient, and adaptable to various modes of operation. It can be used for adsorption, partition, ion-exchange, and gel chromatography, for both large-scale preparations and analytical purposes. Because we do not have the engineering facilities for developing the necessary instrumentation at my present location, we have meanwhile acquired components for the popular high-pressure liquid chromatography. We have adapted it to the analysis of plant steroids and gibberellins and find it highly useful in our work on problems of plant biochemistry. However, like most of us who are active in chromatography, we must devote most of our time to problems in which chromatography plays only a supporting role. When we succeed in solving problems where others have failed, it is often because we can make better use of chromatography than they. REFERENCES 1 E. Heftmann, Science 111 (1950) 571. 2 E. Heftmann, Ed., Chromatography, Reinhold, New York, 1st ed. 1961;

2nd ed. 1967; 3rd ed. Van Nostrand Reinhold, New York, 1975.

130 4 E . Heftmann and D.F. Johnson, Anal. Chem. 26 (1954) 519. 5 F.O. Anderson, L . R . C r i s p , G.C. R i g g l e , G . G . Vurek, E . Heftmann, D . F . Johnson, D. F r a n c o i s and T.D. P e r r i n e , Anal. Chem. 33 (1961)

1606. 6 E. Heftmann, J.M. Krochta, D.F. Farkas and S . Schwimmer, J . Chromatogr. 66 (1972) 365.

131

GERHARD E. HESSE

GERHARD EDMUND HESSE was born in 1908, in Tubingen, Germany. He studied at the Universities of Bonn and Munich and received his Ph.D. in 1932, under H. Wieland, winner of the 1927-Nobel Prize in Chemistry. He received his h a b i z i t a t i o n in organic chemistry in 1937 from the University of Munich. In the period of 1938-1944 he was on the faculty of the University of Marburg/ Lahn. In May 1944, Dr. Hesse accepted a professorship at the University of Freiburg/Breisgau and joined the Institute of Staudinger. Finally, in November 1952, he moved to the University of Erlangen/Nurnberg as professor and head of the Institute of Organic Chemistry. Professor Hesse is the author and coauthor of about 160 papers on organic natural substances, boron- and nitro-compounds and chromatography, and of two books, Adsorptionsmethoden i m Chemisehen Laboratoriwn (1943) and Chromatographisches Praktikum (1968). He is a member of the Bavarian Academy of Sciences. He is the recipient of the Fresenius Award of the German Chemical Society (1972) and the M.S. Tswett Chromatography Medal (1975). Dr. Hesse’s activities in chromatography started in Munich, at Wieland’s Institute. He was one of the first to apply chromatography to the analysis of complex mixtures of natural substances, he wrote the first detailed review of the technique and he pioneered in the development of suitable and standarized adsorbents for liquid column chromatography. In Marburg, he developed a new technique where the chromatographic principles were applied to a system using a gas as the mobile phase. This was the beginning of gas adsorption chromatography. When chromatography started its exponental growth, Dr. Hesse recognized the importance of basic training in the technique and started the organization of intensive postgraduate training courses for practising chemists at his Erlangen laboratories. Thus, he can be credited with the training of scores of analytical chemists in the modern aspects of the technique. A special merit of Dr. Hesse which the editors of this book are most happy to recognize is that he helped Dr. Woelm in having M.S. Tswett‘s

first paper on chromatography (published in a Russian journal in 1903 and available in perhaps 2-3 libraries of the World only) translated into German and English and have it published with the proper explanatory notes?

*See ref. 6 after Dr. Hesse's contribution.

133 I was educated as an organic chemist and have utilized chromatography to solve problems occurring in my investigations in that field. In other words, I am mainly a user of the technique and my principal aim was always to utilize chromatography for preparative purposes, although, naturally, we have also dealt with its use as an analytical method, modifying it according to our requirements. My activities were carried out in the .laboratories of four University institutes and at the Max Woelm Company, in Eschwege, which provided me with the necessary means in the darkest times. The subject of my research work was quite different in the various places, however, chromatography was always part of the methods utilized. Below, I will summarize my activities by dividing it into three groups: liquid chromatography, paper chromatography and gas chromatography *

LIQUID CHROMATOGRAPHY . I started my thesis work in March 1930 under Professor €IWieland, in Munich; the subject of my thesis was the poisons of toads (species Bufo). These substances are related to the well-known heart-poisons from digitalis and other plants and have been used in China as medications for heart failure. Thus, during the Easter holidays, I went with my father - he was a zoologist - on toad-hunting. My goal was to obtain the pure secretion of the animals, by pressing their glands, without hurting them and I succeeded in this. Since then, I have always tried to handle sampling in the simplest way. First I tried to work up extracts of toad-skin which I inherited from my predecessors in the laboratory. However, experiments to use adsorption, similar to the method utilized by Willstatter for chlorophyll were negative. About this time the work of Kuhn's Heidelberg laboratory on carotenoids, in which Tswett's method triumphed, was published and naturally, I immediately tried it. However, I was soon disappointed again. Although the spectra remained unchanged, we often found a chemical change in the substances eluting from the column and sometimes this change could only be seen after the isolation of the pure substance. Thus, it is not surprising that Professor Wieland advised me not to use adsorption in connection with these natural substances and my experiments also seemed to prove the validity of his advise: an experiment with a small amount of bufotalin on aluminum oxide showed that it mostly decomposed on the column. Further investigations proved that all commercial aluminum oxides showed strong basic reactions and the sensitivity of my substances to bases was well known. Washing with HC1 did not help, as always some acidity remained, which could not be removed by water. The reason for this phenomenon was explained by G.-M. Schwab in our Institute who utilized these aluminum oxides in ion-exchange chromatography for the first time ( 2 , Z ) . It is connected to the method of preparation. Aluminum oxide is prepared by heating aluminum hydroxide to about 350 OC while aluminum hydroxide is prepared by precipitation from a sodium aluminate solution and always contains some sodium carbonate. During dehydratation one obtains sodium aluminate centers on the surface of

134 the oxide which, when reacting with HC1, became aluminum chloride centers. Thus, the basic aluminum oxides are cation exchangers while acidic aluminum oxides are anion exchangers. Similar observations have also been reported on silica gels.

Neutral alwninwn oxide I soon found out the way to eliminate the problem. If the aluminum oxide which was reacted with HC1 is quickly heated while still wet, then the aluminum-chlorine bonds hydrolyse and one obtains an almost completely neutral aluminum oxide. I could separate the toadpoisons on this material without any loss and chemical change and I could repeat this with a number of other natural substances. In spite of the good results, the disbelief in chromatography was so well established in Munich that Wieland, when he once found me doing these experiments, told me rather angry, "stop this stupid thing!". As a conclusion of our results with the neutral aluminum oxide, such "neutral" substances were soon offered on the market. However, at the beginning these were not truly neutral but represented simply a mixture of acidic and basic A1203 which, in aqueous suspension, gave a neutral reaction. Such a substance is called today a mixedbed ion exchanger. Naturally, it is still active when used with sensitive substances. It is, however, easy to recognize, by shaking with the aqueous solution of a basic and an acidic dye, e.g. methylene blue and eosin. After washing the excess dye, one investigates the particles under a microscope: part of the particles will be blue while another part will be red, the first representing the alkalicontaining A1203 while the second the chlorine-containing A1203. A truly neutral A120, will show no color.

The speed o f adsorption Depending on the method of preparation, the speed of adsorption of dissolved or gaseous substances on A1203 (or silica gel) is quite different. This is naturally an important criterion for utilization in chromatographic separation which is, after all, based on repeated adsorption and desorption. After much work, we could develop in cooperation with the Max Woelm Company three types of aluminum oxides giving optimum separation (3).

Activity When using non-polar solvents, the adsorption of a number of organic substances on aluminum oxide is so complete that desorption is practically impossible. Sometimes, this problem can be solved only by the selection of a proper mobile phase. However, in general, the selection of the solvent depends on the sample. Therefore, it is desirable to be able to reduce the activity of the adsorbent in a reproducible way. This is possible if we block part of the active centers with a substance which is strongly adsorbed; in this way, these centers are eliminated from any reaction with the substances to be separated. A further advantage of this method is that the activity of sorption will be more uniform. Every solid material has different

135 active sites on the surface of the pores. As a conclusion of this, part of the sample - bound to the strongly active centers - will be delayed resulting in zone broadening. If we eliminate these strong centers, the zone will remain sharper. For example, if we react glowed aluminum oxide with some water - but not enough to react with all the adsorption centers - then this will occupy only the centers with highest activity and will eliminate them from interaction with the sample. The stronger the preliminary blocking, the more uniform will be the remaining centers; needless to say, the total capacity of the adsorbent will also be reduced. H. Brockmann proposed letting the oxide cool down in air after the heating period. During this time, it will adsorb atmospheric humidity and lose some of its activity. The inactivation can be checked with the help of mixtures of azo-dyes dissolved in 1:4 benzene-petrol ether and, based on such tests, one can establish a number of activity grades (I-V). However, the problem with this method is that it depends on the weather, i.e. the humidity content of air. One day, I visited my neighbor Erich Hiickel in Marburg and he offered me a cigarette which was kept in a desiccator. When I was surprised by this way of storage, he told me that he has 40% sulfuric acid in the desiccator. Based on this experience, I tried to use isotonic aqueous sulfuric acid mixtures corresponding to the activity grades of Brockmann ( 4 ) . This was successful and one can adjust these grades in this way very accurately. In case of the aluminum oxide produced by Woelm one only has to add to the commercial product (Grade I) a calibrated volume of water and let the mixture stay for a few hours in a flask with periodic mixing, The water will evenly distribute on the adsorbent and block only the strongest centers.

Drying of solvents Non-polar solvents such as hydrocarbons, halocarbons'and ether can be dried in a very short time by filtration through a column containing very active aluminum oxide ( 5 ) . Besides water, alcohols (e.g. from chloroform or esters) and oxidation products of ether can also be eliminated in this way. For this purpose one should utilize a basic aluminum oxide so that it also eliminates any traces of acids which may also be present. It is convenient to conduct the column effluent directly into the reaction vessel, in order to avoid the possibility of absorbing water again from the surrounding air. This method is widely used by organic chemists e.g., when working with organometallic substances.

Other adsorbents Tswett had already reported on a number of substances which could be used as the stationary phase (6). In our work, we have also applied additional adsorbents such as e.g. Madrell's salt ( 7 ) , iron oxide (8) and microcrystalline cellulose triacetate ( 9 ) , the latter mainly for the separation of racemic compounds. We had great hopes in the possibility of using activated charcoal for aqueous solutions,

136

Fig. 14.1. "Identification" of the substance from the extract of spruce barks which lures the beetle Hylobius a b i e t i s . The "identification" was done by the insect itself, by eating the particular part of the paper where the spot of the substance was located after development. however, the results were unsatisfactory. According to our experience, the characteristics of activated charcoal are difficult to reproduce, the column packing is often non-homogenous and the solvents may even extract some substances from the solid ( 1 0 ) .

Detectors Since in the first period of our activities, our main goal was preparative separation, we did not try to continuously analyse the column effluent. The appearance of a fraction could almost always be recognized by "schlieren" in the fraction collector or by color change. In many cases, we even did not wait until the fractions eluted from the column: we simply cut the column into pieces and eluted separately the individual fractions, as it is done in thin-layer chromatography. We have used 15 cm long x 1 mm diameter capillary tubes, packed with the adsorbent for the separation of smaller amounts ( 2 2 ) . These tubes were placed vertically in a test tube containing the mobile phase: flow in the column was accomplished through capillary action, similar to thin-layer chromatography. A 12 cm length in the column could result in a separation equivalent to that obtained in a 1 meter long conventional column. The final chromatogram was investigated under a lamp, then cut into different segments according to the zones and each segment was extracted separately. PAPER CHROMATOGRAPHY Our only contribution to paper chromatography was the determination of the substance which lures the beetle Hylobius a b i e t i s damaging the spruce trees. This insect can be found on young plants and eats the sugar-containing fluid; it smells from great distances certain substances present in this fluid. We wanted to find out which

137 s u b s t a n c e o r s u b s t a n c e s l u r e t h e b e e t l e s t o t h e tr e e s, and w e i n v e s t i g a t e d t h i s i n a v e r y s p e c i a l way. We chromatographed v a r i o u s e x t r a c t s of t h e b a r k on p a p e r , w e t t e d t h e f i n a l chromatograms w i t h s u g a r s o l u t i o n s and p l a c e d t h e i n s e c t on t h e p a p e r s t r i p s . The i n sect looked f o r t h a t p a r t of t h e s t r i p where t h e s p o t of t h e p a r t i c u l a r s u b s t a n c e was l o c a t e d and t h e n s t a r t e d t o e a t i t . I n t h i s way, t h e i n s e c t d i r e c t l y marked t h e zone of i n t e r e s t ( 1 2 ) . Speaking of p a p e r chromatography, I would l i k e t o mention an o b s e r v a t i o n which gave m e some i d e a s about t h e n a t u r e of t h e sepa r a t i o n p r o c e s s i n o r on t h e p a p e r . Here i n Germany, w e f i r s t h e a r d about p a p e r chromatography a f t e r t h e end of t h e War and I immediatel y t r i e d t o reproduce t h e p u b l i s h e d r e s u l t s w i t h h e l p of f i l t e r p a p e r s produced h e r e , i n Germany; however, my r e s u l t s w e r e n e g a t i v e . A s soon a s I c o u l d r e c e i v e Whatman f i l t e r p a p e r from t h e American Army, t h e r e was no problem i n r e p r o d u c i n g t h e s e r e s u l t s . I b e l i e v e t h a t t h e r e a s o n f o r t h i s d i f f e r e n c e i s t h e o r i g i n of t h e p a p e r s : o u r p a p e r was made of p r e c i p i t a t e d c e l l u l o s e w h i l e t h e Whatman p a p e r i s made of l i n t e r s . I n t h e f o r m e r , t h e m i c r o c r y s t a l l i n e r e g i o n s - representing about 70-% of t h e c e l l u l o s e - a r e d e s t r o y e d . Based on t h i s e x p e r i e n c e , I have t h e f e e l i n g t h a t t h e p a p e r does n o t s e r v e o n l y a s a s u p p o r t of t h e mobile phase. Most p r o b a b l y , t h e i n c l u s i o n . o f c e r t a i n l i q u i d s - d e s c r i b e d f i r s t by S t a u d i n g e r (13) - a l s o p l a y s a r o l e ; t h e s e a r e now i n an o r d e r e d s t a t e and a c t a s a s t a t i o n a r y p a r t i t i o n i n g p h a s e o n l y a g a i n s t c e r t a i n mobile phase m i x t u r e s . GAS CHROMATOGRAPHY L i q u i d a d s o r p t i o n chromatography can o n l y work i f t h e compounds t o be s e p a r a t e d a r e more s t r o n g l y adsorbed t h a n t h e mobile phase. For g a s e s , l i g h t hydrocarbons and many o t h e r Compounds, however, one cannot f i n d any s o l v e n t f u l f i l l i n g t h i s c o n d i t i o n . T h e r e f o r e , t h e o n l y p o s s i b i l i t y i s t o move t h e s u b s t a n c e s w i t h t h e h e l p o f a g a s . On t h e o t h e r hand, one c o u l d u s u a l l y h e a r t h e o p i n i o n t h a t t h i s is i m p o s s i b l e : due t o a r a p i d d i f f u s i o n one c o u l d n o t m a i n t a i n separ a t e d zones i n t h e g a s p h a s e . I n t h i s r e s p e c t , I have t o q u o t e a remark of P r o f e s s o r H. Meerwein, an o l d f r i e n d o f mine, about a young c o l l e a g u e : "The poor man i s t o o e d u c a t e d . A s soon a s he h a s an i d e a , he immediately knows why i t s h o u l d n o t work and t h e r e f o r e , he n e v e r t r i e s a n y t h i n g . " For t h i s r e a s o n , I always had t h e o p i n i o n of f i n d i n g o u t w i t h h e l p of s i m p l e e x p e r i m e n t s whether t h e r e i s a p o s s i b i l i t y of d o i n g something. The i d e a t o u s e g a s f o r t r a n s p o r t i n chromatography was obvious f o r m e and I f i r s t d e v i s e d an experiment t o see whether i t is f e a s i b l e e f i l l e d a t u b e w i t h s t a r c h and i n t r o d u c e d a n i t r o g e n s t r e a m at all. W c o n t a i n i n g bromine and i o d i n e v a p o r s . S u b s e q u e n t l y , w e f l u s h e d t h e column w i t h p u r e n i t r o g e n . The s e p a r a t e d brown (bromine) and b l u e ( i o d i n e ) zones c o u l d be c l e a r l y observed p r o v i n g t h a t w e c o u l d o b t a i n a r e a l chromatogram, i . e . , s e p a r a t e d zones. We c o u l d a l s o c a r r y o u t t h e p r e p a r a t i v e s e p a r a t i o n of some esters, which was i m p o s s i b l e through d i s t i l l a t i o n , i n a very s i m p l e a p p a r a t u s , u s i n g s i l i c a g e l a s t h e column packing and carbon d i o x i d e a s t h e mobile phase ( 1 4 ) .

138

Fig. 14.2. Set-up for the first gas chromatographic separation. Column heating was done with help of steam,

Our first systematic investigations were related to the following problems ( 2 5 ) : separation of substances having the same boiling point, separation of azeotropic mixtures and of cis-trans isomers. Due to the high temperature, the possibility of chemical change of the sample components in the column was critical (16) and therefore, we collected the individual fractions and analyzed each of them. As a conclusion of our results, we could exclude the possibility of splitting water from the alcohol molecules or the cis-trans isomerization of 1,2-dichloroether. While these investigations were being carried out, I was called into the Army. When I finally returned from the Russian front and was able to continue to work, the institutes at Marburg and Freiburg were in ruins, a lot of materials lost and the mechanical shops destroyed or robbed. Now, I needed a detector; however, it was impossible to build or buy one. From the ruins, we tried to collect usable equipment and started to teach in,the existing rooms as the students who returned from the War already lost their best years. According to my knowledge, the investigations of Damkohler ( 1 7 ) using brick particles containing some glycerol may be considered as the first report on gas-liquid partition chromatography. Since, however, glycerol was added for a different purpose, to reduce the activity of adsorption, this work is generally not considered as GC. By chance, we had found an interesting gas separation based on chromatographic principles while adsorbing ozonized oxygen on silica gel cooled to low temperatures (18). We observed above the blue-colored band of the ozone a zone containing nitrogen dioxide which originated from the nitrogen impurity of the sample. Elution was carried out using nitrogen while the column was slowly heated. It was easy to elute all the oxygen and stop the elution prior to the emergence of nitrogen dioxide. This was important because it could interfere in the ozonization of olefinic compounds, acting as a radical chain starter. In conclusion I would like to quote from the explanatory notes published in connection with M.S. Tswett’s first paper on chromatography (6) commemorated in this book:

139

Fig. 14.3. The old chemical laboratory at Erlangen in which my students carried out a number of chromatographic measurements. Note some of the columns on the bench at the right.

“The u l t i m a t e aim of MichaeZ Tswett’s research was not reached by himself or during h i s l i f e t i m e , a t l e a s t not f u l l y . But h i s problem was f i n a l Z y solved by means of the method which he had deveZoped and improved. As foreseen by Tswett, chromatographic adsorption anal y s i s gave and continues t o give a great impetus t o the chemistry of natural Zy occurring substances. ” REFERENCES 1 G.-M. Schwab and K. Jockers, Naturwiss. 25 (1937) 44. 2 G . - Y . Schwab and G. Dattler, 2. Angew. Chem. 50 (1937) 691.

140 3 G . Hesse, Z. EZektrochem. 55 (1951) 60. 4 G . Hesse, H. Engelhardt and W. Kowallik, Z. Anal. Chem. 214 (1965) 81. 5 G . Hesse and H. Schildknecht, Angm. Chem. 67 (1955) 737; G. Hesse, B.P. Engelbrecht, H . Engelhardt and S. Nitsch, Z. Anal. Chem. 241 (1968) 91.

MichaeZ TsGett's e r s t e chromatographische s c h r i f t . Woelm Mitteilungen AL-8, A. Woelm, Eschwege, 1954; G . Hesse and H . Weil, Michael Tswett's First Paper on Chromatography. Woelm

6 G . Hesse and H. Weil,

Publications AL-8, A. Woelm, Eschwege, 1954. 7 G . Hesse, H. Engelhardt and D. Klotz, 2. Anal. Chem. 215 (1966) 182. 8 G . Hesse and M. Alexander, Se'paration i m g d i a t e e t chromatographie 1961, G . A . M . S . , Paris, 1961. 9 G . Hesse and R . Hagel, Chrornatographia 9 (1976) 62; Ann. Chem. (1976) 996. 10 G . - M . Schwab and B . Karkalos, 2. Elektrochem. 47 (1941) 345, 348. 11 G . Hesse, Chemie 58 (1945) 76; Chem. 74 (1950) 649. 12 G . Hesse, H. Kauth and R. Wachter, Z. Angew. Entomologie 37 (1955) 239. 13 H . Staudinger, Organische KoZZoidchernie, 3rd ed., Vieweg, Braunschweig, 1950, p. 270. 14 G . Hesse, H. Eilbracht and F. Reicheneder, Ann. Chem. 546 (1941) 233; H. Eilbracht, Dissertation, University of Marburg/Lahn, 1939. 15 G . Hesse and B . Tschachotin, N a t u m i s s . 30 (1942) 387. 16 G . Hesse, 2. AnaZ. Chem. 211 (1965) 5. 17 G . Damkohler and H. Theile, Chemie 56 (1943) 353; Beih. Z . Ver. dtsch. Chemiker No. 49 (1944). 18 G . Hesse and E. Bayer, 2. Naturforsch. 198 (1964) 875.

z.

141

EVAN C. and MAJORIE G. HORNING

EVAN CHARLES HORNING was born in 1916, in Philadelphia, Pennsylvania, U.S.A. He studied at the University of Pennsylvania and the University of Illinois receiving his Ph.D. in 1940. After serving as instructor at Bryn Mawr College for one year he joined the University of Michigan in Ann Arbor, first as an instructor and then as a research associate. In 1945 he became a member of the Faculty of the University of Pennsylvania, in Philadelphia. In 1950 he was appointed as the chief of the Laboratory of Chemistry of Natural Products at the National Heart Institute, National Institutes of Health (NIH), in Bethesda, Maryland. Since 1961, he has been Professor of Chemistry at Baylor College of Medicine, in Houston, Texas. Between 1962 and 1966 he also served as the chairman of the Department of Biochemistry and since 1966 as the director of the Institute for Lipid Research. Since 1971, he has also been an Adjunct Professor of Biochemistry at Rice University, in Houston. Dr. E.C. Horning is the author and coauthor of over 250 publications and the editor of three books, Organic Syntheses Collective Vohne 111 (Wiley, 1955), E f f e c t s of Drugs on Synthesis and Mobilization of Lipids (Pergamon, 1963) and Gas Phase Chromatography of Steroids (with K.B. Eik-Nes; Springer, 1968). Between 1944 and 1950 he served as the secretary of the Editorial Board of Organic Synthesis and in various periods on the editorial boards of the Journal of Medical Chemistry, Advances i n L i p i d Research, Chemical Reviews and the Journal of Atherosclerosis Research. Presently, he is a member of the editorial advisory boards of AnaZyticaZ Biochemistry, Analytical Letters, Journal of Chromatography, L i f e Sciences and Organic Synthesis. Dr. E.C. Horning has honorary doctorates from the Karolinska Institute, Stockholm and the University of Ghent. He received the Torbern Bergman Award of the Swedish Society of Chemists, the C.W. Scheele Award of the Swedish Pharmaceutical Society, the Warner-Lambert Award of the American Association of Clinical Chemists, the M.S. Tswett Chromatography Medal and the American Chemical Society Award in Chromatography. In 1961 he was elected a fellow of the New York Academy of Sciences.

142 While at NIH, Dr. E.C. Horning and his associates introduced packed columns with very low loading utilizing silanized supports and thus opened an entirely new field for gas chromatography: the analysis of steroids and other biologically important compounds. The activities continued in Houston; in effect, Dr. Horning and his school can be credited with making GC one of the most important tools in biological analysis. In recent years, he further extended the usefulness of chromatography in this field by introducing open tubular columns and applying combined, multi-instrument techniques, involving mass spectrometry. -0-o-o-

MARJORIE GROOTHUIS HORNING was born in 1917, in Detroit, Michigan, U.S.A. She studied at Goucher College, Baltimore, Maryland and the University of Michigan, in Ann Arbor, receiving her Ph. D. in 1943. Between 1945 and 1951 she was active as a research chemist at the University of Pennsylvania and between 1951 and 1961 at the National Heart Institute, National Institutes of Health (NIH). In 1961, she joined the Department of Biochemistry of Bay College of Medicine as an associate profess0 Since 1969 she has been Professor of Biochem istry at this University, in the Institute for Lipid Research. Dr. M.G. Horning is the author and coauthor of over 170 publications. She has served on the editorial board of Drug Metabolism and Disposition, and Toxicol0g-j and kppiied PharmacoZogy and on the Advisory Board of Analytical Chemistry; presently, she is a member of the Editorial Board of the JoztrnaZ of Chromatography, Biomedical Applications. Between 1972 and 1976, she was a member of the Pharmacology-Toxicology Program Project Committee of NIH. Presently she is a council delegate of the American Association for the Advancement of Science, a councilor of the Southeastern Texas Section of the American Chemical Society and a member of the Executive Committee of the Drug Metabolism Division of the American Society for Pharmacology and Experimental Therapeutics. Dr. M.G. Horning received a honorary doctorate from Goucher College, the Warner-Lambert Award of the American Association of Clinical Chemists and the Garvan Medal of the American Chemical Society. Dr. M.G. Horning pioneered in the utilization of chromatography in analytical biochemistry and pharmacology particularly for the study of drug metabolism.

143 In 1950, Marjorie and I moved to Bethesda, Maryland, to join the National Heart Institute (now the National Heart, Lung and Blood Institute) of the National Institutes of Health. I was Chief of the Laboratory of Chemistry of Natural Products, and in establishing the Laboratory I followed the conventional organic chemical views of that period for work on the structure of natural products. Around 19551956 I became interested in chemical work related to atherosclerosis, and in 1956 I was asked by the Institute Director, Dr. James Watt, to establish a liason between the Institute and chemical research groups in the food fats and oils industry. At that time, as now, there was great concern over the effect of cholesterol and food fats and oils on the development of cardiovascular disorders originating from arteriosclerotic lesions, and industry spokesmen had offered to contribute industrial technical and scientific knowledge to research programs supported by the Institute. I attended a meeting of the American Oil Chemists Society in New Orleans in order to meet several industry scientists, and later visited a number of laboratories. A great deal of practical knowledge was available about shelf life, color and odor of food fats and oils, but very little was known about the chemical characterization of lipids in any exact way. The methods, by present standards, were generally primitive. The immediate problem was that of improving the analytical methodology of lipids and of steroids and of providing suitable reference materials for chemical, biochemical and biological studies. I established an informal committee, and with advice and help from Marjorie, who had been trained as a biochemist, I set out to change the state of analytical methodology in the field. The road proved to be long but interesting. The problem was a general one: highly precise and accurate methods for multicomponent quantitative analyses of complex samples of biologic origin were required, and they did not exist at that time. Those of us who were concerned with this problem at the Heart Institute included William J.A. VandenHeuvel, Charles C . Sweeley and Arthur Karmen. In our search for improved analytical methods I learned that several American laboratories were using the new technique of gas chromatography, and with my colleagues I visited or called several scientists I had not known previously. These included S . R . Lipsky (Yale University School of Medicine), E.H. Ahrens, Jr. (Rockefeller Institute, now Rockefeller University) and S. Dal Nogare (E.I. du Pont de Nemours, Inc.). It was clear that gas chromatography was likely to become an extremely valuable analytical method if it could be perfected. All biomedical equipment was home-made; there were major technological difficulties arising from high bleed rates, insensitive detectors, inadequate separations and decomposition of samples; and it was widely believed that only compounds of relatively low molecular weight could be brought through a gas chromatographic column. It was not possible to find a manufacturer of scientific instruments who was interested in making a gas chromatograph for biomedical investigations. Sandy Lipsky suggested that the BarberColman Co. was the best prospect, possibly because they knew very little about scientific instruments but were anxious to sell recorders.

144 I received an emergency call one morning to come to New York to meet with several Barber-Colman representatives who were on the point of deciding that they should not start to market a laboratory gas chromatograph. In New York, the sales manager agreed that if three orders could be obtained in ten minutes, he would recommend trial manufacture of several instruments. I called David Turner (Mt. Sinai Hospital, Baltimore) who agreed to order an instrument; Lipsky and I also agreed to purchase one for our respective laboratories, and the Model 10 (now obsolete) was on its way. Marjorie and I were in London in 1958 for about seven months. I had a Guggenheim fellowship (it had been awarded earlier) and was working with Charles E. Dalgleish in the Department of Chemical Pathology at the Royal Postgraduate Medical School, while Marjorie was associated with George Popjak (at that time at Hammersmith Hospital). We greatly enjoyed our stay in London for many reasons. Through the good offices of Charles Dalgleish and Earl King we met many British chemists and biochemists, and Anthony James very kindly permitted me to use the Martin-James gas chromatographs at Mill Hill. The compounds I was interested in never emerged from the GC columns, for reasons that later became clear but which were rather mysterious at the time, and the work with Charles Dalgleish was completed using paper chromatography. George Popjak very kindly showed me his independently built GC units and his titration detector for acids, as well as the gas density balance. Marjorie also spent some time in Stockholm at the Karolinska Institute, and we had an opportunity to discuss analytical methodology, as well as other problems, with Sune Bergstrom and his colleagues. I also attended the historic 1958 meeting in Amsterdam at the Tropical Institute. After returning to Bethesda, my colleagues and I started our laboratory work on gas chromatographic methods. Our objective was to separate steroids, in part because of the involvement of cholesterol in atherosclerosis, and in part because it seemed clear that if steroids could be separated by this technique, the way would be open to develop new analytical methods for almost all organic compounds except macromolecules. In the early monograph by A.I.M. Keulemans it was pointed out that a relatively large amount of liquid phase was needed for all separations, but this also meant that steroid separations would require many hours. My colleagues and I thought that we could accomplish our objective by using relatively thin films of thermostable phases on deactivated supports, and this proved to be correct ( 2 ) . Some time earlier (1959) I had visited Albert Zlatkis (University of Houston) to discuss GC problems, and he had given me a sample of a linear dimethylsiloxane polymer (SE-30). This polymer provided our best steroid separations, and it is still one of the most useful of all liquid phases. Our methods for preparing GC column packings were published in detail in 1963 (2) but the basic studies were carried out in 1959-1960. I later heard, perhaps incorrectly, that Louis Fieser, who was unquestionably one of the world's best steroid chemists, had said that it was impossible to separate steroids by gas chromatography. However, our results were accepted and the basic procedures are still widely used. Other work, notably by Lipsky and

145 by J.E. Callen (Procter and Gamble Co.) on liquid phases led to the development of linear polyester phases that are still used in fatty acid separations. The number of visitors and collaborators in our laboratory increased. This group included Eero A.O. Haahti (then at Turku University), Charles J.W. Brooks (The University, Glasgow) and Nobuo Ikekawa (now at the Tokyo Institute of Technology) who soon became the leading Japanese authority on the separation of natural products by gas chromatography. During the course of these studies it became clear that GC separations of polyfunctional compounds would not be possible without derivative formation to reduce hydrogen bonding Tapani Luukkainen (University of Helsinki) suggested that trimethylsilyl ether derivatives might be useful; this approach was successful (3). These derivatives came into wide use over the next few years; they are still among the most useful of all derivatives for GC and GC-MS-COM analyses. In 1961 Marjorie and I moved to Houston. Dr. VandenHeuvel (now at Merck Sharp and Dohme, Rahway N.J.) also transferred to Houston, and numerous studies were soon under way in our laboratories in Baylor College of Medicine (formerly Baylor University College of Medicine). The earlier committee was expanded, and there was a remarkably free flow of information among numerous laboratories; a committee report was published in 1964 (4). Reference materials, starting with longchain acids, gradually became available, largely through the efforts of Arthur Rose (Pennsylvania State University and Applied Science Laboratories) and Walter Lundberg and Orville Privett (Hormel Institute). Walter Supina and Nicholas Pelick later formed Supelco, Inc., to aid in supply problems involving lipid work and all forms of chromatography. Other organizations that became interested included Pierce Chemical Co. and Regis Chemical Co. Meanwhile the field of gas chromatographic instrumentation grew rapidly and particularly the success of the Wilkens Aerograph (due to Keene Dimick and his associates) and the F&M gas chromatograph (due largely to A.J. Martin, C.E. Bennett and F. Martinez) soon led to commercially available instruments of many kinds. In Italy, Carlo Erba equipment was developed by Francisco Poy; in England, Pye equipment was developed by numerous individuals; in Japan, Shimadzu gas chromatographs came into wide use with the advice and help of Nobuo Ikekawa. The further development of high sensitivity detectors was a matter of considerable importance. James E. Lovelock spent some time in Houston, at Baylor and at the University of Houston, working on this problem. The argon ionization detector, the electron capture detector, and the hydrogen flame detector came into use within a few years. The most significant instrumental advance made during 1958-1962, in addition to direct developments in gas chromatographic theory and practice, was the development of the "molecule separator" for GC-MS instruments by Ragnar Ryhage (Karolinska Institute). The general concept of a new kind of instrument, based on combining a gas chromatograph and a mass spectrometer, had occurred to a number of scientists. In Sweden, Sune Bergstrom was particularly effective in establishing work on the new instrumental combination; the Swedish work resulted in the first commercial combined gas chromatograph-mass spectrometer

146 (LKB Model 9000). The Robert A. Welch Foundation supported our early work in Houston. At that time there were two prototype instruments in the world - one in Stockholm and one in Houston. Ryhage came to Houston to assemble the instrument; it was a modified Atlas CH4 with a jet-orifice separator, made in the Karolinska shops, and with highspeed scan. At that time we could not have worked as we did without the early and strong support of the Welch'Foundation and our Swedish colleagues. The great advance in aualytical capabilities provided by the development of the combined GC-MS instrument and advanced GC technology soon became evident. One of the first LKB instruments was installed in Milan by Rodolfo Paoletti (University of Milan); others were obtained by Herman Adlercreutz (University of Helsinki) and by several laboratories in the United States. We continued work in Houston on a number of problems; metabolic profile procedures were developed ( 5 ) and methods were found for studying adrenocortical steroids and their metabolites (6-20). This work was started with Bill VandenHeuvel, who carried out most of the early steroid work, and was continued with C.J.W. Brooks, N. Ikekawa, W . L . Gardiner, E.M. Chambaz, B.F. Maume, G.M. Maume, P. Devaux, J . - P . Thenot and others, Early GC-MS methods were largely used for detection and identification purposes, but quantitative procedures were also needed. The work that demonstrated the best approach to quantitative GC-MS analyses wa5 carried out by Bo Holmstedt (Karolinska Institute) ( 1 ; ) in 1967-1968, and this was based in turn on an earlier study by Sweeley, Elliott, Fries and Ryhage ( 1 2 ) . This universally used technique was called "mass fragmentography" by Holmstedt, but is now usually called selected ion detection. The development of computer-based techniques for instrument operation, data acquisition and data analysis led to current GC-MS-COM bioanalytical systems; these provide the most powerful methods now known for the analysis of biologic samples for compounds other than macromolecules. The development of open tubular (capillary) columns occurred in two phases. During the early developmental period of gas chromatography, the studies of Marcel Golay led to many attempts to make satisfactory glass or metal open tubular (capillary) columns. Some degree of success was attained in hydrocarbon separations, but in our laboratory and in many others it was not possible to make generally useful open tubular glass (capillary) columns. Some years later, through the work of Grob and of Zlatkis, and Desty's efforts, it was realized that satisfactory glass open tubular (capillary) columns could in fact be prepared. My colleagues and I returned to this problem; Anton L. German (Technological University, Eindhoven) prepared excellent glass open tubular (capillary) columns and developed a satisfactory sample introduction device ( 1 3 , 1 4 ) . Current instrumental studies include the development of atmospheric pressure ionization (API) mass spectrometry, methods involving HPLC combined with mass spectrometry, and negative ion bioanalytical mass spectrometry. Subpicogram sensitivity of detection is now possible; twenty years ago much larger samples were needed for virtually all analytical work. Current biochemical and biological studies involve new pathways of drug metabolism and drug toxicity.

147

Fig. 15.1. At the 1973 International Chromatography Symposium in Toronto, Ontario, Canada. Left-to-right: J.H. Purnell, T.Z. Chu and E.C. Horning. The scientific and human aspects of work in this field have always been interesting. Our current knowledge of biochemistry and biology could not have been achieved without modern analytical methodology. The rate-limiting factor in the development of any field is the state of analytical methodology in the field, and this fact is not sufficiently understood. Concepts are always critically important. Very few scientists realized in 1955-1960 that the basic principles of chromatography and mass spectrometry were quite general, and that there was no theoretical barrier to separating, identifying and quantifying or studying the structure, of organic compounds in the gas phase. The problems were essentially experimental in nature, but were unfamiliar because of their novelty and their departure from established techniques. The concept of combining a gas chromatograph and a mass spectrometer, to provide a new kind of gas-phase instrumental system, was not considered initially to be a practical approach to bioanalytical problems, largely because it represented a major departure from established techniques and established views. Quantitative bioanalytical methods based upon chromatography and mass spectrometry are now used so widely that their relatively recent origin is often forgotten.

148

Fig. 15.2. At the 1973 International Chromatography Symposium in Toronto, Ontario, Canada. Left-to-right: E.C. Horning, K . I . Sakodynskii and A. Zlatkis. Scientific endeavors, like most human endeavors, do not usually occur in completely harmonious fashion, and both Marjorie and I have been impressed by two opposing aspects of the development of bioanalytical methodology. Relatively few scientists were involved in the early stages of the evolutionary period that started in the United States in 1955-1960, and the informal exchange of information that occurred then and later led to much faster progress than would have been possible otherwise. The rapid rate of development was aided in part by expanding financial support of research by the National Institutes of Health, but it was also due to the enthusiasm of individuals who were working in a new field. At the same time, some scientists and scientist-administrators strongly opposed the new ideas and new techniques. This effect is not limited to scientific studies; it can lead to a strong delaying action in every field when it is present. Fortunately, in this area of work sufficient momentum was acquired to overcome the objections and negative attitudes of the period. In Bethesda and in Houston this work was made possible by the help of many collaborators with varying backgrounds and from many different countries, and by continued contact with instrument makers

149

Fig. 15.3. At the 1975 International Chromatogmphy ?Symposium in Munich, German Federal Republic. Left-to-right: J.E. Lovelock, E.C. Horning, G. Hesse, A. Zlatkis, C.S.G. Phillips and J. Janhk. including Robert E. Finnigan. Very few of our associates had any kind of formal training in analytical chemistry; most were trained in organic chemistry or medicine. When computers came into use, Richard N. Stillwell became our expert in this field. Earlier studies (before 1960) were carried out primarily with W.J.A. VandenHeuvel and C.C. Sweeley, both well known for their many contributions to bioanalytical methodology. Other immediate associates for short or long periods included C.J.W. Brooks, N. Ikekawa, J. Sjovall, K. Sjovall, T. Luukkainen, E.O.A. Haahti, B.G. Creech, R.J. Hamilton, K. Tanaka, B. Holmstedt, P. Capella, C.E. Dalgleish, P.I . Jaakonmaki, R. Tham, E.M. Chambaz, B.F. Maume, G.M. Maume, G. Casparrini, J. Vessman, P.G. Devaux, A.C. Moffat, K. Okuda, N. Sakauchi, S. Murakami, C.D. Pfaffenberger, J.-P. Thenot, A.L. German, D.J. Harvey, G.W. Griffin, J . Szafranek, R.M. Thompson, P. van Hout, K.D. Haegele, Y. Maruyama, R. Fanelli, C. Fanelli, S.-N. Lin, C.U. Oertli, P. Hartvig, R. de Sagher, S.S. Lau, D.I. Carroll, I. Dzidic, W.G. Stillwell and K. Haalpaap. The committee members involved in the 1964 report were E.C. Horning, E.H. Ahrens, Jr., S.R. Lipsky, E.H. Mattson, J.T. Mead, D.A. Turner and W . H . Goldwater. The work was largely supported by the National Heart Institute, the National Institute of General Medical Sciences and the Robert A . Welch Foundation.

150

Fig. 15.4. At the 1975 International Chromatography Symposium, in Munich, German Federal Republic. Left-to-right: M.G. Horning, Esther Zlatkis and A. Zlatkis.

REFERENCES 1 W.J.A. VandenHeuvel, C.C. Sweeley and E.C. Horning, J . Amer. Chem. Sac. 82 (1960) 3481. 2 E.C. Horning, W.J.A. VandenHeuvel and B.G. Creech, in Methods of Biochemical AnaZysis, D. Glick, ed., Interscience, New York, Vol. XI., 1963. 3 T. Luukkainen, W.J.A. VandenHeuvel, E.O.A.Haahti and E.C. Horning, Biochim. Biophys. Acta 52 (1961) 599. 4 E.C. Horning, E.H. Ahrens, Jr., S . R . Lipsky, E.H. Mattson, J.T. Mead, D.A. Turner and W.H. Goldwater, J . Lipid Res. 5 (1964) 2 0 . 5 C.E. Dalgliesh, E.C. Horning, M.G. Horning, K.L. Knox and K. Yarger, Biochem. J . 101 (1966) 792. 6 W.L. Gardiner and E.C. Horning, Biochim. Biophys. Acta 115 (1966) 524. 7 E.C. Horning, M.G. Horning, N. Ikekawa, E.M. Chambaz, P.I. Jaakonmaki and C.J.W. Brooks, J . Gas Chromatogr. 5 (1967) 283. 8 E.M. Chambaz and E.C. Horning, Anal. Biochem. 30 (1969) 7. 9 J.-P. Thenot and E.C. Horning, Anal. L e t t . 5 (1972) 21, 10 J.-P. Thenot and E.C. Horning, Anal. L e t t . 5 (1972) 801. 11 C.G. Hammar, B. Holmstedt and R. Ryhage, Anal. Biochem. 25 (1968) 532. 12 C.C. Sweeley, W.H. Elliott, 1. Fries and R. Ryhage, Anal. Chem. 38 (1966) 1549. 13 A.L. German and E.C. Horning, Anal. L e t t . 5 (1972) 619. 14 A.L. German and E.C. Horning, J . Chromatogr. S c i . 11 (1973) 76.

151

CSABA HORVATH

CSABA HORVATH was born in 1930 in Szolnok, Hungary. He began his studies at the Technical University of Budapest and graduated as a chemical engineer in 1952. Then he became a faculty member of the Department of Organic Chemical Technology and remained there until 1956 when he moved to Germany. In the next four years he worked at Farbwerke Hoechst AG in Frankfurt am Main-Hochst, first in a pilot plant, and later doing research on applied surface chemistry. In 1961 he left industry to continue his studies at the University in Frankfurt am Main and received his doctorate in physical chemistry in 1963. Subsequently he em2grated to the U . S . A . and became a research fellow at the Harvard Medical School. In 1964 he moved to Yale University, New Haven, Connecticut where he has held various faculty appointments and presently is associate professor in the Department of Engineering and Applied Science. Dr. Horvhth is the author and coauthor of close to 100 scientific papers, and numerous patents. Together with B.L. Karger and L.R. Snyder he coauthored the highly succesful book Introduction t o Separation Science (Wiley, 1973). Dr. Horvhth received the Steven Dal Nogare Award in 1978 for his contributions to chromatography. Dr. Horvhth significantly contributed to the development of both gas and liquid chromatography. In the field of gas chromatography he is best known for the development of novel column types. He started to work on the development of high-performance liquid chromatography in 1964 and has made wide-ranging and technological contributions in this area. Besides chromatography Dr. Horvhth's research interests have been in the application -of chemical engineering to life sciences; presently, his research activities are equally divided between chromatography and various aspects of biotechnology.

152 I s i t not s u r p r i s i n g t h a t w e do not have s c i e n t i s t s c a l l e d chromatographers j u s t a s w e have s p e c t r o s c o p i s t s ? Those who have been a c t i v e i n t h e development of chromatography c a l l themselves biochemist, physico-chemist, chemical engineer, a n a l y t i c a l chemist, physician o r by some o t h e r p r o f e s s i o n a l designaticjn. This i s probably so because most workers became involved with chromatography when they wanted t o solve a p a r t i c u l a r s e p a r a t i o n o r a n a l y t i c a l problem r a t h e r than v i a p r o f e s s i o n a l education. In my c a s e i t w a s d i f f e r e n t ; I turned t o chromatography when I wanted t o g e t a Ph.D. Nevertheless, I a l s o conform with t h e custom and c a l l myself a chemical engineer. After l e a v i n g Hungary i n 1956 I had an e x c e l l e n t job with Farbwerke Hoechst AG, t h e g i a n t German chemical company, a t t h e i r c e n t r a l p l a n t i n Frankfurt am Main-Hochst. Nonetheless, i t bothered m e t h a t I d i d not have a Ph.D. s i n c e about t h e t i m e of my graduation from t h e Technical University Budapest a s a l i c e n c e d chemical engineer i n 1952, t h e t r a d i t i o n a l ways of e a r n i n g a d o c t o r a t e were abolished i n Hungary. In o r d e r t o overcome t h e handicap I w a s looking around f o r a place t o work on a Ph.D. t h e s i s . A t t h a t t i m e IstvPn HalPsz, with whom I served on t h e f a c u l t y of the Technical University i n Budapest, w a s Privatdoze n t (Associate P r o f e s s o r ) i n t h e I n s t i t u t e of P h y s i c a l Chemistry a t t h e U n i v e r s i t y of Frankfurt am Main. W e maintained a r a t h e r c l o s e c o n t a c t and I knew t h a t he was s e r i o u s l y i n t e r e s t e d i n gas chromatography. When the t i m e came t o make my d e c i s i o n he convinced m e t h a t GC had a b r i g h t f u t u r e and o f f e r e d m e an opportunity t o do my d o c t o r a l r e s e a r c h with him. In f a c t he s o l d m e on t h e i d e a of chromatography so t h a t I l e f t Farbwerke Hoechst and joined h i s group i n 1961. The main p a r t of my d o c t o r a l work ( I ) w a s based on a suggestion made by Golay ( 2 ) t h a t t h e performance of open t u b u l a r columns could be g r e a t l y enhanced by i n c r e a s i n g t h e i n n e r s u r f a c e of c a p i l l a r y tubes. The experience i n s u r f a c e chemistry, which I gained i n Hungary and l a t e r a t Hoechst working with v a r i o u s c o l o r pigments, came i n very handy i n f i n d i n g a way t o put a t h i n porous l i n i n g i n t o 0.25 and 0 . 5 mm I.D. metal tubing ( 3 ) . Support-coated open t u b u l a r (SCOT) columns indeed found u s e f u l a p p l i c a t i o n s and have been manufactured and s o l d by Perkin-Elmer s i n c e 1965. The o t h e r h a l f of my d o c t o r a l t h e s i s d e a l t with c o a t i n g g l a s s beads with a porous support o r adsorbent l a y e r ( 4 ) . G l a s s beads coated with a l i q u i d f i l m had a l r e a d y been used i n GC and an enhancement of t h e s u r f a c e a r e a by d e p o s i t i n g a porous l a y e r was expected t o have s i m i l a r b e n e f i c i a l e f f e c t s on performance as i n t h e case of c a p i l l a r y columns. This approach, which a l s o went back t o Golay's s u g g e s t i o n , however, has not found any important a p p l i c a t i o n i n GC. In 1963 I received t h e t i t l e of D r . r e r . n a t . i n p h y s i c a l chemistry and s t a r t e d my p r o f e s s i o n a l l i f e a l l over again. A f t e r g e t t i n g married t o an I t a l i a n g i r l , w e could not decide whether we should s e t t l e i n Germany (my place) o r I t a l y ( h e r p l a c e ) ; t h u s , w e immigrated t o t h e United S t a t e s and I became a r e s e a r c h fellow i n t h e Physics Research Laboratory of t h e Massachusetts General Hospital a t Harvard Medical School. I t w a s not e x a c t l y my i n t e n t i o n t o be a r a d i o b i o p h y s i @ i s t but Boston had so much t o o f f e r c u l t u r a l l y and s c i e n t i f i c a l l y t h a t t h e p o s i t i o n looked a t t r a c t i v e . Moreover, I was a l s o i n t e r e s t e d i n

153 finding out what was going on in life sciences and it was one of the best places to do s o . Nevertheless, I remained in contact with gas chromatography through Leslie Ettre who played a major role in exploiting the potential of SCOT columns. My first exposure to research in life sciences was both exciting and shocking. The excitment came from perceiving that the thrust of science is in the biological field and the greatest discoveries, which are going to shape both our looking at the world and our technology in the future, will come not only from the electronic revolution but also from rapid advances in life sciences. The shock was caused by seeing the biochemists spending most of their time doing some kind of chromatography but most of them lacked the knowledge and interest in the physico-chemical processes responsible for the separation in the column or on the sheet. Consequently, improvements came along slowly and for someone familiar with the potential of GC, the methodology employed in the various branches of liquid Chromatography in the early sixties appeared to be clumsy, slow and inefficient. The exception was, of course, Stein and Moore's Amino Acid Analyzer that was like Pharos, a technical wonder, and at least in my eyes a guide showing the direction to further developments in chromatography. It was time to remember Tswett's words: "every scientific advance is an advance in method". My task was to irradiate cholesterol with gamma rays and electron beams. The autoxidation of this substance, which has been, in my opinion unjustly blamed for so much evil, had of course been widely investigated. A new approach could have been based on a much better analytical method for the separation, identification and quantitative determination of the multitudinous reaction products. The idea of using GC had to be discarded because many of the compounds were peroxides which would have to decompose and react with the others at high temperatures. For my project, a liquid chromatographic system having the essential features of a gas chromatograph such as precise control of operating conditions, sensitive detector, and highly efficient column, would have been particularly useful. At that time it became emminently clear that GC was not only a magnificent tool for separating and analyzing volatile and not-so-volatile substances, but also a microanalytical instrument of unsurpassed versatility, sensitivity and reproducibility. Of course, for nonvolatile substances paper and thin-layer chromatography represented the microanalytical branches of liquid chromatography. For someone like me who was experienced in GC, however, the idea of a LC instrument which gives total control over the chromatographic process and stimulates the development of high efficiency columns, was most appealing. In my nafvet6, I submitted, rather poorly written, a proposal to the head of the laboratory to build a "liquid chromatograph" with narrow bore columns along the concepts evolved in GC. Discussions with Leslie Ettre who just heard Ernst Bayer's presentation in Houston on the use of copper capillary columns for amino acid analysis, were most encouraging to pursue such a project which was unfortunately outside of the interests of the laboratory. Therefore, I proceeded using TLC for the separation of the components of irradiated cholesterol ( 5 ) . I had an

154 e x c e l l e n t c o a c h , Kurt R a n d e r a t h , who worked w i t h h i s w i f e E r i k a i n a nearby l a b o r a t o r y , t o l e a r n t h e i n s and o u t s of t h e t e c h n i q u e . Nevert h e l e s s , i n my mind TLC was an i n f e r i o r method and I wished t h a t a I t was p a r t i c u l a r l y l i q u i d chromatograph had n o t been o n l y a phantom. i r r i t a t i n g t h a t , w i t h o u t g e t t i n g a s s u r a n c e s from t h e w e a t h e r b u r e a u t h a t t h e humidity was i n a c e r t a i n r a n g e , i t d i d n o t make s e n s e t o s t a r t TLC work t h a t d a y , s i n c e t h e r e s u l t s w e r e g r e a t l y a f f e c t e d by the moisture content of s i l i c a . One day i n t h e s p r i n g o f 1964, I r e c e i v e d a c a l l from Sandy L i p s k y , who was embarking on an a m b i t i o u s p l a n t o s e t up a s t r o n g GC-MS l a b o r a t o r y i n t h e School of Medicine a t Yale U n i v e r s i t y . The p o t e n t i a l u s e of t h i s t e c h n i q u e , which was i n i t s i n f a n c y a t t h a t t i m e , was enormous a s c o r r e c t l y a s s e s s e d by Sandy, who was w i l l i n g t o do p i o n e e r i n g work. He had a l s o r e a l i z e d t h e p o t e n t i a l of a f u l l - f l e g g e d l i q u i d chromatog r a p h i c i n s t r u m e n t and was a l r e a d y e x p e r i m e n t i n g w i t h a h e a t o f adsorpt i o n d e t e c t o r . A f t e r making a v i s i t a t h i s l a b o r a t o r y i n New Haven, I got a j o b t o d e v e l o p l i q u i d chromatography and w e moved t h e r e w i t h t h e f a m i l y i n t h e f a l l o f 1964. The f i r s t t a s k was t o b u i l d an i n s t r u m e n t a n a l o g o u s t o a g a s chroma t o g r a p h . A s t h e u s e o f e i t h e r c a p i l l a r y o r narrow bore packed columns was e n v i s i o n e d , p a r t i c u l a r a t t e n t i o n had t o be p a i d t o extra-column dead volumes. Indeed a number o f t r i c k s w e r e used t o r e d u c e e x t r a column band s p r e a d i n g . C a p i l l a r y columns had t h e i r a p p e a l s i n c e Golay showed t h a t t h e y g i v e more e f f i c i e n c y p e r u n i t p r e s s u r e d r o p t h a n packed columns. N e v e r t h e l e s s , h i s e q u a t i o n p r e d i c t e d abysmal performance f o r LC under c o n d i t i o n s used i n GC. S i m i l i t u d e a n a l y s i s s u g g e s t e d t h a t 10-30 p m I . D . c a p i l l a r i e s would do t h e j o b , b u t t h e i d e a o f u s i n g such s m a l l d i a m e t e r t u b e s was d i s c a r d e d as i m p r a c t i c a l . On t h e o t h e r hand, t h e r e s u l t s o f e x p e r i m e n t s t o enhance r a d i a l mixing i n l a r g e r b o r e capi l l a r i e s by s e c o n d a r y o r t u r b u l e n t flow and t h e r e b y o b t a i n a d e q u a t e performance were n o t p r o m i s i n g enough. For packed columns t h e u s e of narrow b o r e t u b e s h a v i n g 1 mm I.D. and 1/16 i n . O.D. appeared t o b e most a t t r a c t i v e b e c a u s e of t h e low s o l v e n t consumption. A s t h i s t e c h n i q u e was supposed t o b e a microa n a l y t i c a l t o o 1 , t h e r e was no need f o r wide-bore columns conventionA major problem w a s t h e s e l e c t i o n a l l y used i n l i q u i d chromatography. of s u i t a b l e column m a t e r i a l . According t o t h e t h e o r y t h e u s e of s m a l l p a r t i c l e s augments exchange r a t e between mobile and s t a t i o n a r y p h a s e s s o t h a t s u f f i c i e n t l y low HETP's can be o b t a i n e d a t r e l a t i v e l y h i g h flow v e l o c i t i e s . P a r t i c l e t e c h n o l o g y , however, was n o t advanced enough t o p r o v i d e u n i f o r m l y s i z e d s u b s i e v e p a r t i c l e s and t h e u s e of h i g h column i n l e t p r e s s u r e s e x p e c t e d t o b e n e c e s s a r y w i t h s u c h columns had n o t been e x p l o r e d e i t h e r . F u r t h e r m o r e , no methods w e r e a v a i l a b l e t o pack columns w i t h v e r y s m a l l p a r t i c l e s so t h a t a v e r y uniform p a c k i n g s t r u c t u r e i s obtained. A t f i r s t t h e method, which I developed i n F r a n k f u r t , was used t o o b t a i n a s t a t i o n a r y phase f o r r e v e r s e d p h a s e chromatography. G l a s s beads ( dp-. 80 u m ) w e r e c o a t e d w i t h g r a p h i t i z e d c a r b o n b l a c k i n a s i m i l a r way a s d e s c r i b e d f o r GC ( 4 ) and packed i n t o a l o n g narrow t u b e . For t h e s e p a r a t i o n of l o n g - c h a i n f a t t y a c i d s an a l k a l i n e e t h a n o l w a t e r m i x t u r e was used a s t h e e l u e n t . The e x p e r i m e n t a l s e t - u p gave an

155

3 P

z YI

2

4E UJ

c12 C182 4

C161

L1

Cl8:l

I

0

10

MINUTES

I

20

30

Fig. 16.1. Reversed-phase separation of fatty acids on pellicular graphitized carbon black, in 1964, Column: 1 m long, 1 mm I.D., packed with 100-200 mesh glass beads coated with graphitized car on black. Eluent: mixture of -ii ethanol and 10 M aqueous NaOH. Detector: refractive index monitor with a 10 1.11 flow cell designed by Emmett Watson.

excellent opportunity to test the prototype refractive index detector built by Emmett Watson. A more refined version of this unit later became the popular LDC RI-detector. There were several problems with the column, however. First, carbon particles frequently dislodged into eluent steam and interfered with detection. It took a certain amount of luck to get a troublefree chromatogram.Second, the preparation of the column material was a dirty job as carbon black was all over the laboratory. It was located in the hospital, and I was advised to stop the experiments and use less mischievous column packing in order to avoid severe environmental conflicts. Since the use of liquid ion-exchangers for the chromatographic separation of inorganic ions had been fashionable those days, it was interesting to find out whether this type of "reversed phase" chromatography would be useful for the separation of biological substances. Early results (6) obtained by using our equipment did demonstrate that the approach was feasible and later the technique under the name "ionpair" chromatography indeed enjoyed a wide popularity. The inherent instability of the liquid-liquid chromatographic system was the source of so much annoyance, however, that liquid-solid chromatography became very interesting and the approach to coat glass beads with a stable sorbent layer appeared to be more and more attractive. By using standard screens we could obtain 38-42 1.1m glass beads easily so that uniform, spherical and relatively small particles were available. A s most colleagues in the Medical School used some kind of ion-exchanger in column chromatography, my first attempt was to polymerize styrene-divinyl benzene onto the surface of glass beads, convert the resin layer into an ion exchanger and see if such a

156

Fig. 16.2. Liquid chromatography anno 1965. The reservoir of the eluent was in a thermostated air bath, which could be electrically heated or cooled by liquid CO2 ' The eluent was pumped by a Milton Roy pump through a pre-column (one turn coil in center) and the sample injector into the narrow bore (1 mm O.D.) column which was coiled onto a thin-walled perforated cylinder. The injector was a 10 1-11 sliding valve which was operated by the handle at the bottom and filled by a syringe mounted left outside the oven. The detector was a Hitachi-Perkin-Elmer Model 139 UV-VIS spectrophotometer with a 5 1.11 flow cell made from a Swagelock GC fitting with quartz windows. The positioning of the flow cell was accomplished by two micrometers operated from outside the cell compartment. The instrument was built for column inlet pressure up to 1000 psi and used in the experiments on ion-pair chromatography (6). Later, the instrument was fitted with a simple gradient forming device, as well as with a pump having higher pressure rating and was used for the analysis of nucleotides (8). material would yield reusable and stable columns and a substantial increase in speed of analysis using the high pressure capability of the instrument. My interest in this undertaking did not abate when

157 a physician coming over from the next door laboratory saw a reading of 1200 psi on the pressure gauge of the liquid chromatograph and remarked "you are never going to sell this dangerous stuff to clinical chemists or biochemists". At that time Ben Preiss joined our laboratory. He knew a lot about chromatography of nucleic acid derivatives by using anion-exchange resins as practiced in biochemical laboratories. It-made sense, therefore, to explore this area. We did not have much success with the conventional acetate and formate buffers, but one night I had a nightmare seeing nucleotide and phosphate ions struggle with each other. Next morning we tried phosphate solution as the eluent and got excellent chromatographic results. The ensuing widespread use of phosphate in the eluent still has no solid scientific basis. The almost invariable success with phosphate eluents may be due to the super-hydrogen-bonding or ion-pairing properties of phosphate. Who knows ? The first good opportunity to give a more detailed account of our work was at the Sixth International Symposium on Gas Chromatography and Associated Techniques (LC then was only an "associated" technique" :) in Rome, my wife's home town. There was a discussion section on liquid chromatography which just began to evoke some interest ( 7 ) , and I was the discussion starter. This gave me a chance to present our results on the rapid separation of nucleic acid constituents with narrow-bore columns packed with pellicular anion-exchangers which was published later in Analytical C h e h s t r y (8). The records of the discussion session at the Rome meeting in 1966 stated: "there is no doubt that analysts will soon see a breakthrough in liquid chromatography". It was undoubtedly followed by an exciting period of time as it was felt that "instrumented liquid chromatography" was an idea whose Zime arrived and a number of workers active in GC had entertained the thought of going liquid. However, no breakthrough came soon or later. It has been a development which was quite slow for my taste before LC finally made it as HPLC in the early 1970's. Our approach was quite successful and the instrument was used as a prototype by Picker Corporation to build the first commercial liquid chromatograph. Unfortunately, certain minor changes which would have been necessary for the instrument to be generally useful were not made so that it remained a relatively short-lived "nucleic acid analyzer". The adjectival word "pellicular" coined to describe the spherical annulus configuration of any sorbent has survived, however. In the late 1960's there were several instruments commercially available and by 1970 instrumentation of liquid chromatography had made considerable progress. In March 1970, at the 21st Pittsburgh Conference on Analytical Chemistry, in Cleveland, a special symposium was held on "High Speed Chromatography" and at this symposium, two-thirds of the papers were on LC and only one-third on GC. I was invited to present a paper; I gave the title "High-Performance Liquid Chromatography" to it and in it, I discussed the various attributes of the technique which altogether sum up to high performance. My lecture not only took notice that the new technique was on the way being widely accepted but unexpectedly also contributed to the vernacular of chromatography. The title of the lecture replaced the earlier names of the new-fangled liquid chromato-

158 graphy and "high-performance (or high-pressure) liquid chromatography" and its acronym, HPLC, became an international expression for modern LC. More interesting, however, was the instant success of the word i s o c r a t i c . The expression "elution at constant eluent strength", which was used previously to distinguish this elution mode from its counterpart "gradient elution", was much too cumbersome. After consulting the largest Greek dictionary in Yale's Sterling Library I put together the suitable adjective "isocratic" and made ample use of it during my presentation in Cleveland. At the end of the Symposium I walked to the exhibition and was passing by a booth when suddenly the word isocratic hit my ears. An adroit salesman of a leading manufacturer was explaining the features of a new, relatively inexpensive, instrument to customers: "it is our latest liquid chromatography, especially designed for isocratic elution". He picked up the new word at the lecture and immediately turned the weakness of the cheap instrument, which did not have gradient-elution capability, almost into a sales pitch to impress his customers by using the very scientifically sounding new word I As LC expanded its muscles and the field attracted more and more workers my academic interests changed. I obtained a position in the Department of Engineering and Applied Science at Yale in 1967, when a new field of scientific endeavor "enzyme engineering", (the word was coined a few years later) was just about to be opened up. It looked as exciting as liquid chromatography a few years before and I decided to start doing research with immobilized enzyme reactors. Of course, my experience with pumps, column packings, detectors and the surface chemistry needed for binding enzymes to solid supports was very useful in this undertaking which has greatly profited from my earlier work in chromatography. SCOT columns paved the way to the development of OTHER'S (Open Tubular Heterogeneous Enzyme Reactors) which have found applications in automated analysis and medicine ( 9 ) . Similarly, the replacement of the stationary phase with a catalyst led to the deuelopment of pellicular immobilized enzymes (10). Only after the excitement wore off and enzyme technology established itself in the middle seventies did I return again to chromatographic research at the university with the goal of finding out why HPLC works at all. I am still pursuing this goal. REFERENCES 1 C. Horvhth, Trennsaulen m i t diinnen pordsen Schichten fiir d i e Gaschromatoyraphie", Inaugural Dissertation, Frankfurt am Main, 1963. 2 M.J.E. Golay, in Gus Chromatography 1960, R.P.W. Scott ed., Butterworths, London, pp. 139-143. 3 I. Halksz and C. HorvPth, AnaZ.Chem. 35 (1963) 499. 4 I. Halksz and C. HorvPth, AnaZ.Chem. 36 (1964) 1178. 5 C. Horvhth, J.Chromatogr. 22 (1966) 41. 6.C.G. Horvfith and S.R. Lipsky, Nature 221 (1966) 748. 7 Liquid Chromatography Discussion Section, J.V. Mortimer, reporter, in Gus Chromatography 2966, A.B.Littlewood, ed., Institute of Petroleum, London, 1967, pp. 414-418. 8 C.G. Horvhth, B.A. Preiss and S.R. Lipsky, Ana2.Chem. 39 (1967) 1422. 9 C. Horvhth and B.A. Solomon, Biotech.Bioeng. 14 (1972) 885. 10 C. Horvhth and J.-M. Engasser, Ind.Eng. Chem. Fundam. 1 2 (1973) 229.

159

J.F.K. HUBER

JOSEF FRANZ KARL HUBER was born in 1925, in Salzburg, Austria. He studied at the University of Innsbruck, receiving a Ph.D. in 1960. He was appointed university demonstrator at Innsbruck in 1958. In 1960 he became scientist and in 1963 senior scientist at the University of Technology at Eindhoven, the Netherlands. In 1965 he accepted a position as senior scientist at the University of Amsterdam, became Associate Professor of Separation Science in 1969, and full professor in 1972. In 1973 he was a visiting professor at the Northeastern University of Boston. In 1974 he became Professor of Analytical Chemistry and Director of the Institute of Analytical Chemistry at the University of Vienna. Dr. Huber is the author and coauthor of over 60 scientific papers on chromatography. He has written the chapter on column liquid chromatography in Comprehensive Analytical Chemistry..He has been the editor of the proceedings of three international symposia on column liquid chromatography held in 1973, 1976 and 1977. Recently he edited a book on Instrumentation i n High Performance Liquid Chromatography. He is a member of the editorial board of the Journal of Chromatography, Jour-

nal of Chromatographic Science, Chromatographia, Microchimica Aeta, Analytica Chimica Acta - Computer Techniques and Optimization, and Environmental AnalyticaZ Chemistry. Between 1964 and 1974 he has served as the chairman of the discussion group on analytical separation methods of the Dutch Chemical Society. Presently, he is the chairman of the Arbeitsgruppe on analytical separations of the Austrian Chemical Society and member of the executive board of the Arbeitsgruppe on chromatography of the German Chemical Society. He is a honorary member of G.A.M.S., the French analytical-chemistry society. Dr. Huber's scientific interests are the chromatographic process and the methodology of problem-solving in analytical chemistry. His involvement in chromatography began in 1958 while working with Professor Cremer as a graduate student in Innsbruck, and he particularly credited with the fundamental work - especially in the field of very fine packing materials - leading to the evolution of modern liquid chromatography.

160 In the beginning of my scientific carreer I had the great benefit of working with some of the pioneers in chromatography. I started in gas chromatography in 1958 working for a thesis on the determination of gas-solid adsorption isotherms with E. Cremer at the University of Innsbruck. In 1960 I accepted an invitation from A.I.M. Keulemans to work with him in gas chromatography at the new University of Technology, in Eindhoven. There, in 1363, I started my research in liquid chromatography. In Eindhoven I could take advantage of the presence of M.J.E. Golay and later A.J.P. Martin, who were temporarily associated with this university. Instead of reviewing my various efforts in the field of chromatography, I would like to focus on two major topics, the development of columns with microparticulate packings and the optimization of the chromatographic separation process by multi-column operation.

MicroparticuZate Colwnns From the fundamental papers on the theory of the chromatographic process, especially the paper of van Deemter, Zuiderweg and Klinkenberg (I), it becomes evident, that the particle size is a key parameter for column efficiency in liquid chromatography. Several workers have tried to improve resolution and the speed of separation in liquid chromatography by reducing particle size. The most successful drive in this direction was undertaken by Hamilton, Bogue and Anderson (2) in ion-exchange chromatography. In 1963 I recognized that these attempts represented only the beginning, and that column efficiency and speed could be improved by at least one order of magnitude. Therefore I started to work on systematic investigations and exploitation of the effect of particle size in column liquid chromatography, together with a student, J.H. Quaadgras, who was researching for his master's degree. To begin with, an apparatus suitable for carrying out experiments with short high-efficiency columns had to be constructed. A high pressure, small dead-volume injection port for syringe injection was designed, permitting injection of microliter samples directly in front of the column. In addition, a micro flow detector for UV-absorbance was developed by miniaturizing the flow cell of the LKB Uvicord detector. The modified flow cell had a volume of 5 p1 and a light path of 1 mm. The columns were constructed from thick-walled glass tubes and low dead-volume column fittings with negligible mixing were also developed. The liquid was forced through the column by a reciprocating pump (LKB). With this set-up experiments with columns packed with materials of different particle size were carried out, measuring the theoretical plate height as a function of flow velocitiy for components with different retardation. The results were summarized in the master's thesis of J.H. Quaadgras, in January 1964 (3). The experimental results could not be fully explained by the usual theoretical plate height equation ( 2 ) . Therefore efforts were made to extend and generalize this equation. Preliminary results were reported at a symposium ( 4 ) in Liverpool; however, the completion of this work was retarded by my move to the University of Amsterdam

161

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Fig. 17.1. Chromatogram obtained in 1964 (3); probably, this is the first chromatogram on HPLC. Column: 770 x 1.5 mm glass tube packed with diatomite (37-50 Um) coated with 1,2,3-tris[cyanoethoxdpropane. Mobile phase: 2,2,4-trimethylpentane. Room temperature. Peaks: l=butylbenzene; 2=nitrobenzene; 3=2-aminobenzoic acid methyl ester; 4=3-phenyl-l-propanol; 5=a-hydroxytoluene. The theoretical plate number of peak 3 (capacity factor 0.13) is 4000 which corresponds to a theoretical plate height of 190 Um at a mobile phase velocity of 0.5 mm/sec. in 1965. In Amsterdam the investigations were continued, now together with J.A.R.J. Hulsman, who also prepared his Ph.D. thesis. Our results were presented at the International Symposium on Physical Separation Methods in Chemical Analysis, in Amsterdam, April 10-14, 1967 and published in AnaZytica Chimica Acta ( 5 ) . In this paper a generally valid equation of the theoretical plate height was presented and verified experimentally. The effect of the particle size was clearly demonstrated. The conclusion was drawn that a further decrease of particle size and development of adequate packing procedures promises a further major improvement. In the continuation of this work, efforts were made to improve the packing procedure in order to obtain a better packing geometry and to prepare narrow-sized particle fractions having an average particle diameter of 10 pm or less. Our results were presented at the Fifth International Symposium on Advances in Chromatography, in Las Vegas, January 1969 ( 7 ) and the 5th International Symposium on Separation Methods: Column Chromatography, in Lausanne, October 1969 (8). At that time quite strong opposition was expressed in the discussions to the use of small particles in the 1O-pm range. It was claimed e.g. that particles smaller than 20 pm would not be stable and grow together because of their high surface energy or the preparation of regular packings was considered to be "black magic".

162

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F i g . 1 7 . 2 . E f f e c t o f p a r t i c l e s i z e and p a r t i c l e s i z e range ( 5 , 6 ) . Column: 500 x 2 mm g l a s s t u b e packed w i t h d i a t o m i t e c o a t e d w i t h 1 , 2 , 3 - t r i s rcyanoethoxylpropane. P a r t i c l e s i z e : ( A ) 63-80 Um; (B) 25 u m (37%)'; 25-56 pm-(28%), 56-80 u m (35%); (C) 25-36 u m . Mobile phase: 2 , 2 , 4 - t r i m e t h y l p e n t a n e . Temperature: 25 OC. Sample: x , b u t y l b e n z e n e ; 0 , n i t r o b e n z e n e . By measuring t h e t h e o r e t i c a l p l a t e h e i g h t ( H ) v s . mobile p h a s e v e l o c i t y ( u ) c u r v e s f o r a n o n - r e t a r d e d and a r e t a r d e d component, an e s t i m a t i o n o f t h e c o n t r i b u t i o n s due t o mixing and mass exchange t o t h e t h e o r e t i c a l p l a t e h e i g h t c a n be made. The c u r v e o f t h e n o n - r e t a r d e d component c o r r e s p o n d s t o t h e mixing p r o c e s s , w h i l e t h e d i f f e r e n c e between t h i s c u r v e and t h e c u r v e o f a r e t a r d e d component c o r r e s p o n d s t o t h e m a s s exchange p r o c e s s . From ( A ) i t can be s e e n t h a t f o r l a r g e p a r t i c l e s b o t h c o n t r i b u t i o n s t o H a r e l a r g e . (€3) shows t h a t t h e mass exchange term i s d r a s t i c a l l y reduced w i t h smaller p a r t i c l e s b u t t h a t a t t h e same t i m e t h e mixing t e r m i n c r e a s e s s i g n i f i c a n t l y due t o a d e t e r i o r a t e d p a c k i n g geometry i f a wide p a r t i c l e s i z e r a n g e i s u s e d . Reducing t h e p a r t i c l e s i z e r a n g e r e s u l t s i n an improved mixing t e r m ; t h i s can be s e e n i n ( C ) .

163

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e 0

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Fig. 17.3. Theoretical plate height curve for favorable packing geometry (7). Column: 200 x 2 . 7 mm glass tube packed with diatomit_e (28-32 pm) coated with 1 ,2,3-trisLcyanoethoxd propane. Mobile phase: 2,2,4-trimethylpentane. Temperature: 25 OC. Sample: nitrobenzene. The theoretical plate height (H) value is the sum of four terms which take into consideration four independent processes: mixing by molecular diffusion (HMd), mixing by convection (HMc), mass transfer in the moving phase (HEm) and mass transfer in the stationary phase (HEs). This figure shows that at the theoretical plate height minimum, value of about 3 is achieved which quality of the packing geometry. It is surprising that the potential of microparticulate columns in liquid chromatography was for years ignored by the scientific community although we published a few more papers in 1970-71 supporting our first results. It is true that the fact that packing material in the 10-U-m range was not available represented a serious handicap; such materials had to be individually prepared as compared to the commercially available porous layer beads. There was also an aversion to working at the high pressures required when operating columns with very small particle size packing. Finally, in 1972, papers by other authors started to appear, reporting on results with microparticle columns. In the following period the interest in this type of columns exploded. Improved and simpler packing procedures were developed and many different types of materials became available at the 10-U.m particle size range. Today column liquid chromatography is dominated by microparticulate columns and has expanded tremendously. One can conclude from theoretical considerations, that microparticulate packings should even bring an improvement in gas chromatography. We started in 1973 to examine this prediction and the experimental results supported the theoretical conclusions (9, 1 0 ) . Such columns which have to be operated at a higher pressure drop offer a compromise between the conventional packed columns and capillary columns.

164

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Fig. 17.4. Rapid separation on a microparticle column (8). Column: 230 x 2.8 mm glass tube packed with diatomite (5-15 pm) coated with 1,2,3-tris[cyanoethoxd propane. Mobile phase : 2,2,4-trimethyl pentane. Temparature: 25 OC. Pressure drop: 19.5 bar. Peaks: l=butylbenzene; 2=nitrobenzene; 3=2,6-dimethylphenol; 4=2,4,5-trimethylphenol; 5=2,4-dimethylphenol;6=2,3-dimethylphenol; 7=3,5-dimethylphenol; 8=2-aminonaphthalene; 9=3-met,hylphenol. Comparison of this figure with Fig. 17.1 clearly demonstrates the improvement in separation and speed due to the reduced particle size. The pressure drop still remains moderate.

Column Switching In 1964, during the course of an investigation into the optimal conditions for the gas chromatographic separation of complex mixtures, I came to the conclusion that optimal conditions can be partially approached only if more than one column is used. Therefore, I obtained a custom-made high-temperature switching valve to couple columns in gas chromatography. By using a squalane and a dinonyl phthalate column coupled by this switching valve it was possible to separate, at moderate temperature, a complex hydrocarbon mixtures. The results of this work were presented ( 1 1 ) at an Informal Symposium of the Gas Chromatography Discussion Group, in Leeds, in 1965. However, the switching valve proved to be unreliable in the long term, especially at higher temperature; moreover, at the same symposium D.R. Deans reported on a new flow-switching system, without passage of the sample through the valves. Therefore, this research project was dropped. The problem returned, however, in liquid chromatography. In 1972, I started the investigations on column switching in liquid chromatography and developed together with E. Ecker and M. Oreans of Siemens AG a suitable switching system. The switching valve could be operated at high pressure and was constructed for low mixing. The

165 column-switching system was tested and its application in HPLC demonstrated. In this way a gradual adjustment of the column length and the phase ratio in liquid-liquid chromatography was performed. These results were presented at the First International Symposium on Column Liquid Chromatography, in Interlaken, 1973 ( 1 2 ) . We continued the studies on the optimization of column liquid chromatography by means of column switching by investigating the gradual adjustment of column selectivity. The use of columns with constant mobile phase and different types of adsorbents as stationary phases was studied. The results were presented at the 11th International Symposium on Chromatography in Birmingham, 1976 ( 1 3 ) . In true optimization each variable must be adjustable independently. In adsorption chromatography the adjustment of the phase ratio requires adsorbents with different specific surface areas but constant adsorption per unit surface area. Such materials were developed recently by E. Merck. Their application to the gradual adjustment of the phase ratio by column switching was investigated together with F. Eisenbeiss. The results were presented at the Third International Symposium on Column Liquid Chromatography in Salzburg in 1977 ( 1 4 ) . The effect of chromatographic separation depends on both the achieved resolution and the ratio of the amounts of the components to be separated. A relative enrichment of a component leads to a better separation of this component. Such a relative enrichment can be achieved by column switching if the total of the intersting component is exactly cut out from the effluent of the first column for further separation on a subsequent column and a large fraction of the overlapping components is removed in this manner. This procedure was developed for the determination of trace components in complex mixtures by column liquid chromatography. The results were presented at the Third International Symposium on Column Liquid Chromatography in Salzburg in 1977 ( 1 5 ) . so far column switching in liquid chromatography received only little attention. It is my firm opinion, however, that it represents one of the most powerful separation techniques and has a bright future, especially in routine analysis where optimal conditions are desired. The development of microparticulate columns and of columns switching in liquid chromatography are examples of typical features in the history of chromatography. Reconsideration of the roots of the method was often the starting-point for imDortant new developments. REFERENCES 1 J.J. van Deemter, F.J. Zuiderweg and A . Klinkenberg, Chem. Eng. S c i . 5 (1956) 271. 2 P.B. Hamilton, D.C. Bogue and R.A. Anderson, AnaZ. Chem. 32 (1960) 1782. 3 J.H. Quaadgras, M.S. T h e s i s , University of Technology, Eindhoven, January 1964.

166 Huber, lecture on High Efficiency Columns in Liquid Chromatography, Symposium on Recent Devezopments in Chromatography, CoZZege of Technology, LiverpooZ, May 13-14, 1965. 5 J . F . K . Huber and J.A.R.J. Hulsman, Anal. Chim. Acta 38 (1967) 305; correction of printing errors Anal. Chim. Acta 38 (1967) 581. 6 J.A.R.J. Hulsman, A Contribution to the Optimization of Liquid Chromatography in Columns Especially in Steroid Analysis, Ph.D. T h e s i s , University of Amsterdam, 1969, p. 16. 7 J.F.K. Huber, J . Chrornatogr. Sci. 7 (1969) 85. 8 J . F . K . Huber, in C o z m Chromatography - Lausanne 1 9 6 9 , E.Sz. Kovats, ed., Suppl. to Chirnia (1970) 2 4 . 9 J . F . K . Huber, H.H. Lauer and H. Poppe, J . Chrornatogr. 1 1 2 (1975) 377. 10 H.H. Lauer, H. Poppe and J.F.K. Huber, J . Chromatogr. 132 (1977) 1. 11 J . F . K . Huber, lecture on Two-Dimensional Gas Chromatography, Symposiwn on The I d e n t i f i c a t i o n o f EZuted Components i n Gas Chromatography, Lee& University, September 2 4 , 1965. 12 J . F . K . Huber, R. van der Linden, E. Ecker and M. Oreans, J . Chromatogr. 83 (1973) 267. 13 J . F . K . Huber and R. Vodenik, J . Chromatogr., in press. 14 J . F . K . Huber and F. Eisenbeiss, J . Chromatogr. 149 (1978) 127. 15 J . F . K . Huber and W. Damboritz, Anal. Chem., in press, 4 J.F.K.

167

ANTHONY T. JAMES

ANTHONY TRAFFORD JAMES was born in 1922, in Canton, Cardiff, South Wales. He studied in London, at the Northern Polytechnic and at the University College receiving his B.Sc. with First Class Honours in 1943, and his Ph.D. in 1946 under Professors Sir Christopher J. Ingold and E.D. Hughes. After a Medical Research Council Junior Fellowship at Bedford College, University of London, with Professor E.E. Turner (1945-47) he joined the Lister Institute for Preventive Medicine, in London, where he worked with Dr. R.L.M. Synge. In 1950, he joined Dr. A.J.P. Martin at the National Institute .for Medical Research, in London, where he remained until 1962 when he became associated with Unilever's Research Laboratories, in Sharnbrook, Bedfordshire, serving first as divisional manager and head of the Biosynthesis Unit and later as the head of the Division of Plant Products and Biochemistry. Since 1969 he has been manager of the Biosciences Group at Unilever. Between 1966 and 1971 he was also industrial professor of chemistry at Loughborough University of Technology. Dr. James has been very active in the scientific community. During his student days he was president of the Joint University of Wales and University College Student Representative Council and president of the National Union of Students. He is a member of the Council and the Board of the Science Research Council of the United Kingdom, chairman of the Food Science and Technology Committee of the Ministry of Food and Agriculture Research Council and president of the International Conferences on the Biochemistry of Lipids. Dr. James received a number of honors and awards, among them the Prize of the American Society of Cosmetic Chemists, the John Price Wetherill Medal of the Franklin Institute, the Lipid Award of the American Oil Chemists' Society, the bronze medal of the French Biochemical Society and the M.S. Tswett Chromatography Medal He is the author and coauthor of a number of scientific publications and also wrote a book with M.I. Gurr on lipid biochemistry, published in 1972. Dr. James' activities in gas chromatography started in 1950 when, working with A.J.P. Martin, they jointly developed the technique of

168 gas-liquid partition chromatography. In addition, his major contributions to chromatography are the development of radioactive scanners and new detectors for gas and liquid chromatography and the demonstration of the application of gas chromatography in biochemistry, particularly in the analysis of lipids. In lipid chemistry, Dr. James' major achievements are the elucidation of the mechanism and stereochemistry of the formation of long-chain unsaturated acids in higher plants and mammalian systems and the involvement of acyl lipids in the biosynthesis of polyunsaturated acids.

After receiving training in physical organic chemistry under Ingold and Turner an attraction to biological systems caused me gradually to transfer my interest to biochemistry, firstly via a study of anti-malarials (I). It was here that I first tried chromatographic procedures for the purification of synthetic organic compounds. After joining the Lister Institute and working with Synge on the structure of the antibiotic Gramicidin ( 2 ) , I was first introduced to liquid-liquid chromatography. When A.J.P. Martin arrived at the Lister Institute in 1950, an immediate rapport was struck. I was then working on one of the first applications of borate-complexing for the separation of sugar derivatives (3) and required for this work an automatic fraction collector. No such equipment existed then and with Martin, the first successful automatic mechanical devices were designed and constructed ( 4 ) . Martin then transferred to the National Institute for Medical Research and invited me there as a co-worker, the subject being the development of a counter-current column procedure using crystallisation as the basic distribution system. After a few months, success was limited so Martin suggested that we try to develop the gas-liquid chromatogram. Work with the crystallisation system had provided the basis of long, thin, Celite-packed columns and this geometry was used for the first gas chromatographic columns. Unfortunately, the system was first tried out on separations of short-chain fatty acids and these gave considerable trouble due to dimerisation in the stationary phase. It was not until non-associating substances, volatile amines, were used that success was obtained. Using this information, the short-chain fatty acids were then tried again using a non-volatile acid in the stationary phase. The separations now worked and the procedure, using an elegant microburette designed by Martin, was first described by us in 1951 ( 5 ) . The apparatus was then automated using a recording microburette (the first successful pH-stat) that was controlled electrically. This was published in 1952 (6) in a paper which outlined many of the future developments of the technique. The method was then rapidly applied to other volatile water-soluble compounds, which gave rise to ions in solution, e.g., ammonia and the methylamines ( 7 ) , and to longer-chain amines (8). In this latter paper, the use of two columns having sufficiently different stationary phases to allow distinct types of solute-solvent interaction was exploited for the identification of unknown compounds. In this way primary, secondary, tertiary and cyclic amines could easily be distinguished. In a general paper, we then described the fundamental parameters of gas-liquid chromatography, produced a theory of its operation in terms of the theoretical plate concept, and laid the basis for the future exploitation of the technique, and for the understandidng of the solution effects concerned ( 9 ) . The method was further extended to water-insoluble bases, such as aromatic amines, by using glacial acetic acid as the medium and perchloric acid as the titrant (10). The general technique of gas-liquid partition chromatography (GLC) had by then attracted much attention and N.H. Ray of Imperial

170 Chemical Industries - one of our very early visitors - was encouraged to use infrared and catharometer detectors. During this time attempts were also made (unsuccessfully) by us to condense the zones as they emerged from the column and to continuously record their weight on a highly sensitive, novel microbalance. The need for a moderately sensitive, general-purpose detector based on a simple parameter (unlike the catharometer) was pressing and attention was turned to automatic methods of gas density measurement. This resulted in the gas-density balance, a detector unsurpassed for ease of calculation of response, but unfortunately too difficult for many instrument firms to construct (11). This detector was rapidly exploited in a variety of fields and resulted in series of papers on general descriptions of the technique, e.e;., in Times Science Review ( Z Z ) , Endeavour ( 1 3 ) , Manufacturing Chemist ( 1 4 ) and especially in Research (15). The use of different types of column for structure identification was still further developed, particularly for the hydrocarbon field (16) By 1956 the basis of GLC and its possible exploitation was firmly laid. At that time Martin wished to leave the Institute and in our last joint paper ( 1 7 ) we entered the lipid field and produced the first good separation on long-chain fatty acids. I now turned my attention to the further exploitation of GLC in lipid studies and the use of chromatographic procedures for structure identification; e.g., with Cornforth on the structure of a naturally occurring antagonist of streptomycin (18) and with Pitt-Rivers on the chemistry of the oxidation of diiodotyrosine derivatives (19). The combination of conventional oxidative cleavage of unsaturated fatty acids and GLC of the products was adapted for structure determinations on a micro scale ( 2 0 ) . This procedure and the use of two different stationary phases to obtain log relative retention volume plots were then combined in the analysis and identification of sebum fatty acids ( 2 1 , 22). My attention was also turned to blood lipids and atherosclerosis, a joint study with Lovelock, Trotter and Webb being made on the level and fatty acid composition of blood lipids in normal and diseased patients. This was one of the first such studies that helped to clarify the situation so far as any connection between coronary artery disease and the level of "essential" fatty acids was concerned (23). The only apparent difference between the two groups was found to be in the oleic/stearic ratio. This is now known to be due to the elevation of plasma triglyceride in coronary patients. About this time, I became interested in fatty acid biosynthesis and with Peeters and Lauryssens, I was the first to directly demonstrate formation of long-chain, odd-numbered fatty acids from propionate by the isolated udder ( 2 4 ) . Later attempts at proving the formation of branched-chain acids from isovaleric acid and leucine failed in this system because of their rapid degradation. This was later accomplished by Vagelos and co-workers in rat liver preparations. My collaboration with Jim Lovelock drew attention to the limitations in the sensitivity of the gas-density balance as a GLC detector so far as biological samples were concerned. Attempts were

.

171 then made to construct a much more sensitive system that resulted in the design and operation of the argon ionisation detector ( 2 5 ) . This work was then extended by Lovelock to produce a whole family of valuable detectors. At this time I visited the Rockefeller Institute for Medical Research, in New York City, and introduced GLC to clinical studies in Dr. E.H. Ahrens' group. This resulted in a number of collaborative papers, e.g. with Insull, Webb and their groups (26-28). Studies on fatty acid biosynthesis in whole blood followed and this culminated in the demonstration, in conjunction with Lovelock and Webb, that the major site of synthesis was in the white cell fraction but that there was considerable lipid exchange between all the cellular and plasma lipoprotein components ( 2 9 - 3 1 ) . Whilst still at the National Institute for Medical Research, I developed together with Piper an improved radiochemical detector for GLC ( 3 2 , 3 3 ) which enabled a better attack to be made on fatty acid biosynthesis. From then on my major effort was put into studies of the biosynthesis of long-chain unsaturated acids. This did not, however, mean that I lost interest in chromatographic techniques. After moving to the Unilever Research Laboratory, I developed with Ray Scott and John Ravenhill the moving-wire detector for liquid chromatography ( 3 4 ) and with Werthessen and Beall a moving-tape detector for the same purpose ( 3 5 ) . This really completed my work on chromatographic systems and from then on, I concentrated on lipid biochemistry. REFERENCES 1 A.T.James and E.E. Turner, J . Chem. Soc. (1950) 1915. 2 A.T. James and R.L.M. Synge, Biochem. J . 50 (1951) 109. 3 E.F. Annison, A.T. James and W.T.J. Morgan, Biochem. J . 48 (1951) 477. 4 A.T. James, A.J.P. Martin and S.S. Randall, Biochem. J . 49 (1951) . 293. 5 A.T. James and A.J.P. Martin, Biochem. J . (Proc.) 4 8 (1951) vii. 6 A.T. James and A.J.P. Martin, Biochem. J . 50 (1952) 679. 7 A.T. James, A.J.P. Martin and G.H. Smith, Biochem. J . 52 (1952) 238 8 A.T. James, Biochem. J . 52 (1952) 242. 9 A.T. James and A.J.P. Martin, Analyst (London) 7 7 (1952) 915. 10 A.T. James, Anal. Chem. 28 (1956) 1564. 11 A.J.P. Martin and A.T. James, Biochem. J . 63 (1956) 138. 12 A.T. James, Times Science Review No. 16 (Summer 1955) 8. 13 A.T. James, Endeavour 15 (58) (1956) 73. 14 A.T. James, Mfg. Chemist 26 ( 1 ) (1955) 5. 15 A.T. James, Research (London) 8 (1955) 8. 16 A.'T. James and A.J.P. Martin, J . Appl. Chem. 6 (1956) 105. 17 A.T. James and A.J.P. Martin, Biochem. J . 6 3 (1956) 144. 18 J.W. Cornforth and A.T. James, Biochem. J . 63 (1956) 124. 19 R. Pitt-Rivers and A.T. James, Biochem. J . 70 (1958) 173. 20 A.T. James and J. Webb, Biochem. J . 66 (1957) 515. 21 A.T. James and V.R. Wheatley, Biochem. J . 6 3 (1956) 269.

172 22 V.R. Wheatley and A.T. James, Biochem. J . 65 (1957) 36. 23 J.E. Lovelock, A.T. James, J. Webb and W.R. Trotter, Lancet (1957) 705. 24 A.T. James, G. Peeters and M. Lauryssens, Biochem. J . 6 4 (1956) 726. 25 J.E. Lovelock, A.T. James and E.A. Piper, Ann. N . Y . Acad. S c i . 7 2 (1959) 720. 26 W. Insull and A.T. James, in Advances i n Gas Chromatography, Div. Petrol. Chem. and Div. Anal. Chem., 132nd National Amer. Chem. SOC. Meeting, New York, N.Y., September 8-13, 1957; P r e p r i n t s , Amer. Chem. Soc., pp. Dlll-D113. 27 A.T. James and J.P.W. Webb, Proc. 4 t h Int. Conf. Biochem. ProbZems of L i p i d s , Oxford, 1957 (publ. 1958) pp. 3-8. 28 V.P. Dole, A.T. James, J.P.W. Webb, M.A. Rizak and M.F. Sturman, J . CZin. I n v e s t . 38 (1959) 443. 29 A.T. James, J.E. Lovelock and J.P.W. Webb, Biochem. J . 7 3 (1959) 106. 30 J.E. Lovelock, A.T. James and G.E. Rowe, Biochem. J . 74 (1960) 137. 31 J.P. Webb, A.C. Allison and A.T. James, Biochim. Biophys. Acta 4 3 (1960) 89. 32 A.T. James and E.A. Piper, J . Chromatogr. 5 (1961) 265. 33 A.T. James and E.A. Piper, Anal. Chem. 35 (1963) 515. 34 A.T. James, J.R. Ravenhill and R.P.W. Scott, in Gas Chromatography 1964 (Brighton Symposium), A. Goldup, ed., Inst. Petroleum, London, 1965, pp. 197-209. 35 N.T. Werthessen, R.J. Beall and A.T. James, J . Chromatogr. 46 (1970) 149.

173

JAROSLAV JANI~K

JAROSLAV JANiK was born in 1924, in Uzhorod then in Czechoslovakia. He studied at the Technical University of Prague, finishing his studies in chemistry in 1947; later he received the D.Sc. degree in chemical sciences at the same school. He started his professional career at the West Bohemian Chemical Works, in Most and continued it in 1951 at the Institute for Petroleum Research, in Brno. In 1956, he was appointed to organize the Laboratory for Gas Analysis of the Czechoslovak Academy of Sciences, in Brno, which 10 years later was expanded into the Institute of Instrumental Analytical Chemistry and, in 1974, into the Institute of Analytical Chemistry. He served as the head of this institute from its inception and developed it as one of the leading European centers of research and postgraduate education in chromatography. In 1964, he became an external associate professor of analytical chemistry at the J.E. Purkynk University, in Brno. In 1969, he spent a sabbatical year at the Consiglio Nazionale delle Ricerche, in Rome. Dr. Janfik is the author and coauthor of over 250 scientific papers and patents dealing with analytical and instrumental problems. In the early stage of his activities he was also connected with the geocherhical prospecting for natural gas and crude oil; subsequently his fields of interest centered around research in gas analysis and separation science. His Czechoslovak patent filed in 1952 represents the first world patent on a gas chromatograph. Dr. Jandk is one of the editors and coauthors of a large comprehensive book on liquid column chromatography (Elsevier, 1975) and is serving on the editorial boards of the Journal of Chromatography and the Journal of Chromatographic Science. His international activities are performed under the Scientific Exchange Agreement, the international foundation supporting European research in chromatography, in the COMECON Committee for Scientific Instruments and in the IUPAC Committee for Microchemical Techniques and Trace Analysis. Since 1974 he has been the vice president of the Brno section of the Czechoslovak Chemical Society. Dr. Janfik on three occasions (1954, 1966 and 1975) received the Czechoslovak State Prize and State Distinction; he is the recipient

of the J. Heyrovsk9 Silver Medal in Chemical Sciences, the J. Hanus Medal of the Czechoslovak Chemical Society, the Polish Memorial Medal of Gdansk Technical University and the M.S. Tswett Chromatography Medal. Dr. JanBk’s research activities in the early 1950’s significantly contributed to the acceptance of gas chromatography as a routine analytical tool. His later and present interests are in the fields of methodology and instrumentation of analytical separation methods and gas analysis.

175 The paths which took me to chromatography resulted from varied circumstances, from my own inclination towards an analytical understanding of things to necessity born of working conditions. I studied chemical technology at the Technical University in Prague in specializations relatively different from each other, such as metallurgy and fuel technology. Even as a student I was fascinated by the dynamics of data and the ways in which they can be used to reconstruct the picture of chemical processes and to gain an understanding of their reaction schemes. I had a strong feeling that analytical chemistry might be the very branch to form a common basis for my various interests in chemistry and technology. However, I remember well that classical analytical chemistry did not satisfy me. I was aware that the future of analytical chemistry must lie in chemical characterization and measurements aimed at finding the structure and reactivity of the individual substances. A young man's formative impressions are often imparted through subtle means by wise teachers. One of the strongest impressions made upon me was by my teacher, Professor Rudolf Luke&. When I presented him with the final report on my laboratory work in organic chemistry I was working on the multi-stage synthesis of nicotinic acid from chloroacetic acid - I was rather proud of the analytical evaluation of the individual stages of synthesis, which I had for the most part supplied with small samples of beautiful crystalline intermediates in the small pockets used by philatelists for keeping rare stamps. However, professor Lukeg took me down a peg by observing that it was a very nice piece of documentation, but professors Prelog and Ruzicka in Switzerland were making a complete structural identification of natural substances from the same amount of material. This blow basically predetermined my further interest in microanalysis and led me, logically, to structure elucidation and separation methods. I enrolled immediately in the optional lectures on gas analysis by Professor Frantigek Cuta and those on organic analysis by Professor Miroslav JureEek, and began to experiment with modern chemical analysis using specific organic reagents according to Professor Arnost Oklz. The desire to get to know chemical technological processes through analytical data saved me from accepting a premature post at the university immediately on completing my studies in order to carry out research there. Thus even before I had finished my studies, in 1947, I accepted a job as an analyst in the main laboratories of our biggest chemical plant in Most, based on the hydrogenation of browncoal. This place turned out to be a chemist's paradise, for the rich composition of the low-temperature browncoal-tar and -waste waters, the extensive and varied gas technologies from the production of hydrogen through the high-pressure gasification of coal to the low-temperature distillation of gaseous hydrocarbons, the new plants producing high grade mono- and dibasic phenols, and the beginnings of our petrochemical industry, all provided a young chemist with an unusually wide range of problems and opportunities. There, alongside the existing control laboratories which became my responsibility, I organized a special laboratory for new analytical

176 methods, paving the way for the successful realization of new plants: in it we obtained an analytical profile of the raw materials, and prepared suitable analytical methods to provide an understanding of the mechanism of the associated chemical reaction and evaluate new products. For many technologists analytical chemistry was only a list of recipes, or at best a basis for achieving a prescribed mass balance and at first, our ideas confronted a lack of understanding. However, the proof supported our idea: analytically prepared processes ran more economically in production. On the basis of this philosophy I was later successful in forming a link between the academic institute and industrial practice and I consider this one of the most important factors underlying the overall preparedness of our country for modern analytical methods compared with many other countries, for whom the import of modern instruments is an incomparably simple affair. The chemical plant had a very nice library, covering German and, with the exception of the first war years (1940-1942), English and American literature; this is the reason I read the basic paper of A.J.P. Martin and R.L.M. Synge (1941) only much later. At that time I was able to read the publications of Roth, Ohme and Nikish (1942), Turner (1943), Tiselius (1943) and Claesson (1946) presenting the possibilities of displacement chromatography. It caught my attention, but it seemed a clumsy method in comparison with the effective fractional distillation. Within one year, far-reaching changes came about in our country's political system, which had been followed by certain international consequences. An attempt by our plant's management to expand the activity of the advanced analytical laboratory by buying the then progressive Podbielniak Hyperrobot for the analytical distillation of hydrocarbon gases failed on account of an embargo imposed by the U.S. Government on exports to Czechoslovakia. It is not without interest to recall that this restriction resulted merely in a rapid creation of self-sufficiency by our own development of distillation apparatus, and that in addition a sufficient economic impulse was created for the later development of gas chromatography in our country. One other fact is also interesting in this connection. Even in 1958, when I personally met Dr. Walter Podbielniak, I was obliged to warn him of his optimism concerning the future of the analytical distillation of gases, Even, then, gas chromatography was not always fully appreciated, even by experts in the field. At that time Podbielniak's world-famous company was actually rapidly losing ground. Meanwhile my own positive experience with the elution chromatography of monobasic phenols on a column packed with alumina and a very nice separation of dibasic phenols which, in our research group Dr. Vladimir Mrhz achieved by paper chromatography convinced me in 1949 that there was no reason at all why gases could not be just as effectively separated chromatographically without respect to their chemical character. This conviction was only strengthened by our first conference on chromatographic techniques, held in Prague. It was organized in 1950 by Professor F. Sorm in order to

177

Fig. 19.1. One of the first versions of Janiik's gas chromatograph developed for the analysis of natural gases and gases of similar composition at the Institute for Petroleum Research, Brno, in 1951. make available to us the results of the 1949 Faraday Discussion in London; I received the full text of this meeting only much later, when I had already made contacts with English colleagues. I should also mention that at that time, I was also in charge of the elemental analysis laboratory. Experience with the Dumas volumetric determination of nitrogen was the final inspiration leading me to the construction of my first gas chromatographs. Meanwhile I was transferred to the Institute for Petroleum Research, in Brno, and I finished the development of these instruments there. In the library of the Institute, I could read the papers of Turkel'taub (1950) and Zhukhovitskii (1951) and particularly the papers of Professor Cremer (1951) for whose elution concept of the chromatographic separation of gases I had the highest regard. I managed to eliminate their difficulties in detection and irreversible sorption by using CO2 as the carrier gas, its own sorption being so high and so variable with different sorbents that it allowed a controlled modification of their sorption properties. This was the origin of a Czechoslovak patent ( I ) , actually representing the first patent on a gas chromatograph in the whole world. This patent application was followed by a series of papers, first in Czech ( 2 , 3 ) and soon after, in German ( 4 , 5 ) . Our results were also

178

Fig. 19.2. The final version of the volumetric gas chromatograph, based on Czechoslovak Patent No. 83,991 filed on September 20, 1952. confirmed by the group at the B.P. Research Centre, in Sunbury-onThames, headed by Dr. S . F . Birch (6); later the method also became the basis of Tentative Method No. 169 of the Institute of Petroleum (1959) for the analysis of natural gas and had a considerable influence - mainly in Europe - on the acceptance of gas chromatography as an industrial analytical tool. Today, naturally, the volumetric gas chromatograph of 1952 has already been superseded by other techniques and even in our own institute, it is kept only in the museum. However, one should not forget that this instrument already incorporated some of the key elements retained in every modern gas chromatograph: sampling by injection or through a by-pass loop, columns in series and in parallel, combination of various column packings, modification of the surface of sorbents, and a temperature program. It is still the only system giving analytically absolute results for chemically inactive gases, and the accuracy with which it can determine the hydrogen content of gases is still on a high level. Because of its didactic value this device can still be seen in use for teaching purposes in some universities, even abroad. It is not without interest that until recently a group of automated JanLk's gas chromatographs was still in operation in the BASF laboratories at Ludwigshafen, in the German Federal Republic.

179

Fig. 19.3. A page of my laboratory notebook, dated August 1, 1952 showing the analysis of a mixture of hydrogen, nitrogen and methane. In 1955, I presented a lecture on the microanalysis of gases in Vienna. There I met for the first time Dr. Michael Lederer, and this contact resulted in a continuous active cooperation in connection with the Journal of Chromatography, which Dr. Lederer founded two years later. I recall with pleasure the lively discussions which Dr. Karel Macek and I had with him on the future trends of chromatography during my research stay in Rome in 1969. In respect of the editorial policy he adapted the conclusions of these discussions both into the conception of the journal, and the composition of the editorial board and into administrative matters such as the changeover from monthly to fortnightly, the publication of papers presented at important symposia as original articles in the journal, etc. In this connection I cannot fail to recall a pleasurable and efficient working relationship with Miss Tilly Sijpesteyn and her predecessor, Miss Mil Anton, at Elsevier in Amsterdam, whose influences lie behind the high editorial standard of this international journal. In 1956 I was invited to participate at the first world symposium on gas chromatography organized by the Hydrocarbon Research Group of the Institute of Petroleum. I was then very inexperienced in international contacts and linguistically most unprepared. I was unable to participate personally in the English discussion and I even had problems with the German translation of the remarks, provided by

180 W b

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Fig. 19.4. The title page of the first world patent on a gas chromatograph. Translation of the text: Czechoslovak Republic, Patent Office (top); Class 42 14/05, Issued February 1, 1955; Patent Document No.83,991; (title:) Apparatus for Quantitative and Qualitative Analysis of Hydrocarbons and Other Gases (Gas Chromatograph). (last line:) Applied September 20, 1952 - patent validation from September 20, 1952. Dr. Keulemans. However, I must state that I encountered a great sense of fair play among my English colleagues. I found the doors of Dennis Desty wide open, both at his home and at B.P., I was received by A.J.P. Martin and A.T. James at Mill Hill, warmly welcomed by Courtenay Phillips at Oxford, by Dr. Nickless at Bristol, by John Knox at Edinburgh, and also by many others. A later meeting with A.J.P. Martin and R.P.W. Scott was particularly significant for me. In 1969, the CIBA Foundation organized a discussion on the application of gas chromatography in biology and medicine. This meeting constituted an opening to the possibilities which (gas) chromatography offered in diagnostics followed by our group in studying the composition of substances liberated by living organisms particularly in breath (7). Lou Keulemans, my "interpreter" during the London Symposium later became a dear old friend of mine. Our first meeting was followed by many years of active cooperation between his institute at Eindhoven and ours in Brno (a cooperation continuing to this day under the present

181 leadership of Carl Cramers at the Eindhoven Institute). This cooperation, characteristic of like minds concentrated on the future significance of gas chromatography in biological disciplines, especially in medical chemistry and diagnostics, later grew into a cooperation to propagate European research in chromatography and international postgraduate education. Its basis was to be the international Scientific Exchange Agreement (S.E.A.) founded on the initiative of Lou Keulemans by Clark Hamilton and his wife Trudi. Later there was my personal contact and cooperation with Georges Guiochon (who assumed the S.E.A. leadership after the death of Professor Keulemans) in Paris and Josef Huber, first in Amsterdam, now in Vienna. Before turning from my recollections of the fruitful period at the Institute for Petroleum Research, I should like to mention a very different utilization of the principles of chromatography. In those years I was obliged to get my teeth into the basics of geology and to acquire a deeper understanding of petroleum geochemistry. The application of chromatographic principles to natural processes led me to unusually interesting practical conclusions. I managed to deduce theoretically and prove experimentally that the formation of the composition of underground water in sedimentary rocks, carrying the ion "stamp" of the original, usually marine, environment from which they originated, is controlled by a chromatographic ion equilibrium under the dynamic conditions created by the underground migration of water (8). All at once it was a simple matter to explain the composition and to understand the genesis of underground water, not only in crude oil and natural gas drillings, but also of most of the mineral waters of our numerous spa and other natural springs (Karlovy Vary , Marihskb Liizn6, LuhaEovice , Piegzany, TrenEianskk Teplice, etc.) which previously were considered by foremost geologists as being for the most part unique, and individual natural phenomena. I remember the wonder, initial disbelief and later enthusiasm of professors of geology Radim Kettner and Otto Hynie when I simulated and quantitated the composition of the mineral waters of the Dudince spa in Slovakia during deep-drilling operations, using a frontalchromatographic concept of the ion-exchange process in calcium carbonate-sulphate water arising from mezozoic sediments during its migration through ion-exchanging sodium pelites of tertiary age sediments. During the reconstruction of the spa's wells it was necessary to sink deep bores to the mineral springs and to tap them without the danger of destroying their healing composition. After drilling, the water was substantially different; however, it had the predicted composition, i.e. containing sodium carbonate-sulphatechloride character with an increased amount of magnesium, but without hydrogen sulfide. Only after seven months of continuous flowing of the water, when finally a balance had been achieved with the sodium complexes of the tertiary sediments (representing the stationary phase of a gigantic natural chromatographic column), did the original "natural" composition return. Since then neither drilling for healing mineral water nor drilling for crude oil has been carried out in our country without a hydro-geochemical investigation.

182

Organisation of an i n s t i t u t e In 1956, the Czechoslovak Academy of Sciences caught up with the development trends. On the suggestion of the late Professor Oldiich TomiEek I was made responsible for the organization of a laboratory to carry out research in gas chromatography, the significance of which had already outgrown the narrow interests of the petroleum industry, offering new and surprising possibilities to the chemical industry and chemical research, particularly in the life sciences. This represents the foundation of our institute and I began to organize the team of collaborators. Without their intellectual qualification and the joy they derived from scientific work it would not have been possible to create the environment in which more than forty foreign and domestic scientists have been educated during the last twenty years and where more than five hundred of our specialists from universities, research institutes and industry took postgraduate courses. During that period our team achieved some results which found wide use. Here, I just would like to mention a few: Defined pyrolysis under gas chromatographic conditions, though not offering fully usable analytical results, was a stimulating influence in its time ( 9 ) . The paper on a new technique in the preparation of glass capillaries for capillary gas chromatography became a classical one ( 1 0 ) . The discoveries of the pressure dependence of the flame-ionization detector ( 1 1 ) and the selectivity of the thermionic detector towards sulphur and other elements (22) represented pioneer works. Multidimensional chromatography using gas chromatography as one dimension ( 1 3 ) proved interesting in forensic screening studies and stimulated the development of special microtechniques ( 2 4 ) , which actually have little connection with chromatography. A theoretical treatment of quantitative analysis by gas chromatography ( 2 5 ) will certainly find its users. High-speed analytical isotachophoresis (16), or electrokinetic detection ( 1 7 ) , making possible the study of capillary liquid chromatography, will probably represent the next subject for further research. In 1956 we organized in Brno the first national symposium on gas chromatography, the result of which was the start of commercial gas chromatography instrumentation in Czechoslovakia; today this production is on a large scale for both chromatography and derived techniques, with considerable exports to the Socialist countries and to the so-called third world. At and following this symposium, we made contact with many scientists in the Socialist countries, e-g.,with Professor Schay of Hungary, Professors Kemula, Waksmundzki and Pompowski of Poland and with Professor Leibnitz and Dr. Kaiser of the German Democratic Republic. In 1959, during the first conference on gas chromatography held in Moscow, I personally met Professors Zhukhovitskii and Kiselev and the late Dr. Turkel'taub. Thus, at that time we started a close cooperation with our colleagues from the neighbouring countries. In those years, I was also invited several times to lecture in the German Federal Republic where I was fortunate to establish a close relationship with a number of fellow scientists such as e.g. Professors H. Kienitz, W. Fresenius, I. Halhsz, W. Kroepelin and the

183 late H. Luther. I remember with please the fruitful descussions I had with Drs. Kelker, Horn and Hachenberg in Frankfurt who had detailed experience in the application of our method for gas analysis. It was amusing to find out that utilization of our method was in German simply called "janakieren.I' In 1958 I had a unique opportunity to visit the Chinese People's Republic at the invitation of their Academy of Sciences. I led a summer school on gas chromatography in Dairen (Port Arthur) and subsequently visited a number of universities and research institutes in northern and central China. Dr. Lu Pei Chang was my excellent translator while lecturing in German. I brought home with me a GC text in Chinese and the strong conviction that the Chinese are exceptionally industrious people and if China can only give a basic education to all its inhabitants, there is an inexhaustible reservoir of talents for any degree and/or kind of scientific activity. A later visit to Brno by Shanghai's Professor of Chemistry Yu Wang, from whom I have a poem composed in beautiful and melodic verse, had unfortunately no continuation and contacts have been severed. Also during those years, Japanese chemists and designers began to be extremely interested in gas chromatography. Many experts passed through our institute, but continual contact was established mainly with Professors Shun Araki from Kyoto and Shoji Hara from Tokyo, and with Dr. Tatsuo Haruki, today a representative of Shimadzu Co., concerning the methodology and instrumentation of gas chromatography, and more recently of analytical isotachophoresis. We had fruitful correspondence with Professor Nobuo Ikekawa of Tokyo in the preparation and exchange of teaching films in the field of chromatography. I like to recall the first contact by letter with Professor Zlatkis after the publication of his paper with Kaufman on capillary gas chromatography in Nature (1959); it represented the start of future contacts. Dr. Zlatkis advocated our frontal-chromatographic concentration method for trace components in gases (78) which, since its publication, has been used in both space research and the study of the emanation of trace materials by living organisms (7). It is interesting that this frontal-chromatographic concept of trace gas analysis also led us to a prospective method of organic elemental analysis and to the construction of an original analyser ( 1 9 ) . Dr. Zlatkis whom we met many times since our first exchange of letters was also kind enough to write the Foreword for our book on liquid column chromatography (20). One of the remarkable meetings where we participated jointly was the Symposium in Leningrad in 1972, honoring the centenary of Tswett's birth; the idea of establishing an international chromatography award was born at this meeting and the M.S. Tswett Chromatography Award is the realization of this idea. Dr. Zlatkis also visited us in 1973 during our chromatography symposium in Bratislava where he lectured together with Drs. Evan and Marjorie Horning and Leslie Ettre who is also an old friend of mine. A number of other American guests also visited our institute, e.g. Drs. Barry Karger, R.S. Juvet and W. Supina. In our Institute in Brno we have a Guest Book which we started in 1958 after Professor Tsitsishvili of Tbilisi, during his visit,

184

Fig. 19.5. The team of the Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, in Brno, in 1976, on the occasion of the 20-years anniversary of its foundation. Left to right. S e a t e d : Dr. Stanislav Haderka (electronics, LC detectors; now retired), Dr. Milog KrejEi (chemist, LC), Dr. Jaroslav Jankk (Institute director), Dr. Josef Novik (chemist, G C ) , Dr. Radka Rungtukovi (chemist, information sciences). Standing: Dr. Karel Tesa3ik (chemist, capillary GC and LC), Dr. Vlastimil Rezl (chemist, elemental analysis), Milo8 JaniEek (fine mechanist, workshop), Dr. Haniel Dubskjr (chemist, GC detectors; now at the Institute of Forensic Medicine, in Brno), Miroslav Rusek (chemist, gas analysis), Dr. Petr BoEek (chemist, analytical isotachophoresis), Dr. Milan Dressler (chemist, GC-MS), and Dr. Stanislav Wizar (chemist, computer science and automation). suggested it, saying that one day, it will be an interesting chronicle. This is not the only way in which he was correct: this guest book is not only witness to our contact with the rest of the World

185 but also reflects many interesting remarks in the various entries. I would like to finish my own recollections by quoting two such entries referring to the international character of chromatography and the cooperation between scientist from various countries:

"Gas chromatography i s l i k e an i n t e r n a t i o n a l b u i Zding i n t o t h e construction of which s c i e n t i s t s from every country have p u t a t l e a s t one b r i c k . I' E. Cremer, Innsbruck

"Gas chromatography i s an e x c e l l e n t method t o separate substances and an e x c e l l e n t way t o u n i t e people. I' A.A. Zhukhovitskii, Moscow REFERENCES 1 J. Janhk , Czechoslovak Patent No.83,992 (filed : September 20, 1952; issued: February 1, 1955). 2 J. Janhk, Chem. L i s t y 47 (1953) 464. 3 J. Janhk, Chem. L i s t y 47 (1953) 817, 828, 837. 4 J. JanAk, C o l l e c t . Czech. Chem. Conunun. 18 (1953) 798. 5 J. Janak, Co'Ylect. Czech. Chem. Conunun. 19 (1954) 684. 6 C.L.A. Harbourn, D.H. Desty and S . F . Birch, British Petroleum Co., Report No. 5296 (1955). 7 J. JanPk, in Gas Chromatography i n Biology and Medicine ( C I B A Symposium), R. Porter, ed., Churchill, London, '1969, pp. 74-85. 8 J. Janhk, GeoZ. Prcfce 15 (1959) 107. 9 J. Janhk, Nature 185 (1960) 684. 10 K. Tesa?ik and M. Novotn?, in Gas-Chromatographie 1958, H.G. Struppe, ed., Akademie Verlag, Berlin, 1968, pp. 575-584. 11 P. Bozek, J. NovPk and J. Janhk, J . Chromatogr. 48 (1970) 418. 12 M. Dressler and J. Janhk, J . Chromatogr. S c i . 8 (1969) 451. 13 J. Janhk, J . Chromatogr. 15 (1964) 15. 14 I. Klimeg and J. Janhk, Microchem. J . 13 (1968) 534. 15 J. Novtik, Q u a n t i t a t i v e Analysis by Gas Chromatography, M. Dekker, Inc., New York, 1975. 16 P. BoEek, M. Deml and J. Janhk, J . Chromatogr. 106 (1975) 283. 17 A. KrejEi and K. Slais, J . Chromatogr. 148 (1978) 99. 18 J. Novhk, V. Va8Pk and J. Jandk, Anal. Chem. 37 (1965) 660. 19 V. Rezl, Microchem. J . 15 (1970) 381. 20 Z. Deyl, K. Macek and J . Janhk (eds.), Liquid Cohmn Chromatography, Elsevier, Amsterdam, 1975.

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187

RUDOLF E. KAISER

RUDOLF ERNST KAISER was born in 1930, in Teplice Banov (Teplitz-Schonau) , Czechoslovakia. He studied at the Technical University, Dresden and Karl-Marx-University, Leipzig, German Democratic Republic (DDR) receiving his Dr.rer.nat. degree in 1954. In 1952, he joined the Institute for Chemical Technology of the (East-) German Academy of Sciences, where he soon became responsible for the Section Chemical Measuring Techniques, a field including chromatography. In 1960 he moved from Leipzig, DDR, to Ludwigshafen/Rhein, German Federal Republic, joining the Badische Anilin & Soda Fabrik AG (BASF). He worked there until 1972 when he started his own Institute for Chromatography, in Bad Durkheim. In this Institute, he has trained over 2000 chromatographers from over 16 countries and is now starting to organize courses in Africa as well as the United States. Since 1974, he has also been carrying out development work - together with a number of colleagues from various institutions and companies - on the fully instrumentalized version of thin-layer chromatography. Dr. Kaiser is the author of over 100 papers and a number of books. His first book Gas Chromatographie was published in 1959, in East Germany while his four-volume series Chromatographie in der Gasphase went through three editions in West Germany and was also translated into English. One of the volumes of this series published in 1962 represents the first book on capillary gas chromatography. Dr. Kaiser organized in 1968 the international journal Chromatographia and is now organizing - together with a four-member Editorial Board - a new journal,

Journal of High-ResoZution Chromatography and Chromatography Communications. Dr. Kaiser i s one of the pioneers in gas chromatography who started to be active in this field in 1953. His major contributions are the demonstration of the possibility of carrying out reliable quantitative high-speed analysis with capillary columns and the organization of highlevel training courses. In recent years, he has become involved in the modernization of thin-layer chromatography. Chromatography is Dr. Kaiser's real hobby.

188 In the spring of 1953, in Leipzig (the German Democratic Republic, DDR), my old teacher Dr. Otto Mittelstaedt (the physicist who for the first time correctly measured the ,speed of light) told me that my main job - doing kinetics and organic group analysis of the catalytic oxydation products of hydrocarbons -would be enlarged. "There is a most interesting doctoral thesis work going on at the Bohlen Chemical Complex", he told me, "E. Kogler is separating hydrocarbons with the help of a glass tube packed with cotton wool which is impregnated with some high-boiling oil and by flushing the tube with hydrogen*. It is something called gas chromatography. Visit him, build a similar machine here and carry out separation work with solid n-alkanes up to C3,, which are the basis of the oxidation process because we have difficulty in separating them by distillation". This was an order. I did what I was told. In a few weeks we had a "machine" built; also, my group was increased to a team of six. One of us injected the gasoline sample with help of a milliliter-syringe through a rubber cap into the glass column, the other increased the electric current to the heater stepwise, the third had to look continuously through a very sensitive optical detector, an interferometer, turning the knob to keep the balance of the light-beam and shadow, the fourth recorded the data every ten seconds and finally, the fifth gave the time interval commands. After carrying out three "chromatographic exercises" in a day, we needed three days to do all the drawings, calculation and relaxing. There was no recorder available to us which could do the job. One year later, we organized a small conference on chromatography in Leipzig. Meanwhile, we had learned from Zhukhovitskii and Turkel'taub (Moscow) how to use adsorption columns, thermochromatography and temperature programming and from Professor Cremer (Innsbruck, Austria) how to use the katharometer, the thermal conductivity detector. We built our own katharometers, including the wire to be used in it. In 1955, we had a home-made instrument containing a large aluminium block with five katharometers in parallel and five GC columns which could be used either parallel or in series and could be mechanically switched. The instrument also had flow regulators and everything was in an excellent air-thermostated oven. It was really great. After assembly, it could not be removed from the room: it was too large. However, it worked. Unfortunately, our problem was that carbon dioxide and ethylene gave the same retention times and thus, identification turned out to be too risky. The first London Symposium in 1956 represented a real shock to us: we learned from the lectures that we were doing chromatography in the wrong way: instead of adsorption columns we should use liquid phases. We should also work isothermally and use thermostats with boiling vapors.

*We had no helium and thus, had to use hydrogen as the carrier gas!

189 Meanwhile we carried out systematic studies to improve our thermal conductivity detector. The detector was now shockproof, had practically no baseline drift, had an excellent sensitivity and could be operated equally well with 1 ml/min or 100 ml/min hydrogen flow. With it, we could solve our problem, to separate high-boiling paraffins from C 1 8 to C32 using a 3-m long packed column. We alsb made a movie about the manufacturing of the detector and showed it in a number of places in Eastern Europe, at various international meetings. Then came the 1958 Amsterdam Symposium where I had my second shock: Professor Keulemans told me that my detector was obsolete: we should use an ionization detector! During those years, we also organized our first national conference on gas chromatography, the first training courses for chemists from the industry and research institutes, we had the first doctoral theses on gas chromatography and we experienced unbelievable cooperation. It was a great time to make friends. Our first series of GC instruments had now been used in a number of laboratories and they produced more-and-more results. One of my colleagues separated amino acids, the other phenols, the third amines, and the fourth carried out pyrolysis-gas chromatography. At the beginning, we had to change the minds of a number of sceptical persons but we succeeded. I will never forget the day - it was prior to 1956 - when we had been invited to demonstrate the possibilities of gas chromatography at the Buna Werke, in Schkopau, one of the largest manufacturers ot synthetic rubber in Europe. We started the demonstration by injecting their cleanest butadiene into a polar packed (glass) column which was mounted in the open on a table so that everybody saw what we were doing. In the first few minutes the column exploded because we had too-high a hydrogen pressure. Fortunately, we had a second system in reserve and, 10 minutes later we continued the experiment. When the chromatogram was ready, it was clear that the "purest" butadiene was indeed not pure, containing a number of impurities. In November 1958, D.H. Desty accepted the invitation of the Academy of Sciences and our Institute, in Leipzig, to present a paper on new developments in gas chromatography, I served as his interpreter and also as his bodyguard and I remember in great detail the atmosphere of total silence in an overcrowded lecture hall when he showed his over-100,000 theoretical plate gasoline run using a 100-m metal capillary column and when he discussed the potentialties of glass capillary columns. At that instant, we were more-or-less converted to continuing our work with capillary columns. We made our own flame ionization detectors, amplifiers and electronic integrators and visited Jaroslav Janhk in Brno, in neighbouring Czechoslovakia, to learn how to make our own (copper) capillaries. We soon changed to aluminium capillaries. However, our basic problem remained: we had no fast potentiometer recorders. On the 15th of October, 1959, my guests - E. Cremer (Innsbruck, Austria), E. Smolkovh (Prague, Czechoslovakia), A.V. Kiselev (Moscow), J.F.K. Huber (Innsbruck), J. Janhk (Brno), A.I.M. Keulemans (Eindhoven, The Netherlands), 0. Grubner (Prague)

190 and A.A. Zhukhovitskii (Moscow) tried to establish, together with my wife, a working group on gas chromatography. I still have my guest book with the entries of all these friends. However, we soon learned officially, that for certain reasons, we should not continue any organization along these lines. In March 1960 my coworkers H. Holzhauser, M. Kuhl, M. Hofmann, H.G. Struppe and H.P. Angel6 plus eight girl technicians succeeded in building a temperature-programmed gas chromatograph with a 5-m aluminium capillary column (alkaline-etched and coated) connected to a flame ionization detector with a home-made high-speed amplifier and an electronic integrator. With this equipment, Angel6 and I flew to Moscow, to a research institute, where finally, we had a high-speed recorder available to us. In the first run, we could separate phenols within a few seconds on this (porous-layer) capillary column. About that time M. Mohnke, my coworker since 1952, (in the previous years we have jointly developed urea-inclusion liquid column chromatography for the separation of paraffin waxes by temperature-programmed l.iquid elution) etched glass capillary columns with aqueous HC1 and NHbOH solutions and utilized this column for the separation of hydrogen isotopes. We summarized our results in a paper, but this was delayed and then published by changing the name of one of the authors. I meanwhile changed my affiliation. In 1960, I joined the Badische Anilin & Soda Fabrik, in Ludwigshafen am Rhein, German Federal Republic. Within a few weeks my new coworker, H. Fiedler, succeeded after a little instruction, in preparing high-resolution capillary columns and was able to carry out high-speed separations of isomeric aromatic compounds, utilizing home-made instruments. Despite the results, I have had colleagues who could not believe the excellent, reliable quantitative and routine analytical data we obtained in such systems and they continued for 10 more years in their disbelief in the power of this new analytical technique. As far as I know, there are still some although now fewer and fewer - colleagues around who do not believe in high-resolution chromatography; this is, however, more the present than history. At the end of 1960, at a scientific meeting at Dechema, in Frankfurt/Main, my former director from Leipzig, Professor Leibnitz questioned the validity of our own former results! At that time, however, I had sufficient additional high-quality data - obtained under industrial analytical conditions - available to show that high-precision quantitative capillary gas chromatography was indeed the technique of the future. By 1964, we had the first automated instrument ready for subppb level analysis of trace impurities in air and water using a special technique basically invented by Zhukhovitskii and Turkel'taub in Moscow. Our results divided the people into three groups: those, who did not believe in the data, those who did believe in them and those who while believing in them, did not like them. At that time, I met D.H. Deans of I . C . I . for the first time and soon combined his column switching technique with capillary columns and sub-ambient

191 conditions. W. Stoll, my coworker carried out the first routine analysis at the level of trace impurities in water and in outdoor air. Backflush, foreflush, on-column enrichment, column transfer, multidetector systems, the use of the first thin-layer glass capillaries impregnated with natural microparticles, superclean carrier gases, as well as concentration and trapping with help from mobile heating fields became our daily tools and technique. I left BASF in 1972 to start my own Institute for Chromatography, in Bad Durkheim. Within a year, my coworker, Rudi Rieder, together with a number of skilled technicians, succeeded in making a dream into a reality: putting the separation system into a "black box", a separation cassette. No longer troubled with installation, separate sample collection and concentration, gas tubing or pneumatic switching: we prepared separation cassettes which do the job and which now also work in miniaturized liquid column chromatography under high pressure. During the past few years, we somewhat modified the techniques of classical thin-layer chromatography and, together with D. Janchen of CAMAG (Muttenz, Switzerland) we succeeded in revitalizing the instrumentation of this technique. It was J. Blome's (Au/Iller) idea to the use circular development from the center (as had been originally done in thin-layer chromatography); Ute B. Hezel (K. Zeiss, Oberkochen) and J. Ripphahn (E. Merck, Darmstadt) developed the . (E. method to evaluate such plates quantitatively while €IHalpaap Merck, Darmstadt) can be credited with the new high-efficiency readymade plate materials. As a result of this collective work, we could now enter a new field: anti-circular multicomponent parallel analysis, 48 samples in 4 minutes, It took only a short time to further improve the technique and to reintroduce the power of a third phase: having the gas phase impregnating the liquid dynamically and the solid phase in a real two-dimensional simultaneous separation. We believe that this is the future technique for routine mass analysis of non-volatile compounds. We are now working on its full automation. Optimization in chromatography is of great importance but sometimes people think that they do not have enough time for it. In my opinion, the present-day computerized techniques will allow us to optimize even the polarity without changing the column in gas chromatography. Everything else will be done by computers, with the help of a very simple multicomponent theory, the abt-concept. Chromatography represents a fascinating field of work. We have lovers, believers, and non-believers, active side-by-side. And we also have the great family of real friends, many of them now with white hair, accompanied by the next generation who will take over from us. After so many years, the scientific meetings are still attracting a large number of scientists and the interest is continuously growing. Chromatxraphy is indeed a wonderful technique. However, the next analytical technique which separates and identifies over 10-orders of magnitude in concentration and the number of compounds in a few seconds will come. Whether it will be a sort of chromatography or not, I don't know. I hope it will

192 still be chromatography. Independently of this, I wish all the future pioneers to have the same exciting and happy time in its development as we had in our occupation, love and hobby: chromatography!

193

ARTHURKARMEN

ARTHUR KARMEN was born i n 1930, i n t h e C i t y o f N e w York. H e s t u d i e d a t New York Univers i t y r e c e i v i n g h i s A.B. d e g r e e i n 1950 and h i s M.D. d e g r e e i n 1954. A f t e r i n t e r n s h i p and r e s i d e n c y i n medicine, he was a s s o c i a t e d w i t h t h e N a t i o n a l Hearth I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h ( N I H ) , i n Bethesda, Maryland. From 1963 t o 1968 he was w i t h t h e D i v i s i o n o f Nuclear Medicine a t John Hopkins U n i v e r s i t y , B a l t i m o r e , Maryland. S i n c e 1968, he h a s been a c l i n i c a l p a t h o l o g i s t and d i r e c t o r of l a b o r a t o r i e s , f i r s t a t N e w York Univ e r s i t y Medical C e n t e r and t h e n a t t h e A l b e r t E i n s t e i n C o l l e g e o f Medicine. H i s p r e s e n t p o s i t i o n is t h a t of p r o f e s s o r and chairman o f t h e Department of L a b o r a t o r y Medicine. D r . Karmen i s t h e a u t h o r and c o a u t h o r of about 90 p a p e r s i n v a r o u s f i e l d s a s s o c i a t e d with a n a l y t i c a l chemistry, c l i n i c a l chemistry and medicine. While i n medical s c h o o l , he developed methods f o r t h e measurement of t r a n s a m i n a s e s and dehydrogenases i n serum. A t t h e N I H , he s i g n i f i c a n t l y c o n t r i b u t e d t o t h e development o f m i c r o a n a l y t i c a l methods p r i m a r i l y u s i n g gas chromatography, i n c l u d i n g t h e development o f i o n i z a t i o n d e t e c t o r s , d e t e c t o r s f o r r a d i o i s o t o p e s and methods f o r a p p l y i n g t h e s e d e v i c e s i n s t u d i e s o f l i p i d metabolism. H e is p a r t i c u l a r l y known i n t h e f i e l d of gas chromatography f o r t h e development of t h e r m i o n i c d e t e c t o r s i n c l u d i n g a f a m i l y of d e t e c t o r s which can be "tuned" t o be s e l e c t i v e f o r c e r t a i n heteroatoms. H i s c u r r e n t research i n t e r e s t s inc l u d e methods and i n s t r u m e n t s f o r t h e m i c r o a n a l y s i s of enzymes and d r u g s i n serum u s i n g c e n t r i f u g a l a n a l y z e r s , g a s chromatography and mass s p e c t r o metry.

194 Most of my work in chromatography has centered around detectors for gas chromatography, adapting GC detectors for liquid chromatography, and measuring I4C and 3H in chromatographic effluents. Yet, when preparing to write this, my first use of chromatography came to the forefront of my recollections. That work involved paper chromatography, I'd like to tell its story not because it made a great contribution to the development of chromatography, which it did not, but because of the very personal reason that it had a reasonable impact in another field, and as a result, made all my subsequent adventures possible. I think it also exemplified some of both the power and limitations of chromatography. In 1952 I was a medical student who wanted to try his hand at research. I had a minimum of relevant laboratory experience and very little to offer except a long summer vacation, an "elective" period during which I was supposed to learn something and just enough enthusiasm to be willing, if necessary, to work without pay. Opportunities were very few. Dr. Felix Wroblewski offered me one. He had conceived the idea that when heart muscle was destroyed, as in myocardial infarction, the enzymes in the muscle might be released intact into the circulating blood where their measurement might aid in diagnosis. The enzymes he thought might be worth investigating included serum esterase, "fibrinolysin", deoxyribonuclease, and one that was reported to be in somewhat higher concentration in heart muscle than in other tissues, glutamic-oxalacetic transaminase (GOT) Dr. Wroblewski had, after a brief time in a biochemistry laboratory at Sloan Kettering Institute in New York, entered into the private practice of internal medicine. His menthors in biochemistry did not consider his idea very promising since it was assumed that most enzymes within cells were bound to sub-cellular particles and therefore would be destroyed in situ and notre1eased.M~ faculty advisor did not think much of the plan as a learning experience for me, since Dr. Wroblewski had no laboratory, could not supervise my work, and had only a very uncertain hypothesis. The idea, however, seemed reasonable to me, and since the time involved was mostly my vacation, I agreed to work at it. He obtained permission for me to use an empty laboratory assigned to a surgeon and made $200 available to me, in cash, from a grant from a pharmaceutical company. I mention this because I believe this was the first and last time I had really unrestricted funds with which to try to do research. A literature review that lasted at least a day and a half revealed several references to the measurement of deoxyribonuclease

*

*Abbreviations used for enzymes: GOT = glutamic-oxalacetic transaminase (aspartate aminotransferase, AST); GPT = glutamic-pyruvic transaminase (alanine aminotransferase, ALT); NAD = nicotinamideadenine dinucleotide; NADH2 = reduced form of nicotinamide-adenine dinucleotide; LDH = lactate dehydrogenase.

195 in serum, many papers that emphasized the difficulties in working with the proteolytic enzyme systems of serum and three methods for measuring transaminase but no reference to its presence in blood. I set up to measure deoxyribonuclease, because a recipe and the equipment were available, but soon became disenchanted with viscosimetry; the solutions of DNA substrate contained particulates that clogged the capillary of the viscosimeter. With somewhat fading enthusiasm, after about a week of working all alone, I decided to switch to studying transaminase (GOT) on the somewhat cynical thesis that its measurement in serum would be novel even it not useful. GOT catalyzes the reversible reaction: Glutamate + oxalacetate $ aspartate + alphaketoglutarate. The methods that had been described for measuring it in tissue involved : (a) monitoring the absorbance of a reaction mixture at 280 nm. The absorbance of oxalacetate is appreciably greater than that of alphaketoglutarate. If we follow the reaction from right to left, the rate increase of absorbance is the measure of the GOT. (b) Incubating aspartate and alphaketoglutarate for a fixed period and then adding aniline citrate to decarboxylate the oxalacetate. The carbon dioxide released is a measure of the GOT. (c) Incubating the substrates for a fixed time, precipitating the protein, and chromatographing the amino acids of the proteinfree filtrate on paper. The quantity of the second amino acid formed, measured by reacting it with ninhydrin, is the measure of the GOT. After several polite inquiries in laboratories at both Sloan Kettering and at New York University, where I was enrolled as a student, I learned that ultraviolet spectrophotometers and Warburg gasometric equipment were too delicate to be entrusted to a medical student without an appropriate prior apprenticeship in science. A post-doctoral fellow, Dr. Jacques Fresco, who became my friend and chief scientific advisor, felt I would do least damage using paper chromatography. He loaned me a cylindrical glass tank with which to play, and his reprint of a review by A.J.P. Martin; he also gave me a five-minute course in the practice of descending paper chromatography as he understood it. I purchased the substrates, buffer and a pound of phenol, the recommended developing solvent, at a chemical supply house in Manhattan, for cash, without evoking any curiosity that I was aware of. It was New York, after all, and home synthesis of mood-elevating substances had not yet come into vogue. Approximating the technique Fresco described, I spotted some glutamic and aspartic acids and a mixture of the two on a paper strip, set it up to develop and went home, looking forward to seeing my first chromatogram with great anticipation as well as some skepticism. The recommended period of development was 16 hours, It did not occur to me that this was long. I instinctively discovered what is apparently well known to all bacteriologists: that working with reactions that proceed unattended

for 16 hours can result in regular working hours and an active social life. The following day I sprayed the paper with ninhydrin and found that, as advertised, the separation of aspartic and glutamic acids was complete. I was blissfully unaware that the spots were too wide or that I was achieving poor resolution because I was applying too much amino acid over too large a n area. It would have been difficult to convince me that I had anything but high-performance chromatography. Then, after a partially successful attempt to remove the evidence of working with ninhydrin from my fingers, I put on a white coat, which made all sorts of atrocities possible, and performed my first venipuncture on a lady who, I am sure, never became aware of the significance of her role in science. With the small quantity of serum obtained, I set up a number of incubations and controls and spotted my second paper that evening. The following day, after much incantation while spraying the paper, and especially while heating it, I saw sufficiently more aspartate in the strip representing the complete incubation mixture than in any of the controls to demonstrate conclusively that transaminase activity was present. Reassured by this quick success, I contentedly spent the remainder of the summer and early fall working out a method for measuring the enzyme activity quantitatively and demonstrating that it had the same chemical characteristics as the enzyme in tissue. Since a-ketoglutaric acid was more readily available than oxalacetate, I decided to follow the reaction by incubating aspartate and a-ketoglutarate and measuring the glutamate formed. The success of this experiment helped confirm the presence of the enzyme. Using a-ketoglutarate also made the identification of glutamic-pyruvic transaminase (GPT) in serum easy. I simply substituted alanine for aspartic acid in the incubation mixture. In the compendium on enzymes I used as chief reference, on the page following the one containing the statement that GOT was in highest concentration in heart muscle, was a statement that GPT was in high concentration in liver. I reasoned that GPT might prove helpful in determining whether an elevated serum GOT reflected heart or liver damage. Finding GPT in serum and setting up a method for measuring it was the work of one afternoon. To get enough product to measure by the crude technique I used, I increased the incubation time to 16 hours. To increase the number of assays I could do, I purchased a 30-gallon fish tank and, with my father's help,fitted it out with a rack made of aluminium angle to hold eight glass butter dish tops so that I could develop sixteen 4-inch papers at once. I was thus able to turn out 16 enzyme assays, in triplicate, every three days. Even so, to complete this part of the project in the time I had left required that I work 14-hour days, 7 days a week. I yearned for access to either a Warburg apparatus or an ultraviolet spectrophotometer, either of which, I was sure, would increase my output. Reporting this work at a "student research day" the following spring gave me that opportunity. Dr. Severo Ochoa, then Professor of Pharmacology at New York University, was impressed enough with my interest to offer me the opportunity to work in his laboratory

197 during my next elective time in the winter of 1953. With the guidance of several of his post-doctoral fellows, I learned, at long last, how to use the Warburg apparatus and the ultraviolet spectrophotometer. With these devices, I found, to my disappointment, that the high serum bicarbonate and protein content made the gasometric and the spectrophotometric assay at 280 nm impractical. Much of the work in Dr. Ochoa's laboratory at that time involved purification and characterization of enzymes. To assay the enzymes his people characteristically used second enzymes as reagents. Enough of the appropriate dehydrogenase was added to catalyze the oxidation or reduction of one of the products of the reaction of interest by NAD or NADHp as fast as it was formed. This second reaction, and therefore the first as well, could be followed in the spectrophotometer because of the high absorbance of NADH2 and the low absorbance of NAD at 340 nm. Malate dehydrogenase, which catalyzes the reduction of oxalacetate to malate, happened to be in Dr. Ochoa's refrigerator. It therefore seemed reasonable to try adding malate dehydrogenase and NADH2 to the GOT substrate and to try to measure transaminase by the rate of oxidation of NADHp. When I tried the experiment, I observed that NADHp was oxidized by the serum without addition of the transaminase substrates. For most of a week I struggled to find the cause of this reaction which would have made coupling to a dehydrogenase difficult or impossible. Then I found that the reaction would stop if I added enough NADHp and waited long enough, as though what was causing it was exhausted. I tried dialyzing the serum and found this would eliminate the reaction. But then so did adding more NADH;! and waiting, and that was easier. After waiting, I completed the reaction mixture and measured GOT. To find out whether the NADHp was being oxidized by pyruvate in serum, I looked for and found lactic dehydrogenase activity and, for good measure, malic dehydrogenase activity as well. That afternoon it seemed as though all one had to do to find any enzyme in serum was to look with a sensitive enough method. The assay of GOT based on these observations, took 15 minutes. Much to my delight the normal range was the same as what I had observed using paper chromatography the previous year. Then, as a sort of climax to all this work, I measured GOT in the serum of a patient in Bellevue Hospital in New York City who had had a myocardial infarction the previous day, and found that it was several fold higher than any I had measured until then. It returned to normal over the next few days. I spent the next few weeks haunting several hospital emergency rooms to get more specimens to prove that the rise and fall of serum GOT after myocardial infarction was reproducible and therefore had diagnostic value. One of the obstacles to progress by amateurs in those days was the unavailability of purified enzymes to be used as reagents. Dr. Ochoa's scientists prepared their own, with great effort. There was lactate dehydrogenase (LDH) in his refrigerator, but I wasn't able to put together a similar assay for GPT because the lactate dehydrogenase itself had GPT activity. The development of this test that has proved so useful for following liver disease had to await

somebody's making this reagent available in more pure form. Many such enzymes became commercially available once the diagnostic implications of GOT became known. After several exciting months of daytime classes and evenings in the lab, I wrote up all the work, graduated from medical school and started my internship. Dr. Wroblewski and his coworkers began publishing many papers that defined the clinical usefulness of these serum enzyme measurements. I followed the rapidly growing literature with some interest, but I wasn't part of it. As far as clinical enzymology was concerned, I disappeared from view. After two years of house staff training, I was given the opportunity to work at the National Heart Institute, National Institutes of Health (NIH), in the laboratory of Dr. Daniel Steinberg. We had an interest in common. The previous year he had restudied my spectrophotometric transaminase assay, adapted it to a simple spectrophotometer, and had measured GOT in the serum of many patients in the Washington area, including President Eisenhower when he had his coronary. I tried to join in some studies of fatty acid metabolism that were on-going in that laboratory, but found analytical methods so difficult that I looked for otherwork. Then, one day, one of my colleagues, Dr. DeWitt Goodman, told me he had seen an apparatus at Rockefeller Institute that could analyze fatty acids. The story unfolded that the device, a gas chromatograph, had been constructed under the supervision of an English visiting scientist (Dr. A.T. James) who had brought with him a detector (a gas density balance) that was the "only detector with enough sensitivity to do the analysis". It was also said that instrument companies in the States had not been able to make the device. For some reason this proved to be an irresistible challenge. I dropped some promising work on a synthetic substrate for measuring trypsin in serum when it was almost ready for publication, and started to look for help in trying out several ideas for detectors. Scientists at the National Hearth Institute - Drs. Steinberg, Robert Gordon, Evan Horning, and several others, almost to a man - referred me to the NIH's renaissance man - physician, physicist, and instrument maker - Dr. Robert L. Bowman, who had just recently completed the design of chloridometer, a freezing point instrument, and a spectrophotofluorometer that did much to introduce the measurement of fluorescence into biology. He listended with enthusiasm but pointed out that my ideas were not feasible. He did this with such kindness that I was immediately prepared not only to accept that his ideas about detectors were better but also to work with him. This began one of the more pleasant and productive times of my scientific career. During the next six years, we developed ionization detectors such as the radio-frequency glow discharge detector, and non-radioactive, self-sustained electric discharge argon and helium ionization detectors. For a long while we were s i x months behind Jim Lovelock but we didn't follow him. Along with ionization detectors we designed methods for measuring radioactivity in GLC effluents,

199 and applied them in studies of fat metabolism. We started work with adapting GLC detectors for LC and with the alkali flame detector for halogens and phosphorus. Those were interesting days in other labs at NIH as well, Evan Horning's group showed us how to analyze steroids, drugs and other high boiling compounds by GLC while Sidney Udenfriend's group studied catecholamine metabolism using this method. Others developed methods for GLC of amino acids and sugars. During these same years, the use of the serum enzymes for medical diagnosis grew exponentially until GOT, GPT, and LDH became some of the most commonly used tests. I remained no part of it. Then, one careful group pointed out the obvious: that good temperature control was essential for accurate enzyme measurement. They suggested that the technique I had described, using a spectrophotometer on a bench top was "subject to error". Another group, introducing what they considered to be an improved method, which used a temperature controlled bath, claimed it was "less subject to error than Karmen's method." They claimed this in full page advertisements on the back covers of many clinical journals. I framed a copy of the advertisement to show my family. They had done me a great favor. A year or so later, to my surprise, GOT and GPT were being measured in "Karmen Units". With the explosion in laboratory testing that followed the introduction of automation, my name became a household word in medicine, although I believe it was generally assumed that I was long dead. I watched all this with some amusement and continued to work with chromatography and radioisotopes. Then one of my friends pointed out that clinical pathology, with its newly growing emphasis on instrumentation and automation, might be the most logical field of application of my research interests. When I sought to enter the field, even after all the years of productive work in GLC and instrumentation, the work with GOT and other enzymes was my primary credential. It was a good one: I was offered several prestigious jobs. In 1968 I entered clinical pathology. I reappeared on the clinical enzyme scene like a Rip Van Winkle or Ulysses. I was invited to speak at scientific meetings as a world famous expert. People seemed to be coming out just to look, amazed that I was still alive. I had to study hard to try to live up to my apparent reputation. As part of my job now, I became embroiled in solving the problems associated with providing large numbers of assays to a demanding medical community; our clinical chemistry laboratories alone analyze more than 1500 patient samples a day, up to 20 tests on each. Designing and operating a system that will do this reliably requires attention to many factors in addition to analytical biochemistry. Yet that too was involved. To provide accurate enzyme assays that would be comparable in three different laboratories required a reworking of many of the methods to smooth out edges that were still rough after all those years. Somehow our staffs expected it of me.

200

In recalling this story for this volume, I thought often about the crucial role of paper chromatography in it. It was so easy to apply, even an amateur could use it successfully, even if not optimally. It offered practically incontrovertible evidence that the reactions I was looking for had occurred. It was less subject to interference than any of the other methods. It did not require rare or unstable reagents. The same system could be used to study several different things. It cost so little to set up that I was able to complete an entire research project for less than $150. The same could be said for all the early chromatographic methods, those before the advent of High-Priced Liquid Chromatography and HighPriced Thin-Layer Chromatography. It was thus an ideal tool to be used in the important preliminary exploratory work. After the objective had been defined, the faster, simpler to perform, but inherently more expensive and complicated specific assay turned out to be better. Many of us observed the same phenomenon after we devised highly specific assays for hormones and drugs using GLC, assays that were much more useful than the wet chemistry methods they sometimes replaced. When the need for many assays arose, immunoassays, even those using far more expensive reagents, often became the methods of choice. For those of you who prefer more realistic endings to stories, I would only add that fame in enzymology, like fame in chromatography, is short lived. Our profession apparently has negative feedback build into all its mechanisms for dispensing scientific recognition. In recent years committees of enzymologists met and decided to change the names of the enzymes. Apparently to reflect the currently popular direction for following the reactions, GOT is now to be called aspartate aminotransferase, or AST, and GPT alanine aminotransferase, or ALT. They also recommended standardized assay conditions and the reporting of results in "International Units". Who, pray tell, was "International"?

201

JUSTUS G. KIRCHNER

JUSTUS GEORGE KIRCHNER was born i n 1911, i n Cedar Rapids, Iowa. H e r e c e i v e d a B.S. e m Zaude i n c h e m i s t r y from C r e i g h t o n U n i v e r s i t y a t Omaha, Nebraska i n 1935 and h i s Ph.D. from Iowa S t a t e C o l l e g e , a t A m e s , Iowa, i n 1939. I n t h e f a l l of t h a t y e a r he took a postdoct o r a t e assistantship at the California Instt u t e of Technology. A y e a r l a t e r he became a Research Fellow a t t h e I n s t i t u t e . I n 1945 he accepted a p o s i t i o n w i t h t h e United S t a t e s Department of A g r i c u l t u r e F r u i t and Vegetable Laboratory i n Los Angeles and remained w i t h t h e l a b o r a t o r y when i t r e l o c a t e d i n Pasadena, C a l i f o r n i a . I n 1954 he j o i n e d Tenco, an Ins t a n t Coffee Company, and a f t e r o r g a n i z i n g a Research Department, became D i r e c t o r of Research i n 1955. I n 1956 he w a s named D i r e c t o r of Research and Development f o r t h i s company which became a D i v i s i o n of t h e CocaCola Company i n 1961. I n 1968 h e moved t o A t l a n t a as S e n i o r S c i e n t i s t f o r t h e C o r p o r a t e D i v i s i o n of t h e Coca-Cola Company. D r . Kirchner ret i r e d i n 1976. D r . Kirchner i s t h e a u t h o r and c o a u t h o r of o v e r 30 s c i e n t i f i c p a p e r s and t h e a u t h o r o f two books and t h r e e book c h a p t e r s . Thin-Layer Chromatography, an e x t e n s i v e t r e a t i s e of t h e s u b j e c t , w a s p u b l i s h e d i n 1967; t h e e x t e n s i v e l y r e v i s e d second e d i t i o n i s scheduled f o r p u b l i c a t i o n i n 1978. I n 1975 he was p l e n a r y l e c t u r e r on t h i n - l a y e r chromatography f o r t h e F e d e r a t i o n of A n a l y t i c a l Chemistry and S p e c t r o s c o p i c S o c i e t i e s ' meeting. D r . Kirchner founded t h e present-day system of t h i n - l a y e r chromatography, t h e r e s u l t s of which were p u b l i s h e d i n 1951-1954. I n t h e s e publ i c a t i o n s he i n t r o d u c e d f o r t h e f i r s t t i m e a number of i m p o r t a n t t e c h n i q u e s now u n i v e r s a l l y u t i l i z e d i n t h i n - l a y e r chromatography.

202 In 1939 I was hired as a research assistant to Dr. Zechmeister at the California Institute of Technology, but s nce his arrival in this country was delayed, I worked with Dr. A.J. Haagen-Smit for one year. On Dr. Zechmeister's arrival the following year I was offered the opportunity of remaining with Dr. Haagen-Smi h to work on the chemistry of the aroma and flavor principles in pineapple, or of transferring to Dr. Zechmeister's laboratories. Work on the chemistry of flavor and aroma was in its infancy at that time, and I decided to remain with it. It was while on this project that I first became acquainted with chromatography, and developed a column-chromatographic separation method for thep-phenylphenacylesters of acids ( 1 ) . The publication of the work on pineapple flavors (2, 3 ) then led to an offer to work on the chemistry of orange and grapefruit flavors at the Fruit and Vegetable Laboratory of the United States Department of Agriculture. This was indeed a challenge at that time because gas chromatography had not been developed and the minute amounts of flavoring and aroma materials required a micro method for isolating and identifying these constituents. Even though large quantities of juice were processed (3,000 gallons each of fresh, freshly canned, and stored canned orange juice, 2,760 gallons of fresh grapefruit juice, and 2,470 gallons each of freshly canned and stored canned grapefruit juice) the amount of these flavor materials was exceedingly small. This problem of quantity is exemplified by the sulfur-containing ester found in pineapple flavor ( 3 ) where the fruit was found to contain 112 to 250 mg of sulfur per kilogram of fruit. My first thoughts were to use paper chromatography which had been used with such excellent results with amino acids, but it was soon evident that paper was much too mild an adsorbent to accomplish the required task. Flood ( 4 ) and Hopf ( 5 ) had used alumina-impregnated paper for spot tests and this soon led us to the development of the first silica gel impregnated paper for use in chromatography (6). This was produced by soaking filter paper in sodium silicate solution and then precipitating silicic acid in the fibers by immersing in hydrochloric acid. This showed promise and was used to separate some 2,4-dinitrophenylhydrazones of aldehydes and ketones which could not be separated on unimpregnated paper. However, the impregnated paper was still not the answer to our needs, its capacity was still not great enough. During this time when Chemical Abstracts were much less expensive than they are today, I would clip out abstracts which were of interest to me and file them away for future use. On this particular day I had come across the abstract of Meinhard and Hall's work (7) on drop chromatography for the separation of inorganic ions. I clipped the abstract and laid it on the corner of my desk. Later in the day one of my assistants came in very much discouraged with his attempts at separating terpene constituents by paper chromatography. I picked up the abstract and said "Here, let's make layers of silicic acid on strips of glass and develop them in an ascending manner analogous to paper chromatography." This latter step proved to be the key to the development of the successful system, now

203

Fig. 22.1. Fractionating citrus oils at the U.S.D.A. Fruit and Vegetable Laboratory, in Pasadena, California, circa 1950.

better known as "thin-layer chromatography", existing today. The method was first published in 1951 (8) and was used successfuly by a large number of research workers prior to 1956 (9a) when Egon Stahl began publishing his first work. Drop thin-layer chromatography had first been demonstrated by Beyerinck in 1889 ( 2 0 ) which even predates Tswett's excellent work as well as the work of Reed in 1893 ( 1 1 ) . However, this drop method as demonstrated by Beyerinck and practiced by Izmailov and Shraiber ( 2 2 ) as well as others was too limited in its scope for our puropose. After we had proved to our satisfaction that the method would work, the next task was that of finding the best adsorbent and the optimum conditions for making thin-layer chromatography reliable and reproducible. Of the sixteen adsorbents tested, silicic acid was selected a5 the best for our purpose. This was combined with Amioca starch (National Starch Products Co.) to give a satisfactory layer. Later ( 1 3 ) this binder was replaced to advantage with Clinco 15 modified starch (Clinton Foods, Inc.) or by a 2:l mixture of cornstarch and Superior AA tapioca flour (Stein Hall and Co., Inc.), and at a still later date ( 2 4 ) we found that the starch binder could be reduced to advantage from 5% to 24% in the mixture.

204

Fig. 2 2 . 2 . Separation of p-cymene, pulegone and cinnamaldehyde on silicic acid thin-layers with 15% ethyl acetate in hexane, as shown under ultraviolet light on fluorescent chromatostrips. (8). (Reproduced with permission of the American Chemical Society). From top to bottom: p-cymene, pulegone, cinnamaldehyde. From left to right: l=blank (spot due to traces of impurity in solvent not removed by distillation); 2=p-cymene (143 pg), pulegone (3.3 ug) and cinnamaldehyde (3.3 pg); J=p-cymene (358 pg), pulegone ( 8 . 2 pg) and cinnamaldehyde (9.1 pg); 4= p-cymene (1.4 mg), pulegone (32.8 pg) and cinnamaldehyde (36.4 pg). One of the earliest discoveries we made and reported was that in order to obtain reproducible results, conditions had to be carefully controlled (standardized). To obtain these results, the silicic acid was screened in order to remove the coarser particles and the layers were dried and held under carefully controlled conditions.

To further increase the reliability of results, it was established at that time that it was necessary to run standards to show that the layers were properly prepared and to serve as a reference for comparing RF values. Because most of the compounds with which we were working were colorless, it was necessary to develop methods for locating compounds. For ultraviolet absorbing compounds we adopted the technique of Sease (15) of incorporating inorganic fluorescent material with the adsorbent. Numerous spray reagents were then developed for other compounds, and in some cases these not only located the compound but also indicated the type of compound that was present. For example,the 2,4-dinitrophenylhydrazine spray disclosed the presence of carbonyl compounds and the dianisidine spray showed the presence of aldehyde groups. One of the more useful sprays was the 0 . 0 5 % fluorescein spray with subsequent exposure of the thin-layer to bromine vapor to detect unsaturated compounds or other compounds which would react with bromine vapor under the conditions used. There were some compounds, as for example, camphor, which could not be located by any of the methods thus far devised. I originated the idea of using plaster of Paris as a binder so the compounds could be sprayed with sulfuric acid containing an oxidizing agent. Thus on subsequent heating, the spot location was revealed by the charred areas. This was the origin of the so-called silica gel G, because when plaster of Paris sets, it takes up water of crystallization to form gypsum. Later this charring technique was developed by others into a quantitative analytical method. One of the techniques which wedevelopedand used for fractionating larger amounts was that of using thin-layer chromatography to monitor the fractions as they were eluted from a packed chromatographic column ( 1 6 ) . In this way by the time the column was completely eluted, it was possible to determine which fractions should be combined and which fractions were single compounds under the conditions of the separation used. Another column application was the use of thin-layer principles for the preparation of a self-supporting column unencumbered by a containing envelope ( 1 7 ) . This was designated as a "chromatobar", as it consisted of cylindrical or square bar of silicic acid bound with gypsum around a glass rod which added strength. This column was developed in an ascending manner and could be removed and checked for development by spraying with a visualizing agent on one side. The thin layer of sprayed material could then be scraped off and the column returned for further development. In addition to the use of narrow strips of adsorbent, we also introduced the use of square coated plates in our first publication (8); thus multiple samples could be run on a single plate or twodimensional chromatography could be carried out on an individual sample so as to give improved resolution. An important feature of our work included the introduction of the use of reactions on the thin-layer plate ( 1 8 ) . We used oxidations, reductions, dehydrations, hydrolysis reactions, and the preparation of derivatives. These could all be used to help in identifying a

206

Fig. 22.3 (left). Dr. Kirchner at the CAMAG First International Thin-Layer Chromatographic Symposium demonstrating preparative thin-layer chromatography without a backing plate to support the layer so that it is accessible from both sides for sample application and visualization. For details, see ref. 21. Fig. 22.4 (right). Separation of 100 mg each of six dyes on a 4 x 8 x 8 in. silica gel self-supporting layer with no backing plate. Top to bottom: Yellow OB, narrow line due to second solvent front, Sudan I , Sudan 111. Sudan 11, Methyl Red and Crystal Violet ( 2 1 ) . specific compound because the reaction products had different RF values than the original compounds. This technique is also useful in separating critical pairs of compounds that can not normally be separated by chromatography. This reaction technique proved to be very versatile as it may be applied to the initial spotted sample, or it may be applied after the chromatogram has been developed in one direction and prior to the development in a second dimension. Biphenyl is used by the citrus industry to prevent molding during shipping and storage of citrus fruit. Because there was a need for an accurate method for detecting and measuring the amount of biphenyl absorbed by the citrus fruit, we had the opportunity to apply the thin-layer technique to this problem and so introduced

207

the use of this versatile technique in the quantitative field. By using a spectrophotometric method on the eluted spots we were able to obtain results of +2.8% on amounts ranging from 0.1 to 600 ppm. This helped to establish the reliability of our thin-layer technique at that time. Another technique that we introduced because of our work with thin layers, was the chromatographic preparation of terpeneless oils ( 1 9 ) . I had noticed in running thin-layer chromatograms in hexane on silicic acid that this solvent moved the hydrocarbons, but not the oxygenated components which remained behind at the origin. A check of numerous compounds confirmed this observation and it was then a fairly simple matter to apply the technique using packed columns. On entering into industrial work in 1954, the opportunity was not available to continue research in chromatography although I managed to keep somewhat active in the field by producing a text on thin-layer chromatography ( 9 ) , which has just recently been revised (20). However, there was also an opportunity to develop a preparative thick-layer method ( 2 1 ) . Up to this time the thin-layer methods consisted in running numerous thin-layer chromatograms and combining the separated components until a sufficient quantity of material had been collected. The quantity that could be separated on an individual plate was limited by the thickness of the layer which ranged from 1 to 5 mm thick. The thicker the layer, the more difficult is is to prepare a layer without developing cracks on drying. For the 5 mm layers this resulted in only 50% of the layers being usable. It occurred to me that a thick-layer plate could perhaps be prepared based on the chromatobar technique. The first attempt at the preparation of a 1/4 in. layer without a backing plate was successful; however, the layers were rather fragile, especially when dry, with a tendency for the corners to break. This then led to the use of a stainless steel frame with 0.025 in. stainless steel wires stretched across the frame and imbedded in the layer to help support the latter. These wires in no way interfered with the chromatographic development. The layers were cast in a mold that had been carefully machined so that the layer thickness was extremely uniform. The main problem to be overcome was the tendency of the wet layer to stick to the top and bottom of the mold. Various materials and techniques were tried to solve this difficulty, and this was finally accomplished by covering the top and bottom of the mold with thin sheets of plastic so that the stainless steel plates could be easily removed; then the plastic was rolled back to expose the undisturbed layer. Layers ranging in thickness from 1/8 to 1/2 in. thick were successfully prepared and used. REFERENCES 1 J.G. K,irchner,A.N. Prater and A.J. Haagen-Smith, Ind. E n g . Chem. 18 (1946). 2 A.J. Haagen-Smith, J.G. Kirchner, A.N. Prater and C.L. Deasy, J . Amer. Chem. SOC. 67 (1945) 1646.

208 3 A.J.

Haagen-Smith, J . G .

Kirchner, C . L .

Deasy and A . N .

Prater,

J . h e r . Chem. SOC. 67 (1945) 1651. 4 H. F l o o d , Anal. Chem. 120 (1940) 327. 5 P.P. Hopf, J . Chem. Soc. (1946) 785. 6 J . G . K i r c h n e r and G . J . Keller, J . Amer. Chem. Soc. 7 2 (1950) 1867. 7 J . E . Meinhard and N.F. H a l l , Anal. Chem. 21 (1949) 185. 8 J . G . K i r c h n e r , J . M . Miller and G . J . Keller, Anal. Chem. 23 (1951) 420. 9 J.G. K i r c h n e r , Thin-Layer Chromatography, I n t e r s c i e n c e P u b l i s h e r s , N e w York, 1967; a, p. 5. 10 M.W. B e y e r i n c k , 2. Phys. Chern. 3 (1889) 110. 11 L. Reed, Proc. Chem. Soc. 9 (1893) 123. 12 N . A . Izmailov and M.S. S h r a i b e r , Farmatsiya ( S o f i a ) 3 (1938) 1. 13 J . G . K i r c h n e r , J . M . Miller .and R.G. R i c e , A g r . Food. Chem. 2 (1954) 1031. 14 J . G . K i r c h n e r and V.P. F l a n a g a n , Gordon Research Conference, Colby J u n i o r C o l l e g e , N e w London, N. H., August 1962. 15 J . W . S e a s e , J. Amer. Chem. Soc. 69 (1947) 2242. 16 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 24 (1952) 1480. 17 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 2 3 (1951) 428. 18 J . M . Miller and J . G . K i r c h n e r , Anal. Chem. 25 (1953) 1107. 19 J . G . K i r c h n e r and J . M . M i l l e r , Ind. Eng. Chem. 44 (1952) 318. 20 J . G . K i r c h n e r , Thin-Layer Chromatography, 2nd e d . , W i l e y - I n t e r s c i e n c e , N e w York, 1978. 21 J . G . K i r c h n e r , J. Chromatogr. 63 (1971) 45.

209

J. JACK KIRKLAND

JOSEPH J A C K KIRKLAND was born i n 1925, i n Winter Garden, F l o r i d a . H e s t u d i e d a t Emory U n i v e r s i t y , A t l a n t a , Georgia, r e c e i v i n g h i s A.B. and M.S. d e g r e e s i n c h e m i s t r y i n 1948 and 1949 r e s p e c t i v e l y . A f t e r working f o r t h e H e r c u l e s Powder Company i n 1950-1951, he l e f t t o e a r n a d o c t o r a t e i n a n a l y t i c a l c h e m i s t r y a t t h e U n i v e r s i t y of V i r g i n i a i n 1953. S i n c e t h a t t i m e he has been w i t h E . I . du Pont de Nemours & Company a t t h e Experimental S t a t i o n , i n Wilmington, Delaware. I n t h e C e n t r a l Research & Development Department he c u r r e n t l y i s engaged i n b a s i c res e a r c h i n t h e chromatographic s c i e n c e s . D r . K i r k l a n d a u t h o r e d and c o a u t h o r e d about 50 s c i e n t i f i c p u b l i c a t i o n s i n chromatography. H e is t h e e d i t o r of Modern Pract i c e of Liquid Chromatography (1971) and c o a u t h o r ( w i t h L.R. Snyder) o f Introduction t o Modern Liquid Chromatography (1974). H e i s c o p r o f e s s o r o f two American Chemical S o c i e t y s h o r t c o u r s e s , Modern Liquid Chromatography and Solving Problems w i t h Modern Liquid Chromatography and i s t h e c o a u t h o r ( w i t h Snyder) o f t h e t a p e d ACS Audio S h o r t Course, Modern Liquid Chromatography. H e i s on t h e e d i t o r i a l a d v i s o r y b o a r d s of Analytical Chemistry and t h e Journal of Chromatographic Science and i s a p a s t member o f t h e Instrument a t i o n Advisory P a n e l of Analytical Chemistry and o f t h e a d v i s o r y p a n e l f o r t h e A n a l y t i c a l D i v i s i o n , N a t i o n a l Bureau of S t a n d a r d s . H e r e c e i v e d t h e American Chemical S o c i e t y Award i n Chromatography ( 1 9 7 2 ) , t h e ACS Delaware S e c t i o n Award (1973) and t h e Steven Dal Nogare Memorial Award i n Chromatography from t h e Chromatography Forum of t h e Delaware V a l l e y (1974). I n 1974, he r e c e i v e d a honorary D.Sc. d e g r e e from Emory U n i v e r s i ty. D r . K i r k l a n d ' s involvement i n g a s chromatography s t a r t e d i n 1954; he p i o n e e r e d i n p r e p a r a t i v e GC and t h e u s e of s p e c i a l s u p p o r t s . H i s major c o n t r i b u t i o n t o chromatography h a s been i n t h e f i e l d of l i q u i d chromat o g r a p h y , mainly i n t h e development o f new packing materials.

210 Although I was briefly exposed to chromatography in my Ph.D. dissertation work, the origin of my enthusiasm for this technique actually began late in 1954 in Du Pont's old Grasselli Chemicals Department as a result of an event with the late Dr. Stephen Dal Nogare. I was wrestling with a difficult problem on the analysis of various alkylamines. I learned that Dal Nogare was researching a weird, new form of chromatography, one in which the mobile phase was a gas, and that he was able to make some remarkable separations with this technique. I begged for some help with my problem and he agreed to conduct a short investivation. The next morning I took him some representative samples and explained the analytical needs. I was amazed when he called me that afternoon to say that my needed information was available. In about three hours he had been successful in solving a problem that I had worked on for weeks using conventional methods, without any usable results! This experience convinced me of the power of gas chromatography, so I quickly become involved. With the help of Dal Nogare, I soon had an instrument and was producing valuable chromatograms. Since at that time many of the analytical studies in which my group was involved lacked suitable high purity analytical standards, we became interested in the possibility of preparative gas chromatography to produce standards, and to assist in the isolation and characterizing of unknown samples. We subsequently were successful in developing equipment and techniques for separating gaseous, liquid and solid samples. However, many frustrating hours were spent before we finally understood that the vaporizer of a preparative gas chromatograph required a large heat capacity for the rapid volatilization of gram-size solid samples! The large-scale GC apparatus was largely constructed of glass, which fortuitously provided unique advantages. For instance, if a liquid sample was not properly vaporizing, we could see it percolating down through the top of the column. To prevent breaking the glass column blank (6 ft. lengths of heavy-wall, 31 mm I.D.), we utilized a form of "dump" or "bulk" packing with a minimum of column tapping and vibration, which resulted in a bed structure which others have subsequently shown by definitive studies to be best. Plate heights obtained with our large I.D. glass columns were essentially identical to those for analytical columns operated under the same condition. I never really understood why others reported much poorer efficiencyfor wide-bore preparative GC columns, relative to narrow-bore analytical columns. In this case, ignorance obviously was bliss. A paper on some of this preparative GC work was presented at the First International Symposium on Gas Chromatography sponsored by the Instrument Society of America at the Kellogg Center on the Michigan State University campus in East Lansing, Michigan, in August, 1957 ( I ) . It was during this symposium and meetings at Michigan State in succeeding years that many important concepts in GC were initiated. Some said that there was little else to do in East Lansing, therefore, the symposium attendees had plenty of time to discuss and argue gas chromatography. I clearly remember the spirted exchanges on GC theory between Stephen Dal Nogare, James C .

211 Sternberg (Beckman), J. Calvin Giddings (University of Utah), Howard J. Purnell (Cambridge, at that time), the late Richard Kieselbach (DU Pont) and others. Fortunately, Marcel J.E. Golay (Perkin-Elmer) and Nobelist Archer J.P. Martin (England) were there to keep everyone on the right track, and lend dignity to the proceedings. These Michigan State meetings started a unique tradition in chromatography which led to many lasting friendships, and at the same time, had enormous impact on the development of chromatography. Because of the nature of the work in my Department in the late 1950's, there was much interest in characterizing essentially nonvolatile compounds, for example, high-boiling carboxylic acids, and sulfonic acids and salts. This resulted in an intersting era in my GC experience, because it gave me an opportunity to use some of the organic chemistry so patiently taught me by my professors. I realized that the vapor pressures of compounds could be much increased by making volatile and stable derivatives, so that this approach ultimately led to analyses that were very useful at that time (but now often can more profitably be done by modern liquid chromatography). Nevertheless, the new concept of derivation in GC were very important in those days, and such approaches resulted in our being able to solve problems which otherwise were insoluble by previous techniques (2, 3 ) .

In the late 1950's I was interested in the possibilities of doing trace analysis by gas chromatography, particularly for analyzing pesticide residues and metabolites. I found that such GC analyses could successfully be carried out with thermal conductivity detectors, by injecting large sample volumes and using column programmed-temperature techniques, or by use concentrating pre-columns with a traveling furnace to displace trace components into the chromatograph for subsequent analysis. These techniques were useful at the time, but all this technology was quickly outdated by the development of the sensitive flame ionization detector (1958), and other selective detectors that revolutionized trace analysis by GC and allowed us to solve very important problems in residue analyses. In the early 1960's studies with compounds that were corrosive to siliceous supports led me to study fluorine-containing polymers as solid supports in GC ( 4 ) . The very fragile nature of some of these supports required that new packing techniques be developed. Visitors to our large walk-in refrigerator often were startled to see us sedately packing columns inside, since improved handling characteristics were experienced at low temperatures for many of the polytetrafluoroethylene supports investigated. Of course, we were often accused of taking advantage of the refrigeration strictly for comfort during the hot, muggy summer days in Delaware. It was during the study of the fluorine-containing polymers that the importance of the wetting behavior of liquids on highenergy versus low-energy surfaces came to my attention. Because of the low surface energy of fluorine-containing polymers, many of the relationships involving retention and band broadening as a function of the type and amount of liquid phase had to be re-evaluated. This experience was subsequently quite useful in later studies in liquid chromatography.

212 In 1964 I attended the Symposium of the Gas Chromatography Discussion Group at Brighton, England, to present a paper on some modified gas chromatographic adsorbents and supports, including gas-solid porous layer beads for packed columns and wall-coated gas-solid capillary columns ( 5 ) . During this trip I also visited the laboratory of Professor Keulemans at the University of Eindhoven, The Netherlands, to discuss chromatography. In one of the research laboratories Dr. J . F . K . Huber was carrying out some experiments in "high-pressure" liquid chromatography (100-200 psi) with a converted UV spectrophotometer as a detector, a column of liquid-coated diatomaceous earth particles, and a simple pump. He demonstrated some remarkable separations of compounds that could not be handled by gas chromatography because of their low volatility and/or lack of stability at higher temperatures. Exposure to these exciting experiments made my trip to Europe a complete success, for Josef Huber's studies gave me new hope for a technique I dearly wanted to practice. The reason was that in the late 1950's I had attempted to use "pressurized" liquid chromatography for non-volatile compounds with very little success. I now attribute my virtual failure to a recalcitrant differential refractometer which I attempted to use as a continuous detector, faulty column packing technique, and a poor understanding of extra-column band-broadening effects in chromatography. (Unfortunately, I had not yet had the advantage of Sternberg's classic paper on this subject published later (6).) Dr. Huber's work infused me with optimism and energy. Upon my return to the U.S.A., I easily convinced my forward-thinking research manager, Dr. Warren K. Lowen, to allow me to conduct experiments in high-pressure liquid chromatography. Based on an (incomplete) understanding of the basic chromatographic process, I judged that three main areas of the separation process needed attention before LC separations could be optimized: column performance, sample injection, and detection. To study the first two variables I realized that a sensitive, low-volume detector was a prime need, so my attention was initially focused on this area. It appeared that a highly sensitive, stable, UV photometer earlier developed within Du Pont as an on-stream process analyzer, should be adaptable for the desired detector. The optics of a borrowed unit was modified, and a low volume cell (20 111) was constructed and installed. The resulting instrument (7) was so successful that the need of a practical detector for efficient LC columns no longer existed. Sample injection difficulties were overcome rpS adapting GC know-how. Syringe-injection through low volume cleanly swept ports proved to be simple, convenient, and practical at the pressures which were utilized at that time (s1000 psi max.). Optimizing column performance was much more difficult. However, based on published theory and my experiences in porous-layer beads for GC, it appeared that a configuration of a superficially porous or porous layer bead support should allow improved column efficiency for LC. Fortunately, I had already prepared some unique superficially porous packings for GC, based on technology taught me by my good friend and our resident expert in silica chemistry, Ralph K . Iler,

213

Fig. 23.1. First LC separation on superficially porous particles. Column: 500 x 0.14 cm, 177-250 um particles with 0.5% B,B'-oxypropionitrile. Mobile phase: dibutyl ether, 3.2 ml/min, 550 psi. Temperature: ambient. Sample: 0.5 p1 of a 0.5 mg/ml solution each of 3-(p-chloropheny1)-1,l-dimethylurea and 3-(3,4-dichlorophenyl)1,l-dimethylurea. Detector: UV, 254 nm, 0.1 absorbance full scale.

who had the laboratory next door, To test the basic concept of porouslayer packings, I prepared a column of stainless steel tubing (5 long and 0.14 cm I . D . ) of 60-80 mesh superficially porous beads coated with a stationary liquid. After equilibration with mobile phase, I was able to separate two chlorophenyl dimethylurea in a little over two minutes. I was elated that, even with these relatively large particles, such similar compounds could be easily separated at relatively good column efficiency while operating at a relatively high mobile phase velocity. This experiment was convincing that superficially porous (porous-layer or pellicular) particles had excellent mass transfer characteristics in LC, and thus should be generally useful for high-performance separations. Particles were then synthesized with optimized overall size, porosity of porous shell, and shell thickness for use as a support for liquid-liquid chromatographic separations (8). Based on these and other studies in LC, Du Pont's Instrument Products Division in the late 1960's began to market liquid chromatography instrumentation including column and column packing. The superficially porous particles were then made available as ZipaxR controlled porosity chromatographic support.

The availability of a high-performance packing for LC led to a series of studies in which the experimental aspects of preparing efficient columns was investigated. The results of some of these studies were reported during meetings of the international Advances i n Chromatography symposia ( 9 - 1 1 ) . This brings to mind an early meeting of this symposium series in which Dr. Herman Felton of Du Pont, as a session chairman, gave a parody on the late singer-comedian, Allan Sherman's, "Hello Mother, Hello Father", about some of the internationally-known chromatographic experts attending the meeting. What was not known by the attendees was that Felton's song was developed that day at lunch in a kosher delicatessen over a hot pastrami sandwich, with the collaboration of Leroy Hollis (DowCorning) and myself, and that this performance was given from the podium on a dare! Our distinguished European guests were particularly startled. Fortunately, the symposium organizer, Professor Zlatkis, took all of this in good humor, and the symposium went on without additional sound-effects to be another one of the many successful "Advances" that has been so important in the development of the chromatographic sciences. Specific needs for high-speed ion-exchange chromatography to solve some of the problems in my Department led me to realize that polymers with ionic functionality might be coated on the porous layer of superficially porous particles to obtain packings of improved mass transfer, due to the relatively thin film of stationary phase available for chromatographic interaction. Subsequent development of such new packing materials ( 1 2 ) allowed the solution of difficult problems involving pesticide residues and metabolites. Because of the disadvantages in maintaining mechanically-held liquid phases on supports for liquid-liquid chromatography, I next mounted efforts to develop chromatographic packings based on controlled surface porosity supports with co-valently bound organic stationary phases. The polymolecular bonded-nhase packings that were subsequently developed (with Paul C. Yates) had three-dimensional, non-extractable, thermally and hydrolytically stable organic phases with a variety of functional groups that proved useful for both LC and GC ( 1 3 - 1 5 ) . These packings had significant practical advantages over LC packings with mechanically-held stationary phases, eliminating the necessity for pre-columns or pre-saturating the carrier with stationary phase to prevent loss by "bleed". Such packings quickly became routinely used in our laboratories, and versions of these packings were subsequently marked by Du Pont's Instrument Products Division under the PermaphaseR trademark. In the spring of 1970 the Chromatography F Q r w of the Delaware Valley and Du Pont's Instruments Products Division co-sponsored a state-of-the-art course in modern liquid chromatography in Wilmington. Many of the recognized experts in LC presented in-depth lectures at this meeting, and the 265 attendees were treated to a superb program. Arrangements had been made for these lectures to prepare chapters to be published as the first book in modern LC, with Herman Felton as editor. Much to my surprise (and chagrin), Felton disclosed to US during the meeting that he was leaving Du Pont and would not be able

215 to edit the intended book. He insisted that if the book was to be completed, I would have to assume editorship. With much reluctance and inexperience, I took charge, and the book was subsequently published on schedule ( 1 6 ) . The popularity of this book is evidenced by editions that now are in six languages. Much improved performance in LC columns had been predicted many years ago (e.g., by Calvin Giddings, John H. Knox, and others), but in 1970-1971 only Josef Huber, then at the University of Amsterdam, had been generally successful in preparing and using columns of small particles. I had long been interested in the feasibility of high-efficiency LC with small particles, and in 1970 it was my good fortune again to have the assistance of Ralph Iler to solve some of the practical problems in reaching this goal. During coffee break one day, I explained to Ralph the need for LC particles of less than 10 I.rm (preferably spherical), with known but variable pore structures, with a narrow particle size distribution, and high mechanical strength. He responded with a proposal that such particles probably could be made by a coacervating silica sol particles with a urea-formaldehyde polymer in a homogeneous precipitation reaction. Following this proposal, he quickly synthesized some samples for demonstration. I was excited about the possibilities of these materials when scanning electron micrographs and other physical characterizations were obtained. I began a program to follow up this exciting lead, so that we could learn to prepare the particles with a variety of pore sizes reproducibly. I also initiated column packing studies and quickly found that the balanced-density wet-fill techniques previously used on bonded-phase superficially porous particles ( 1 4 ) could also be used with these new microparticles. Other wet-fill packing techniques were also found useful in preparing high-performance columns for adsorption, liquid-partition and size-exclusion chromatography. A paper on the preparation and application of columns of these porous silica microspheres was given as my ACS Chromatography Award Address at Boston in April of 1972 ( 2 7 ) . Regarding nice things that have happened to me, I should include my long, and I hope fruitful, relationship with Lloyd R. Snyder in many scientific endeavors. In late 1970 Lloyd contacted me to see if I was interested in being a co-professor of a short course in liquid chromatography for the American Chemical Society. I agreed that this should be a useful project, and after much preparation, we gave our first session of Modern Liquid Chromatography at Chicago in June, 1971. In spite of all the "opening day" problems, including air-conditioner failure in 90 OF weather, non-functioning slide projectors, overly-long lectures, etc., the attendees were enthusiastic, and this basic course has continued on the ACS Short Course schedule throught this writing. Largely as result of our experience with this course, Lloyd and I prepared an audio course for the ACS, and next a text on LC, which was published early in 1974 ( 1 8 ) . In that year we also initiated a new Short Course for experienced LC workers, Solving Problems i n Modern Liquid Chromatography, so that almost 2500 students have now suffered through the "Snyder-Kirkland Road

216 Shows”. My close association with Lloyd has been one of the highlights in my personal scientific experiences, and I believe, one that has been helpful in the development of chromatography. The advent of the porous silica microspheres and the ability to make efficient columns of small particles opened up 8 new area of research, and several papers describing the experimental aspects of these particles in liquid-solid and liquid-liquid chromatography resulted (see e.g. ref. 19). Columns of these porous silica microspheres are marketed by Du Pont’s Instrument Products Division as ZorbaxTM chromatographic packing. The possibilities of using small porous spherical particles for size-exclusion chromatography (SEC) were not fully exploited until my transfer into the Central Research & Development Department in 1973. Here I began working with Wallace W. Yau, Donald D. Bly, Henry J. Stoklosa and later, Charles R. Ginnard and Paul E. Antle and our goal was to optimize both the theoretical and experimental aspects of high-performance liquid-size exclusion chromatography utilizing the porous silica microspheres. Some aspects of this work are still continuing. This extensive study has involved a different aspect of my chromatographic experiences. Previously, I had worked essentially alone. However, the size-exclusion studies have involved somewhat of a team-research effort and it has been most stimulating. My association with these co-workers has resulted in several papers defining the optimized parameters, limitations, and experimental aspects of high-performance size-exclusion chromatography (e.g. refs. 20-22). A molecular weight analysis technique that was previously relatively slow and imprecise now has been converted to a rapid method (15-20 min) with considerably improved molecular weight precision. During the time that the size-exclusion studies were being carried out, I also had occasion to pursue certain aspects of other favorite topics, bonded-phase packings (23) and high-performance preparative liquid chromatography ( 2 4 ) . My relationship with Joseph J. DeStefano in this latter work was particularly satisfying, since I had directed his research as a doctoral candidate at the University of Delaware on the same subject. In my past 23 years in chromatography my work always has been interesting, at times exciting. Chromatography has allowed me to travel to places that I probably otherwise would never have visited and to develop friends that I probably never would have met. My experiences in chromatography often have also been satisfying, in that the application of some of my efforts have been identified as useful. There still remains much to be done in chromatography and I’m looking forward to the challenge and the interesting experiences that will result. REFERENCES 1 J.J. Kirkland, in Gas Chromatography (1957 Lansing Symposium), V.J. Coates, H.J. Noebels and I . S . Fagerson, eds., Academic Press, New York, 1958, pp. 203-222.

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J.J. Kirkland, Anal. Chem. 32 (1960) 1388. J.J. Kirkland, Anal. Chem. 33 (1961) 1520. J.J. Kirkland, AnaZ. Chem. 35 (1963) 2003. J.J. Kirkland, in Gas Chromatography 1964 (Brighton Symposiwn), A. Goldup, ed., Inst. of Petroleum, London, 1965, pp. 285-300; see also J.J. Kirkland, Anal. Chem. 35 (1963) 1295; 37 (1965) 1458. J.C. Sternberg, in Advances i n Chromatography, Voz. 2 , J.C. Giddings and R.A. Keller, eds., M. Dekker, Jnc., New York, 1966, pp. 205-270. J.J. Kirkland, Anal. Chem. 40 (1968) 391. J.J. Kirkland, Anal. Chem. 41 (1969) 218. J.J. Kirkland, J . Chromatogr. S c i . 7 (1969) 7. J.J. Kirkland, J . Chromatogr. S c i . 7 (1969) 361. J.J. Kirkland, J . Chromatogr. S c i . 10 (1972) 129. J.J. Kirkland, J . Chromatogr. S C i . 8 (1970) 72. J.J. Kirkland, J . Chromatogr. S c i . 9 (1971) 206. J.J. Kirkland and J.J. DeStefano, J . Chromatogr. S c i . 8 (1970) 309. J.J. DeStefano and J.J. Kirkland, J . Chromatogr. Sci. 1 2

(1974) 337. 16 Modern Practice of Liquid Chromatography, J.J. Kirkland, ed. , Wiley-Interscience, New York, 1971. 17 J.J. Kirkland, J . Chromatogr. S c i . 10 (1972) 593. 18 L.R. Snyder and J.J. Kirkland, I n t r o d u c t i o n t o Modern Liquid Chromatography, Wiley-Interscience, New York, 1974. 19 J.J. Kirkland, in Gas Chromatography 1972 (Montrem Symposium) S.G. Perry, ed., Appl. Science Publishers, Barking, 1973, pp. 39-56. 20 D.D. Bly, H.J. Stoklosa, J.J. Kirkland and W.W. Yau, Anal. Chem. 47 (1975) 1810, 21 J.J. Kirkland, J . Chromatogr. 125 (1976) 231. 22 W.W. Yau, C.R. Ginnard and J.J. Kirkland, J . Chromatogr. 149 (1978) 465. 23 J.J. Kirkland, Chromatographia 8 (1975) 661. 24 J.J. DeStefano and J.J. Kirkland, Anal. Chem. 47 (1975) 1103 A , 1193 A.

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219

ANDREJ V. KISELEV

ANDREJ VLADIYIROVICH KISELEV was born i n 1908, i n Moscow, Russia. A f t e r h i s u n i v e r s i t y s t u d i e s he was a p p o i n t e d a s an a s s i s t a n t i n 1931, an a s s o c i a t e p r o f e s s o r i n 1935 and a p r o f e s s o r i n 1951 a t t h e M . V . Lomonosov S t a t e U n i v e r s i t y o f Moscow. Meanwhile he r e c e i v e d h i s c a n d i d a t e d e g r e e i n 1938 and t h e d o c t o r of chemical s c i e n c e s d e g r e e i n 1951. I n t h e 1 9 4 0 ' s he o r g a n i z e d t h e L a b o r a t o r y o f Adsorption ( s i n c e 1960: L a b o r a t o r y o f Adsorption and Chromatography) i n t h e Chemistry Department o f t h e Univers i t y . I n 1946 he a l s o j o i n e d t h e I n s t i t u t e of P h y s i c a l Chemistry o f t h e Academy of S c i e n c e s of t h e U.S.S.R. where, i n 1960, he o r g a n i z e d t h e Laboratory o f t h e Chemist r y of Surfaces. D r . K i s e l e v i s t h e a u t h o r and c o a u t h o r of o v e r 760 s c i e n t i f i c p a p e r s and seven books on t h e c h e m i s t r y o f s u r f a c e s and p o r o s i t y , thermodynamics and s p e c t r o s c o p y of a d s o r p t i o n from g a s phase and s o l u t i o n s , g a s and l i q u i d ( a d s o r p t i o n and e x c l u s i o n ) chromatography, i n t e r m o l e c u l a r i n t e r a c t i o n s and molecular s t a t i s t i c s of a d s o r p t i o n , and r e t e n t i o n i n m o l e c u l a r ads o r p t i o n chromatography. H i s book Gas Adsorption Chromatography w r i t t e n t o g e t h e r w i t h D r . Yashin was t r a n s l a t e d t o E n g l i s h , French and P o l i s h , and h i s book on Infrared Spectra of Surface Compounds c o a u t h o r e d w i t h D r . V.L. Lygin was t r a n s l a t e d i n t o E n g l i s h . I n 1974 D r . K i s e l e v r e c e i v e d one of t h e f i r s t M.S. T s w e t t Chromatography Medals. H e a l s o r e c e i v e d t h e t i t l e of Honored S c i e n t i s t o f t h e Russian S o v i e t F e d e r a t e d S o c i a l i s t Republic. D r . K i s e l e v ' s i n t e r e s t s are d i v i d e d between t h e t h e o r y o f s u r f a c e s , t h e t h e o r i e s of a d s o r p t i o n and chromatography and t h e i r a p p l i c a t i o n s . H e used chromatography n o t o n l y a s an a n a l y t i c a l method b u t a s a method f o r s t u d y i n g i n t e r m o l e c u l a r i n t e r a c t i o n s and t h e s t r u c t u r e o f molecules and a d s o r b e n t s .

In retrospect, I can summarize my activities in three groups: investigation of adsorbents, theory of the selectivity in adsorption (gas and liquid) chromatography, and investigations on liquid chromatography.

investigation of Adsorbents I started my activities in 1931 in the field of surface chemistry, porosity, thermodynamics of wetting and adsorption using static experimental methods, particularly vacuum and calorimetric techniques for the determination of adsorption isotherms and the heat of adsorption of gases and vapours. I have also studied the adsorption from solutions by static methods. Later, I used spectroscopic methods but also under static conditions. Although at the end of the 1930's and the beginning of the 1940's I was already interested in frontal chromatographic.methods, mainly as possible means for studying adsorption from solutions (i.e. in frontal liquid chromatography), I did not at that time use the chromatographic method itself in investigations. By the end of the 1940's and in the early 1950's we had definite successes in developing adsorbents with a nearly homogenous surface, in other words, adsorbents suitable for the elution-type gas chromatography. This was promoted by our investigations on the geometrical and chemical modification of porous and non-porous silica - my first paper on surface silanol groups was published in 1936 ( I ) - as well as by the results of Beebe, Smith and other American scientists on the graphitisation of carbon blacks and the investigation of their adsorption properties. (See the review (2).) In this connection I should mention the All-Union Conference on Chromatography held in 1950 where I reviewed our activities on the structure of silica gel and its influence on the adsorption properties (3). We continued our activities in this direction and in 1953, I gave a lecture on the "Influence of pore size and chemical nature of silica gel on the adsorption properties" ( 4 ) in which I summarised our investigations of the adsorbents suitable for chromatography although in these investigations the chromatographic method itself was not yet used. Thus, in this period we have studied and developed adsorbents suitable for chromatography but were using non-chromatographic methods in our investigations. I started to use chromatography around the middle of the 1950's and our first paper in this field, "Adsorption of hydrocarbons and the chromatographic separation of their mixtures and petroleum products" was published in 1957 ( 5 ) . The main attention in this paper was directed towards liquid adsorption, particularly to the displacement chromatography of hydrocarbon mixtures; besides this, the necessity of systematic investigations on the influence of the chemistry and geometry of the adsorbents' surface and of the nature and concentration of the sample components on the separation: "TO develop the theoretical concepts of adsorption (chromatographic) separation of hydrocarbon mixtures it is necessary to study these factors systematically, starting from the simplest system and gradually progressing to more and more complicated systems. On the other hand it

221 is necessary to systematize the work on the chromatographic separation of mixtures of pure hydrocarbons as well as of petroleum products in order to reveal the general, though qualitative regularities which then have to be investigated in detail by the chromatography of model systems”. In the reports presented in 1959 at the First All-Union Conference on Gas Chromatography (6) and in 1959 and 1961 at the Symposia held in the German Democratic Republic (7) we dealt with the nature of adsorption of hydrocarbons on graphite, oxides, hydroxides, chemically modified adsorbents and on adsorbents with gradually widening pore size, in other words the adsorbents which have by now found a broad application in the different versions of gas and liquid chromatography. In 1961 we studied gas chromatography on glass capillary columns with a chemically modified surface using wall treatment to increase the efficiency of separation after the deposition of silicone oil. At the ,same time investigations were also carried out with surfaceporous particles for packed columns and with glass capillary columns having a thin porous layer on the wall (8). This permitted us to shorten the analysis time and to improve the efficiency. By then it was clear to us that an adsorbent-type thin surface-modifying layer

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Fig. 24.1. Chromatograms obtained at 25OC: (a) Packed column containing surface-porous glass particles; (b) Packed column containing throughout-porous glass particles; (c) Porouswall glass capillary columns. Packed column: 100 cm x 4 mm, carrier gas (H2) flow-rate 68 ml/min. Capillary column: 10 m x 0 . 5 mm, porous-layer thickness 0.1 mm, carrier gas (N2) flow-rate 3.5 ml/min. Peaks: 1 = methane; 2 = ethane; 3 = ethylene; 4 = propane; 5 = propylene; 6 = n-butane; 7 = isobutane.

222

Fig. 24.2. During the 1962 Hamburg Symposium: (left to right), A . V . Kiselev, J.F.K. Huber and J. Janhk. serving as support must have - un ike the usual supports used in gas-liquid chromatography - sufficiently high specific surface area and that on the surface of such an adsorbent it is necessary to deposit the functional groups which provide high selectivity. "As a result of the modification the adsorbent must be not only weakened but, due to the functional groups of the modifier, it must also be sufficiently good for separation. If only 1% of the activities in GLC were directed for this purpose this would give good results" ( 7 ) . However at that time and even later the companies producing materials for chromatography continued to make hundreds of different stationary liquid phases, and only a few French (Guiochon et al.) and Italian (Bruner et al.) researchers - in addition to our laboratory - successfully utilized adsorption and chemical modification for the separation of complex mixtures (see ( 9 , Z O ) and references cited therein). Only the requirements of high-performance liquid chromatography which arose much later compelled many companies to turn to the geometrical and chemical modification of adsorbents, macromolecular sieves and supports (e.g. for the immobilisation of enzymes). In our laboratory, Yu.S. Nikitin et al. prepared a series of nacroporous silicas (aerosils, silochroms, and macroporous silica gels) with pore sizes up to 1 pm, N.V. Kovaleva et al. prepared carbochroms (macroporous carbon particles having properties close to those of graphitized thermal carbon blacks but mechanically more stable), while T.B. Gavrilova et al. prepared different diatomaceous earth-type supports with extreme macroporousity, as well as metal sulfides. We also carried out the chemical and adsorption modifica-

223

6

LAI8

22 26

30min

i

3

h

Cmin

Fig. 24.3. Chromatograms obtained on surfacemodified adsorbents. (a) Silochrom with a polymer film obtained on its surface. Column length: 250 cm, temperature: S O W , carrier gas (N2) flow-rate: 16 ml/min. Peaks: 1 = 1,2,2-trifluoro-1,1,2-trichloroethane; 2 = 1,2-dibromotetrafluoroethane; 3 = chlorallylene; 4 = 1,1,2-trifluoro-2-chloro-l-bromoethane; 5 = l,l,l-trifluoro-2-chloro-2-bromoethane (halothane); 6 = l,l,l-trifluoro-1,2-dibromochloroethane. (b) Graphitized thermal carbon black with an adsorbed dense monolayer of the sodium salt of sulphonated cobalt phthalocyanine. Column: 120 cm x 3 mm, temperature: 62OC, carrier gas (N2) flowrate: 40 ml/min. Peaks: 1 = cyclopentane; 2 = cyclopentene; 3 = cyclopentadiene. tion of these adsorbents, particularly by depositing on their surface different quantities (up to a dense monolayer) of salts of sulphonated cobalt phthalocyanines, various thin polymer and pyrocarbon films (see ( 9 , Z O ) and references cited therein). We also worked with different porous and macroporous organic copolymer adsorbents. Together with Yashin and others we have investigated the structure, adsorption and chromatographic properties of such adsorbents while with Belyakova, Muttik and others we have utilized the hydrophobic character of the matrix of macroporous copolymers for the adsorption of acidic gases ( C o p , SO2) from water containing dissolved atmospheric gases. We also studied the geometric structure of the macroporous matrix and the chemical nature of grafted base-type functional groups. In the purification of air it is important to have the high adsorption values as well as the ease of re-

224

0

>

4

Fig. 24.4. Frontal chromatogram of C 0 2 and SO2 on macroporous polymers containing functional groups of basic character (as indicated in the figure). The length of the adsorbent layer in the column was 5 cm with 1.6 cm i.d. Temperature: 2OoC, flow-rate: 220 ml/min. The concentration of the two compounds in the carrier gas (N2) was 2 ~01%. generation. In the case of COP both conditions are fulfilled by grafting ethylene diamine and some other polyamine groups; on the other hand, in the case of SO2 it is better to graft monoethanol amine and some other weaker base groups.

Theory of S e l e c t i v i t y i n Adsorption GC and LC In the 1930's we were already interested in the connection between the energy of adsorption or wetting and the structure of the adsorbent and the adsorbate molecules. However from the middle of the 1950's we restricted ourselves to the experimental determination of heats of adsorption and to thermodynamic calculations. By that time Iliin, Barrer and others had already published papers in which attempts were made to calculate the potential energy of adsorbate-adsorbent intermolecular interactions in adsorption, @, but only for simple molecules. In 1956 we carried out the calculations of @ for a series of hydrocarbons on graphite (11). The groups CH3, CH2, CH and -C- as well as the C atoms in graphite lattice were' taken as the force centers; in other words, at that stage we used for @ a group-atom approximation. At the beginning of the 1960's we began molecular-statistical calculations of the thermodynamic characteristics of the retention of different hydrocarbons on graphitized thermal carbon black (GTCB) In 1965 D.P. Poshkus gave a simple molecular-statistical expression for the retention volume at zero sample size ( 1 2 ) . Very soon after this publication we began to use for Q, a more convenient atom-atom approximation. At this time we understood that the combination of

225

Fig. 2 4 . 5 . Dependence of the Henry constant K1 (equal to retention volume per unit surface area of the adsorbent, A = 1 and zero sample size, V A , ~ ,ml/m2) on the temperature for adamantane and diamantane, on graphitized thermal carbon black. Dots represent chromatographic measurements while the plots correspond to calculated data. gas chromatography (i.e,, experimental determination of the Henry constant, K1 in a rather wide temperature range for a few representative hydrocarbons of different classes) with the molecular-statistical theory of adsorption and.the theory of intermolecular interactions in atom-atom approximation, = 10, 'permits us to find the atom-atom potential functions @c...c and @H...c of the iatermolecular interaction of the carbon and hydrogen atoms of the hydrocarbons with the carbon atoms of graphite. As described in our papers, the comparison of the results of theoretical calculations Kl(4) with chromatographic measurements for ethane, ethylene, acetylene and benzene permitted us to introduce the correction factor $ into the results of approximate quantummechanical calculations of dispersion attraction constants. This then permitted investigation of how the electron configuration of the carbon atoms in a hydrocarbon molecule influences the f3 value ( 1 3 , 1 4 ) . Later, Guiochon and coworkers carried out the molecular-statistical calculations of K 1 . The accumulation of more accurate chromatographic data on K 1 values in our laboratory by Scherbakova, Kalashnikova and others and in Guiochon's laboratory, in Paris, by Vidal-Madjar and others, as well as the more accurate determinations of the atom-atom potential functions @c...c (for different electron configurations of the carbon atom) and @H...C made it possible for us to predict the separation of a number of isomers of complex hydrocarbons ( 1 5 , 1 6 ) and to calculate the K 1 values at different temperatures for complex hydrocarbons, in good agreement with chromatographic measurements.

To go further it is necessary to heighten the precision of chromatographic measurements and to improve the model of @.However, it is now already possible not only to calculrle the K1 values knowing the structure of the molecule but also to solve the reverse problem, that is to use the gas chromatographic measurements of K1 at different temperatures on GTCB together with the theory of intermolecular interactions in atom-atom approximation as a method of investigation of the molecular structure itself. The determination of the deflection angle of the CH3 groups from the benzene ring plane in hexamethylbenzene (15), which is in accordance with electronographic data, may be mentioned as an example. Adsorption on ionic adsorbents, especially on porous zeolite crystals, is a more complicated problem both for the gas chromatographic measurements of K 1 and for its molecular-statistical calculation. Investigations in both directions were carried out in our laboratory (K.D. Shcherbakova, A . A . Lopatkin et al.). The porous crystal symmetry, the replacing of integration by summation and use of atom-ion approximation permit us to calculate the K1 values for alkanes ( 2 7 , 1 8 ) and cyclanes (taking into account the polarisation of the molecule by the electrostatic field created by zeolite ions), for nitrogen, oxygen, argon, carbon dioxide and ethylene (28) (taking also into account the quadrupole orientation contribution to @ ) . It is interesting to note that in the case of adsorption of cyclopropane on zeolite (in much higher degree than on GTCB ( 2 3 ) ) the gas chromatographic K1 values are greater than the values calculated for sp' electron configuration of carbon atoms in the molecule. Here the reverse problem was also investigated and the value of "effective" quadrupole moment of the cyclopropane molecule was determined in correspondence with what organic chemists sometimes call the "aromatic" character of this molecule.

Investigations in Liquid Chromatography Besides investigations on the frontal chromatography of liquid hydrocarbons and on selective adsorbents and macromolecular sieves for liquid chromatography we paid special attention to the connection between liquid adsorption chromatography and the adsorption from solutions. In the studies carried out with Yu.S. Nikitin, Ya.1. Yashin and others we have found the energetical proofs that the same substance can adsorb positively on both non-specific and specific o r , so-called, bifunctional adsorbents, if we correspondingly regulate the intermolecular interactions of the components to be separated with an eluent ( 1 9 ) . This permits the separation on the basis of the nature of functional groups as well as on the basis of the molecular mass particularly in the case of adsorbents with deposited pyrocarbon (20). We can use liquid chromatography to separate non-stable metalorganic compounds and to study the reaction directions of these compounds ( 2 1 ) The studies carried out together with V.Ya. Davydov and others on the liquid chromatographic separation of heart glycosides (22) showed that at the same structure of the aglycon part of these molecules the retention time decreases with the increase of their sugar type-part. These studies also showed the importance of column temperature regulation.

227

4

1.

2.

12

,J

3. 4.

on

8

0

6

12

OH

du

T,min

Fig. 24.6. Chromatogram of heart glycosides, at 5OoC. Column: 25 cm x 3 mm packed with 7 pm particles of LiChrosorb. Eluent: 7:3 water-ethanol, flow-rate: 0.6 ml/min. UV detector at 217 nm. Glycoside 1 has the most hydrophobic aglycone part of the molecule; glycosides 2, 3 and 4 have the same aglycone part but differing from that of glycoside 1; glycosides 5 and 6 also have the same aglycone part but now differing from that of the other glycosides. In the field of liquid chromatography of macromolecules, I only want to mention the paper authored together with Riabinina and Eltekov (23). In this we showed the possibility of polymer separation according to their molecular masses using the molecular sieving (size exclusion) effect on organic modified silica as well as the effect of adsorption which increases with the molecular mass of a linear polymer in the case of hydrogen bonding of its functional groups with the hydroxylated silica surface. Today, the molecular theory of selectivity in liquid-solid chromatography is only at its beginning ( 2 4 ) . Further progress depends on the possibility of working out simplified molecular models and being able to carry out sufficiently accurate determinations of the .H atom-atom potential function.

228

Fig. 24.7. A.V. Kiselev (right) in a discussion during a lecture in Leipzig, Fall 1975. Seated, with glasses: W. Engewald. Finally I would also like to mention the attempts I have made to classify adsorbents by their geometric structure and the intermolecular actions ( 9 ) . The latter method has recently been extended to the classification of the various chromatography techniques as well (25).

Persona1 Zontacts I cannot finish my recollections without specifically mentioning the very close and valuable contacts I had with many well known chromatographers, particularly with Professor A.I.M. Keulemans (from 1959 until his recent death) and with Professor Guiochon (since 1962) both being interested not only in the analytical aspects of chromatography but also in its thermodynamic aspects. REFERENCES 1 A.V. Kiselev, KoZloidn. Zh. 2 (1936) 17. 2 A . V . Kiselev, in Advances in Chromatography, Voz. 4, J.C. Giddings and R.A. Keller, eds., Y . Dekker, Inc., New York, 1967, p. 113. 3 A . V . Kiselev, in Issledovanija v Oblasti Khromatografii (Investigations in the Field of Chromatography), U.S.S.R. Academy of Sciences Press, Moscow, 1952, p. 71. 4 A . V . Kiselev, in Trudy Komissii po A n u l i t i c h e s k o j Khimii (Transactions of the Commission on Analytical Chemistry), U . S . S . R . Academy of Sciences Press, Moscow, 1955, vol. 6, p. 46.

229

i Svoistva N e f t e j i Benzokerosinovikh Frakcij (Composition and Properties of Oils and

5 A.V. Kiselev and E.A. Mikhailova, in Sostav

Gasoline Fractions), U.S.S.R. Academy of Sciences Press, Moscow, 1957, p. 35. 6 A.V. Kiselev, in Gazovq’a Khromatografija, Trudy I-oi Vsesojusnoj Conferencii PO Gazovoy Khromatografii (Fevral 1959) (Gas Chromatography, Transactions of the First All-Union Conference on Gas Chromatography (February, 1959)), U.S.S.R. Academy of Sciences Press, Moscow, 1960, p. 45. 7 A.V. Kiselev and K.D. Schtscherbakova, in Gas Chromatographie, Abhandlungen der Deutschen Akademie der Wissenschaften zu Berlin, Akademie-Verlag, Berlin, 1962: Part1 (1959) p. 207, Part I1 (1961) p. 241, Part 111 (1961), p. 263. The quotation is from p. 284. 8 A.V. Kiselev, in Gas Chromatography 1962 (Hamburg Symposium), M. van Swaay, ed., Butterworths, London, 1963, p. XXXIV. 9 A.V. Kiselev and Ya.1. Yashin, Gas Adsorption Chromatography, Plenum Press, New York, 1962; La Chromatographie Gas-Solide, Masson, Paris, 1969. 10 A.V. Kiselev and Ya. I. Yashin, Ads0rbcionnq.a Gazovq’a i Zhidkostnw*a Khromatografija (Adsorption Gas and Liquid Chromatography), Khimija Press, Moscow, 1978. 11 A.V. Kiselev, Proceedings of t h e Second International Congress on Surface A c t i v i t y , Butterworths, London, 1957, vol. 2, p. 168. 12 D.P. Poshkus, Zh. Fiz. Khim. 39 (1965) 2962; D.P. Poshkus and A.J. Afreimovitch, J . Chromatogr. 58 (1971) 55. 13 N.N. Avgul, A.V. Kiselev and D.P. Poshkus, Adsorbcija Gazov i Parov na Odnorodnikh Poverkhnostjakh (Adsorption of Gases and Vapours on Homogeneous Surfaces), Khimija Press, Moscow, 1975. 14 A.V. Kiselev and D.P. Poshkus, J . Chem. SOC. Faraday Trans. 11 72 (1976) 950. 15 A.V. Kiselev, D.P. Poshkus and A.J. Grumadas, J . Chem. SOC. Faraday Trans. 11 74 (1978). 16 W. Engewald, E.V. Kalashnikova, A.V. Kiselev, R.S. Petrova, A.L. Shilov and K.D. Scherbakova, J . Chromatogr. 152 (1978) 453. 17 A.G. Bezus, A.V. Kiselev, A.A. Lopatkin and Pham Quang Du, J . Chem. SOC. Farady Trans. 11 74 (1978) 367. 18 A.G. Bezus, A.V. Kiselev and Pham Quang Du, Doklady Akad. Nauk SSSR 237 (1977) 126; A.V. Kiselev and Pham Quang Du, Dokl. Akad. Nauk SSSR 238 (1978) 128. 19 N.K. Bebris, R.G. Vorobieva, A.V. Kiselev, Yu.S. Nikitin, L.V. Tarasova, 1.1. Frolov and Ya.1. Yashin, J . Chromatogr. 117 (1976) 257. 20 N.K. Bebris, A.V. Kiselev, Yu.S. Nikitin, 1.1. Frolov, L.V. Tarasova and Ya.1. Yashin, Chromatographia 11 (1978) 206. 21 G.N. Bortnikov, T.N. Brevnova, A.V. Kiselev, N.P. Makarenko, N.F. Tcherepennikova and Ya.1. Yashin, J . Chromatogr. 124 (1976) 337. 22 V.Ya. Davydov, A.V. Kiselev, I.V. Mironova and Yu.M. Sapoznikov, Chromatographia, to be published; 23 A.V. Kiselev, T.I. Riabinina and Yu.A. Eltekov, DokZ. Akad. Nauk SSSR 200 (1971) 1132.

230 A.V. Vernov, A.V. Kiselev and A.A. Lopatkin, Vestnik Moskovskogo Gosudarstvennogo Universiteta, S e r i j a 2, Khimija (Trans. of Moscow State University, Ser. 2, Chemistry) 18 (1977) 350. 25 A.V. Kiselev, Chromatographia 11 (1978) 117. 24

231

ERVIN s.KOVLTS

ERVIN s z . KOViTS was born i n 1927, i n Budap e s t , Hungary. H e f i r s t s t u d i e d a t t h e Techn i c a l U n i v e r s i t y o f Budapest, g r a d u a t i n g i n 1949 a s a chemical e n g i n e e r . Soon a f t e r , he l e f t h i s n a t i v e c o u n t r y and moved t o Switz e r l a n d , where he had t o r e p e a t p a r t of h i s u n i v e r s i t y s t u d i e s i n o r d e r t o have h i s d e g r e e a c c e p t e d ; i n 1951 he r e c e i v e d from t h e E i d g e n o s s i s c h e Technische Hochschule (ETH) - t h e Swiss F e d e r a l I n s t i t u t e o f Technology - i n Z u r i c h t h e chemical e n g i n e e r i n g diploma and two y e a r s l a t e r t h e d o c t o r a t e . H e continued h i s a s s o c i a t i o n w i t h t h i s u n i v e r s i t y a s a member o f t h e Lab o r a t o r y o f Organic Chemistry and l a t e r of t h e L a b o r a t o r y o f Organic Chemical Technology u n t i l 1967, when he moved t o t h e Ecole P o l y t e c h n i q u e F b d b r a l e i n Lausanne.At p r e s e n t he i s p r o f e s s o r a t t h i s u n i v e r s i t y . Dr. Kovhts is t h e a u t h o r and c o a u t h o r o f o v e r 50 s c i e n t i f i c p u b l i c a t i o n s . I n 1969, he s e r v e d a s t h e chairman o f t h e S c i e n t i f i c Committee o r g a n i z i n g t h e v e r y s u c c e s f u l Symposium on Column Chromatography h e l d i n Lausanne, October 7-10. I n 1977 he r e c e i v e d t h e M.S. T s w e t t Chromatography Medal. The f i r s t work of Dr. Kovhts a t t h e ETH was on h e t e r o g e n o u s c a t a l y s i s i n t h e g a s phase and t h e c a l o r i m e t r y o f combustion. Toward t h e end of 1955 he became i n v o l v e d i n p r e p a r a t i v e and a n a l y t i c a l g a s chromatography and i n t h e i d e n t i f i c a t i o n o f t h e components i n complex mixt u r e s , mainly e s s e n t i a l o i l s . H e p l a y e d a key r o l e i n d e v e l o p i n g a f u l l y a u t o m a t i c p r e p a r a t i v e g a s chromatograph. Dr. Kovhts i s b e s t known f o r h i s a c t i v i t i e s i n d e v e l o p i n g t h e r e t e n t i o n i n d e x system f o r gas chromatographic i d e n t i f i c a t i o n o f i n d i v i d u a l compounds, H e showed t h e c o r r e l a t i o n of t h i s system w i t h v a r i o u s chem i c a l and p h y s i c a l c h a r a c t e r i s t i c s o f t h e of t h e s u b s t a n c e s , and t h e p o s s i b i l i t y o f u s i n g t h i s system f o r t h e c h a r a c t e r i z a t i o n o f t h e pol a r i t y of s t a t i o n a r y p h a s e s . Recognizing t h e importance of w e l l - d e f i n e d and s t a b l e s t a t i o n a r y p h a s e s , Dr. Kovhts h a s devoted most o f h i s r e c e n t a c t i v i t i e s t o t h e development o f such m a t e r i a l s .

232 In 1953, when I finished my doctoral work with Hans H. Gunthard, Leopold Ruzicka was the head of the Laboratory of Organic Chemistry at the Eidgenossische Technische Hochschule (ETH), the Swiss Federal Institute of Technology, in Zurich. Dr. Ruzicka was one of the 1930 laureates of the Nobel Prize in Chemistry, which he received for his work on terpenes and sex hormones. At the agepof 66 years, he no longer worked in the laboratory, but directed his institute with an astonishing drive and vitality, looking for new lines of research and for young workers whom he could charge with such projects. My training with Hans H.Giinthard (at present head of the Physical Chemistry Institate) had been a mixture of organic and physical chemistry. After my degree I continued to work with him. In the fall of 1955, when I was about to join the Koninklijke/ Shell research laboratory in Amsterdam, Ruzicka asked me to come to his office. First he explained that he didn't think I should go to Shell, and that he had a project for me entitled "Essential Oils". It consisted of applying and adapting physico-chemical methods to the analysis of complex mixtures: "You have one hour to think it over", he said. By the time I came back to his office to give him my answer, the financing of the project had already been arranged with the firm of Firmenich in Geneva, and the next day I moved to the "private laboratory" next to his office and began working. The first move was the elaboration of a working plan. Having read the papers of A.J.P. Martin on chromatography, it was clear that we should utilize his results. It was therefore planned to apply classical liquid chromatography to the separation of the oils in sub-groups, preparative gas chromatography to the separation of components and analytical gas chromatography to the examination of the isolated fractions. As the first test mixture, mandarin-peel oil, a relatively cheap citrus oil, was chosen. It was planned to apply spectroscopic methods in order to distinguish the structure of the isolated pure components. The final aim was to analyse the "queen of essential oils", oil of roses. Today, this plan would seem obvious. It was not so, however, at that time. A gas chromatograph was not available, and NMR spectroscopy was not a standard analytical tool. However, the work in the introductory period was greatly facilitated by the help of Edgar Heilbronner (now professor of physical chemistry at Base1 University) and Willi Simon (now professor at the ETH in Zurich). The initiative for the project, automated preparative gas chromatography, came from Heilbronner while all plans for the automation were from Willi Simon. A few months after the beginning, Adolph Wehrli entered my group, wanting to prepare a doctoral thesis. Thus, the actual working group consisted of Ernst Baumann, an excellent laboratory technician, Adolph Wehrli and myself. After an introductory period we had our apparatus working. Because the first gas chromatograph was oil-thermostated, we sometimes had to clean up a few kilograms of oil from the basement, and as a result we soon changed to an air thermostat. The preparative gas chromatograph became a sort of monster, but it worked ( I ) . Very soon we arrived at a first subdivision of the oil of mandarin. It was

233 first separated into groups by a type of displacement chromatography on silica gel, then every sub-group was divided into distillation fractions. (It is interesting to note, that we subdivided the oil on a silica gel partly deactivated by polyethylene glycol, a method advocated today for the treatment of gas chromatographic supports).

Fig, 25.1. View of the preparative gas chromatograph developed by us in 1955-1956 ( I ) . We systematically characterised these fractions by gas chromatography on two stationary phases, Apiezon L and a polyethylene glycol. First we applied the relative retention method to peak positions, but we had to use different standards for the different fractions, making a general view and comparison of the some fifty fractions not impossible, but very difficult. As the number of fractions was multiplied when we began to isolate substances by preparative gas chromatography, it became clear that we needed a better system for defining peak positions. This topic became the subject of Wehrli's doctoral thesis.

234 For a system of characterization, we first looked for formal or real analogies adaptable to our problem. The centigrade temperature scale seemed to be a good example. Originally, this scale was based on two fixed points, the melting and boiling temperature of water. From a thermodynamic point of view there was no need of two fixed points, and the temperature scale was defined by attributing a given value to the triple point of water (-273.1600 OK). Therefore we first tried to take only one fixed point by taking the retention volume of a unique substance, n-decane, as standard for every temperature. It was very soon realized that secondary standards were needed, and so we adopted the n-alkanes. By using the nearly linear relationship between the carbon number and the logarithm of the retention volume of n-alkanes, the logarithm of the retention relative to decane, r l 0 ,could be calculated from the retention relative to a secondary standard. The logarithm was then multiplied by the absolute temperature, pV = T log r l 0 , in order to diminish and linearize the temperature dependence of the "pv values" ( 2 ) . This system, however, had several disadvantages. Its main drawback was that the retentions of all paraffins relative to decane had to be determined in advance with as high a precision as possible and therefore the p v value for every substance changed with a new, more precise, determination of the data for paraffins. Therefore, we came back to the more primitive centigrade analogy of using two fixed points. As fixed points we took the elution time of two consecutive paraffins and divided the distance between them on a logarithmic scale into one hundred parts. We thus had a series of standards which could be used at every temperature. I remember our excitement with Wehrli when we recalculated all of his data in the new system. All at once we could very easily deduce rules for the prediction of "retention indices" as we named our new system. The temperature dependence was nearly negligible so we could compare chromatograms made at different temperatures. The system was exactly what we needed for our analytical work. This is the origin of the retention index system ( 3 - 5 ) . Like most of our work, this was initially also published in a Swiss journal, in German; this might have been the reason that for some time, not too many laboratories utilized this system. The situation, however changed around the middle of the 1960's and I am happy to note that today, the retention index system is almost universally accepted as an expression of relative retention for both qualitative identification and liquid phase characterization. With the aid of the retention index system, we soon finished the analysis of the mandarin-peel oil (6) and of the oil of lime ( 7 ) , and we began to work on the much more complex oil of roses. This latter work was unfortunately not released for publication by Firmenich, with the exception of synthetic studies of a few new components ( 8 - 9 ) . Nevertheless, with the aid of the damascenon (10) it was possible to prepare a synthetic oil of roses of nearly the same odor as the natural one. A s a student I swore that if there was one branch of chemistry on which I would certainly never work, it was analytical chemistry,

235

Fig. 25.2. Chromatogram on a wide-bore stainless steel capillary ( 3 1 ) . Column: 100 m x 2 mm I.D., Apiezon L; column temperature: 130.7 OC; carrier gas: helium, 25 ml/min; Sample amount: 0.1 mg; katharometer detector; number of theoretical plates: 35,000. Peaks: l=p-cymene; 2=limonene. This column had excellent properties; primary alcohols and even phenols showed no adsorption, and retention indices were the same as on packed columns. Our deactivation procedure consisted of etching the tubing with K2Cr207/conc. sulfuric acid, and then washing with Na2HC0,. All of our quantitative analyses of essential oils were made by using this column which we were never be able to reproduce: During my doctoral thesis I excluded surface chemistry as a second topic. Today in our group we are working exactly in these two fields! However, it is also true that the appearance of analytical chemistry has changed profoundly, mainly because of the introduction of gas chromatography and NMR spectroscopy, two methods which have given an important impetus to the evolution of analytical chemistry. It has also become an interdisciplinary subject, a combination of applied organic and inorganic chemistry together with applied physical chemistry. Modern organic chemistry and progress in medical research could not exist without modern analytical chemistry. In my opinion, the interaction should be even more important. To take chromatography as an example, it is to be regretted that for the time being organic chemists use chromatography purely as a separation method, and do not profit from the knowledge which is acquired by chromatographers concerning molecular interactions.

236 In such a historical review one should also think of Fortuna; without her help, the research would not have been possible. At the very beginning of our work, for example, we chose a hydrocarbon and a polyethylene glycol as the stationary phases - and both types are widely used today. I also had the good luck to have skilled and enthusiastic coworkers without whom much of the work would not have been possible. REFERENCES 1 E.sz. Kovlts and E. Heilbronner, Chimia 10 (1956) 288.

E. Hei,lbronner,E.sz. Kovhts and W. Simon, Helv. Chim. Acta 40 (1957) 2410. 3 E.sz. Kovkts, Helv. Chim. Acta 4 1 (1958) 1915. 4 A . Wehrli and E.sz. Kovlts, Helv. Chim. Acta 42 (1959) 2709. 5 E.sz. Kovgts, Z. Anal. Chem. 181 (1961) 351. 6 E. Kugler and E.sz. Kovlts, Helv. Chim. Acta 46 (1963) 1480. 7 E.sz. Kovits, Helv. Chim. Acta 46 (1963) 2705. 8 G. Ohloff, W. Giersch, K.H. Schulte-Elte and E.sz. Kovlts, Helv. Chirn. Acta 5 2 (1969) 1531. 9 G. Buchli, E.sz. Kovhts, P . Enggist and G. Uhde, J . o r g . Chem. 33 (1968) 1227. 10 E. Demole, P . Enggist, U. Sauberli, M. Stoff and E.sz. KovPts, HeZv. Chim. Acta 5 3 (1970) 541. 11 H. Srickler and E.sz. Kovits, J . Chromatogr. 8 (1962) 289. 2

237

EDGAR LEDERER

EDGAR LEDERER was born i n 1908, i n Vienna, A u s t r i a . H e s t u d i e d c h e m i s t r y a t t h e Univ e r s i t y of Vienna r e c e i v i n g h i s Ph.D. i n 1930. From September 1930 onwards he worked a t t h e K a i s e r Wilhelm I n s t i t u t f i i r Medizin i s c h e Forschung, i n H e i d e l b e r g , Germany, under t h e d i r e c t i o n o f P r o f e s s o r Richard Kuhn. A f t e r t h e advent o f Nazism i n Germany, he went t o France where he worked f o r two years i n various research laboratories. In 1935-1937 he was r e s e a r c h d i r e c t o r a t t h e Vitamin I n s t i t u t e o f L e n i n g r a d , U.S.S.R. I n 1938, he r e t u r n e d t o F r a n c e , was appoint e d an " a t t a c h 6 de r e c h e r c h e s " a t t h e C e n t r e N a t i o n a l de l a Recherche S c i e n t i f i q u e (C.N.R.S.) and s t a r t e d t o work a t t h e Ecole Normale S u p b r i e u r e . Being a n a t u r a l i z e d French c i t i z e n , he was m o b i l i z e d a t t h e o u t b r e a k of t h e Second World War, b u t was a b l e t o l e a v e t h e French Army i n March 1940, subsequent t o h i s w i f e g i v i n g b i r t h t o t h e i r fourth child. After the armistice,he joined Professor Fromageot, i n Lyon. Having s u r v i v e d t h e bombing o f t h e l a b o r a t o r y and v a r i o u s p o l i c e and Gestapo r a i d s he f i n a l l y moved t o P a r i s a g a i n i n 1947. Meanwhile he was a p p o i n t e d Martre de Recherches a t t h e C.N.R.S. i n 1945, and he became D i r e c t e u r de Recherches i n 1952. In 1954, he s t a r t e d t o t e a c h a t t h e Sorbonne and became f u l l p r o f e s s o r o f biochemist r y i n 1958. T h i s c h a i r was t r a n s f e r r e d i n 1963 w i t h p a r t o f t h e a s s o c i a t e d team t o t h e F a c u l t b d e s S c i e n c e s a t Orsay. S i n c e 1960, he h a s a l s o been t h e d i r e c t o r o f t h e I n s t i t u t de Chimie d e s S u b s t a n c e s Natur e l l e s du C.N.R.S. a t Gif-sur-Yvette. D r . L e d e r e r h a s p u b l i s h e d o v e r 300 o r i g i n a l p a p e r s d e a l i n g w i t h v a r i o u s a s p e c t s of chromatography and i t s a p p l i c a t i o n s , and w i t h i n v e s t i g a t i o n s o f n a t u r a l p r o d u c t s . H e a l s o a u t h o r e d and c o a u t h o r e d s e v e r a l books on chromatography, c a r o t e n o i d s and l i p i d b i o c h e m i s t r y ; t h e French monograph p u b l i s h e d i n 1949-1952 and i t s E n g l i s h e d i t i o n p u b l i s h e d i n 1955 w r i t t e n i n c o o p e r a t i o n w i t h M. L e d e r e r , h i s c o u s i n , b e l o n g t o t h e c l a s s i c s o f chromatography. D r . L e d e r e r h a s r e c e i v e d a number o f awards f o r h i s achievements. H e i s a c h e v a l i e r o f t h e Le'gion d'Honneur and an o f f i c e r o f t h e Ordre N a t i o n a l du M b r i t e ; he h a s honorary d o c t o r a t e s from t h e U n i v e r s i t i e s o f Aberdeen ( S c o t l a n d ) and Liege (Belgium) and h a s been e l e c t e d as t h e

238 member o f t h e academies o f s c i e n c e s o f a number o f c o u n t r i e s , among them t h e Royal I r i s h Academy, t h e German L e o p o l d i n a , t h e A u s t r i a n Academy o f S c i e n c e , t h e Academia P a t a v i n a (Padova), t h e I s t i t u t o Veneto d e l l e A r t i e S c i e n z e ( V e n i c e ) , and t h e Accademia d e l l e S c i e n z e F i s i c h e e Matematiche ( N a p l e s ) , and a l s o of v a r i o u s s c i e n t i f i c s o c i e t i e s . H e r e c e i v e d t h e F r i t z s c h e Award and t h e g o l d medal of t h e American Chemic a l S o c i e t y ( 1 9 5 1 ) , two awards from t h e Acadhmie d e s S c i e n c e s o f P a r i s (1950 and 1 9 6 0 ) , t h e August Wilhelm von Hoffmann g o l d medal of t h e German Chemical S o c i e t y ( 1 9 6 4 ) , t h e P a u l K a r r e r g o l d medal of t h e Swiss Chemical S o c i e t y ( 1 9 6 4 ) , t h e g o l d medal o f t h e C e n t r e N a t i o n a l de l a Recherche S c i e n t i f i q u e (1974) and t h e M.S. T s w e t t Chromatography Medal (1976). H e i s on t h e e d i t o r i a l b o a r d o f Tetrahedron, Phytochemistry, Lipids, Nouveau Jow-naZ de Chimie and t h e Journal of Chromatography. D r . Lederer's involvement i n chromatography s t a r t e d i n 1930, i n H e i d e l b e r g , where he i n t r o d u c e d T s w e t t ' s c h r o m a t o g r a p h i c method t o t h e i n v e s t i g a t i o n of n a t u r a l s u b s t a n c e s . H e c o n t i n u e d h i s i n t e r e s t i n t h i s t e c h n i q u e e v e r s i n c e . I n a d d i t i o n , he h a s been a c t i v e i n v a r i o u s f i e l d s o f g l y c o l i p i d s , b a c t e r i a l l i p i d s , n a t u r a l p i g m e n t s , mass s p e c t r o m e t r y of p e p t i d e s , m y c o b a c t e r i a l c e l l w a l l s , immunostimulants, b i o l o g i c a l t r a n s m e t h y l a t i o n r e a c t i o n s , b i o l o g i c a l problems r e l a t e d t o n a t u r a l subs t a n c e s o f p l a n t , animal and m i c r o b i a l o r i g i n .

239 Having obtained n;y Ph.D. at the University of Vienna, in July 1930, with a thesis on the structure and synthesis of indole alkaloids, prepared under the direction of Professor Ernst Spath, I arrived in Heidelberg in September, for postdoctoral work in the Laboratory of Chemistry of the Kaiser Wilhelm Instltut fur Medizinische Forschung, directed by Professor Richard Kuhn, the former pupil of Richard Willstatter who, in 1915, had received the Nobel Prize in Chemistry*. Kuhn and his assistant Alfred Winterstein had just published a paper on the chemical similarity of the pigments of egg yolk and the yellow '*xanthophy1lst1 of green leaves, previously studied by Willstatter. The local newspapers had referred to this work with sensational headlines: "Heidelberg Chemists Produce Egg Yolk From Grass ! '' The reality was, of course, somewhat different: the structure of natural carotenoids was just starting to be defined chemically, following the synthetic work of Kuhn and Winterstein at the Eidgenossische Technische Hochschule (ETH) in Ziirich (Kuhn was a professor at this university prior to accepting the position at Heidelberg-) on diphenyl-polyenes and the parallel work of Professor Paul Karrer at the University of Ziirich. Only two hydrocarbons (both C 4 ~ H s 6 )were known at that time, carotene, the pigment of carrots, crystallised for the first time by Wackenroder in 1831, and lycopene, the red pigment of tomatoes described in 1911 by Willstatter and Escher. Carotene had also been crystallized from green leaves by Arnaud in Paris in 1889, and in 1907 Willstatter and Mieg had proposed the formula C L , ~ H ~ for ~ Othe ~ xanthophylls, hydroxylated carotenoids isolated from green leaves and for "lutein", the pigment of egg yolk; in 1929, Karrer isolated an isomeric pigment, zeaxanthine, from yellow corn. In Kuhn's lab there were two marvelous new instruments: a visual spectrophotometer with which the absorption bands of carotenoids could be measured rather accurately, and a polarimeter with a quartz-cadmium lamp, emitting a strong Cd-line at 643 nm which allowed the determination of optical rotations of the strongly yellow, orange or red solutions of carotenoids. I started to work with these instruments, checking absorption spectra, and optical rotations (and melting points) of various preparations of carotene from carrots, lutein from egg yolk and xanthophylls from leaves. For the latter pigments, rotations varied from +136O to +192O; zeaxanthine had distinctly different properties, and lutein seemed to lie between the two:

.

* Kuhn himself received the Nobel Prize for Chemistry in 1938, for his work on carotenoids and vitamins.

xx When in 1926 the ETH in Ziirich asked Willstatter to come back as Professor of Chemistry, he replied "I recommend to you my pupil Richard Kuhn, 26 years old, who is much better than I"; so Kuhn became full professor in Zurich at 27; two years later he moved to Heidelberg.

240

m. p. Xanthophyll from green leaves Lutein from egg yolk Zeaxanthine from yellow corn

173-174O 195- 196O 201-2020

+136-192O + 720 - 70'

Fig. 26.1. A happy day in Kuhn's laboratory in Heidelberg, sometime in 1931. Identified persons are: l=R. Kuhn; 2=Mrs. Kuhn; 3=A. Wassermann (later emigrated to England); 4=H. Brockmann (became professor at Gottingen); 5=Miss G. Stein (became Mrs. Brockmann); 6=M. Hoffer (emigrated to the U.S.A. and joined Hoffmann-La Roche); 7=Th. Wagner-Jauregg (emigrated to the U.S.A, but later returned to Switzerland); 8=H. Roth (head of Kuhn's analytical laboratory; became professor at Greifswald and Braunschweig); and 9=E. Lederer. One day, Kuhn suggested to me that perhaps the lutein of egg yolk could be a mixture of leaf xantophylls with zeaxanthine; this hypothesis had to be tested, but how?

241 Fortunately I had read the book of Palmer "Carotenoids and Related Pigments" ( 1 ) where Michael Tswett's method was mentioned. Despite the publication of Tswett's book on the chromophylls in the plant and animal kingdom in 1910 ( 2 ) his method had been ignored by chemists; firstly, because it had only been published in Russian, and secondly because Tswett was only known to botanists. Furthermore, Willstatter and Stoll (3) had formally condemned the method, because they had been unsuccessful in the preparative purification of chlorophylls by "Tswett columns"; they had apparently not noticed that Tswett (2) had already stated that the chlorophylls are destroyed by too "aggressive" adsorbants and had recommended the use of powdered sucrose, or of inuline. However, several biochemists - Palmer and Eckles in the U.S.A., Dh6r6 and Vegezzi in Switzerland, and Coward in England - had used Tswett's method with success for separating small quantities of natural pigments for spectroscopic studies, and Erwin Chargaff just recently had studied the absorption spectra of bacterial caroteinoids after separation by chromatography ( 4 ) . Richard Willstatter had a German translation of Tswett's book and had given the manuscript to Kuhn, his favorite pupil. So, when I asked Kuhn about Tswett's work, he could give me this translation and I was able to read details of Tswett's methods in this manuscript. Finally, in December 1930, I performed the following experiment: a solution of a mixture of 0 . 5 mg lutein and 0 . 5 mg of zeaxanthine in carbon disulfide (a rather inappropriate solvent!) was poured onto a large column of powdered calcium carbonate and then abundantly washed with the same solvent; a large orange band became visible on the column, with distinctly different hues in the upper and lower part. After scraping off the upper and lower layers with a spatula, the pigments were eluted with methanol and, lo-and-behold, the upper zone had the spectrum of lutein (Amax 508 and 475 nm in CS2) and the lower the spectrum of zeaxanthine (Amax 517 and 403 nm in CS2). Then came the crucial experiment: 30 mg of lutein of egg yolk ( [a]Fd=+lOOO CHC13) were adsorbed OR a column of 7 cm diameter; after deve opment, the upper and lower coloured zones where eluted, the intermediary part rechromatographed and again the upper and lower zones separated with a spatula. After a third chromatography, the eluate of the upper zone gave crystals with [a] +145O, corresponding to that of leaf xanthophyll, whereas the crystay$ from the lower part were optically inactive (still impure zeaxanthine). It was thus proven (a) that egg yolk lutein is a mixture of oxygenated carotenoids; (b) much more importantly: that Tswett's method could be used f o r preparative separations. In agreement with Willstatter we then proposed the following nomenclature ( 5 ) : the name xanthophyll designates all oxygenated C40 carotenoids, whereas the name lutein is retained for the major constituent of leaf xanthophylls (C,,0H5602 m . p . 19Z0, [aiFip+1450) ; after some stubborn resistance by Karrer and coworkers, s was finally adopted by a nomenclature committee of IUPAC. In the mean while I had continued work on carotene from carrots. Karrer and coworkers (6) had just published a paper (a most admirable piece of work, due to his intuition and genius!) proposing the

242

correct structure for carotene, (which was soon to be known as @-carotene), a compound without any asymmetric carbon atom. This was in apparent contradiction to the rotations I found with various from +15O to +600) ; preparations of carotene from carrots ([a] two possible explanations were considered:'$ither the presence, in our preparations, of dextrorotatory impurities, or the incorrectness of Karrer's formula. The problem was all the more important, as

KURN. WINTEIISTEIN UND L.BDXULR.Zur Kmntaia der Xanthophylle. (Hopps-Syhr't 2. P h y c d . Cbsm.. 197 (1931) 158.) Zerlegung d a Dotterhbetoff~in seine Komponenten. Gemiuha von Lutein und zsurmthin. eina 0.5 mg Lotein und 0.5 mg Zeuanthio mrden in IOCC s c h w e f e l k o h l ~ f fgels Dicsc Ldsuag saugten wir I . l y m dnrch ein mit Wciumcarboxut (gsNlt. E.Merck) gef(LlltczR o b voll 15 cm Uage nnd 10mm lichta Weite. dm untsn durch siaen Wattebaurch abgeschkxua war. Dnmh Nschrufhen mit S c h ~ c l k o ~ (insgcaunt f f 20-3occ) gelang ea. die Admrptioawchichtan mehme Zentimeter audaander LU ziehen. DPI erhdtene ChrOmutqF.mm *t in der Figur darg&dlt. Die Schichttn 1-4 wurden getrennt her8uagenomnu-onnd mit Metbaa01 d d .N.ch dem Filtrierm und Verdampfen faaden wir in Schwefeltohkrrstoif folgeade Ahorption#bauden : I . Schicht: 508 476 3. Schicht: 513 481 2. Schicht: 5477 4. Schicht: 516 483 Lutein: 508 475 Zeaxmtbirn: 517 483 In den Schichten I und 4 waren je 10-15% d a g-tm Fubtoffea vorhaaden. 12.

(8)Fr8kti&W#b#

(b) Fraktioniaung d a Xanthophyllpr&puatea811s bulgarkhen Adsorption und Elution wurden Hahaerciern (Spektrum: 509.5.476 q). wie bei dem k0mtlichen Farbrtoffgemiach 8qeffAhrt. 1. Schicht: 507.5 476 3. Schicht: 510 477 z. Schicht: 510 477 4. Schicht: 512 478

I I

(c) Fraktionierung eina Xanthophyllprap%rated aua holhdischen Habwrrian (Spektrum : 509.476 mp). I.

Sehicht: 507.5

z. Schicht: 509.5

477 477

3. Schicht: 510.5 478.5 4. Sehicht: 514 480.5

II

aa In etwas grbsamm Mnsatob wurde cia Priparat pus bulgarischen Eidottern voll [alca =

+ ioo0 (Chloroform)fraktioaiert. Far 30 ny FarbstoB. die in 500 cc Schwefdkohlenstoff

g e h t waren. verwendeten wir ern Adaorptrolurohr von 7 cm Durchmawr. Die Luteinund Zswnthiaumc wurden 5cm auseinandergezogen, die mittlaen Schichten eluiert. an frkhem Cplciumcorbonot adaorbiert. Die Mittebchichten d a eweiten Rohre wurden wider eluiert ood der darin enthaltme Forbstoff ein drittca wal dunh ein Cdciumcarbonnt-Rohr ggchickt. Die vereiayltea Schichten I und die ve-n Schichten 4 eathielten etwa je 10%dea paeewandten Farbstoff~a.Nach dem Eluimn mit Methanol und ubctfahren in Schwefelkohleast.off faaden wir: Schichtco I : 508 476 Schrchten 4: 513.5 479 mp Die Luteiafraktion ( I ) t e e noch dem Umkryatnllisieren 1a

(a)ca = (+0.16"1oo) : (o.ozz.o.5) = +145O (Chloroform). was einem Luteingehalt von etwa po% entspricht. Dic Zeaxanthinfraktion (4) war kaum optisch aktiv. Sie schmolr in noch unreinem Zustande bu 195-196. (korr).

Fig. 26.2. Page from the first publication by Kuhn, Winterstein and Lederer ( 5 ) .

243

Euler et al. (7) had shown two years before that carotene was a provitamin A . Having previously, during my Ph.D. work, used various methods of precipitation (of picrates, picrolonates, etc..) for purification, I tried a reaction described by Willstatter and Escher (8): the addition of a solution of iodine to a carotene solution gives a black, amorphous precipitate of carotene iodides. They do not seem to have regenerated carotene from this precipitate; when I tried this, by treatment with thiosulfate, another pigment was obtained, much darker than carotene, which we called isocarotene. Karrer and Schwab showed later ( 9 ) that it was, in fact, a dehydrocarotene. The mother liquor of the precipitate of iodide was also treated by thiosulfate and the orange solution thus obtained kept at room temperature; after a few days magnificent crystals had formed, which after recrystallisation gave a pigment C40Hs6, m.p. 173O with and [ a ] cd+3800! this we named a-carotene. It was easy, then, to show that chromatography on alumina (especially on the so-called "Fasertonerde" of Wislicenus) could separate a- and 6-carotene; Tswett ( 2 ) had already written: "It is very probable that carotene of leaves is not a unique pigment, but a mixture of two or more homologues, which could be separated by the method of adsorption, using adequate adsorbents".

Fig. 26.3. Crystals of a- and $-carotene, from benzene-methanol. (x225, between crossed Nicol prisms ( 2 3 ) .

244

We then showed that the optically inactive @-carotene was the main carotene of leaves of spinach, nettles, etc. (10) and Kuhn and Brockmann (11) found, with my preparations, that a- and f3-carotene were both active as provitamin A . The first paper mentioning the preparative use of chromatography is dated February 17, 1931, and was published in Die flaturnissenschaften under the title "Fraktionierung und Isomerisierung des Carotins" (12) (fractionation and isomerization of carotene). This very short, preliminary communication mentions chromatography only in the sentence: "Durch f r a k t i o n i e r t e Adsorption oder f r a k t i o n i e r t e

FaZlung m i t Jod gelingt e s eine sehr stark optisch a k t i v e Komponente (a-Carotinl und eine i n a k t i v e f$-Carotinl zu erhazten. I' [With help of fractional adsorption or fractional precipitation with iodine, it is possible to obtain a component which is optically very active (a-carotene) and one which is inactive (6-carotene)l. This note was followed by a detailed paper "Uber das Vitamin des hachstums. I. Mitteilung. Zerlegung des Carotins in seine Komponenten" (On the vitamin of growth. First communication. Separation of carotene into its components) which was sent to the Beriehte der Deutschen Chemisehen GeseZZschaft on March 18, 1931 ( 1 3 ) . Eight days earlier the paper "Zur Kenntniss der Xanthophylle" (On the knowledge of the xanthophyls) containing the description of the preparative separation of lutein and zeaxanthin by chromatography had been sent to Hoppe-SeyZer's Z e i t s c h r i f t fiir PhysioZogische Chemie (51, I should mention here that scientific papers were unusually written at Kuhn's home, on Saturday afternoons or Sundays; once he expressed his satisfaction at the absence of Mrs. Kuhn, who had joined her parents in Switzerland for a few days, by saying "when I am alone at home, I am happy, I can go to bed with my boots and the Chemisehes ZentraZbZatt. A few months later I tried to isolate the yellow pigments of the flowers of dandelions picked from the lawn in front of the Institute. Here, chromatography on calcium carbonate immediately gave two distinct yellow zones, lutein and a new pigment which we called taraxanthine ( 1 4 ) (this was later shown to be mainly luteinepoxide ( 1 5 ) ) . Chromatography was then very quickly applied by many other authors: first by Brockmann ( 1 6 ) and Winterstein (17-19) in Kuhn's laboratory, then by Karrer and his colleagues ( 2 0 ) in Zurich and by Zechmeister in P6cs (Hungary) (21) who was the first to publish a monograph on chromatography, in 1937 (22). Looking back, my personal contribution to the "renaissance" of Tswett's method has probably been best characterized by Kuhn himself who, as President of the German Chemical Society, in 1964, gave me the A.W. Hofmann gold medal with the following words (23): "In HeideZberg, an meinem I n s t i t u t , haben S i e a- und B-Carotin getrennt 2nd die Methode der Chromatographie zu neuen Leben erneckt," (In Heidelberg, in my Institute, you separated a- and &Carotene and resuscitated the method of chromatography).

245 REFERENCES 1 L.S. Palmer, Carotenoids and Related Pigments. The Chemical Catalog co., New York, 1922. 2 M.S. Tswett, KhromophyZii v RastiteZnom i Zhivotnom Mire

3 4 5 6

7

(ChromophyZZs i n the PZant and Animal Kingdom), Izd. Karbasnikov, Warsaw, 1910. R. Willstatter and A . Stoll, Untersuchungen iiber ChZorophyZZ. Springer, Berlin, 1913. E. Chargaff, ZbZ. BakterioZ. 129 (1930) 121. R. Kuhn, A . Winterstein and E. Lederer, Hoppe-SeyZer's Z. PhysioZ. Chem. 197 (1931) 141. P. Karrer, A . Helfenstein, H. Wehrli and A. Wettstein, HeZv. Chim. Acta 13 (1930) 1084. B.V. Euler, H.V. Euler and H. Hellstrom, Biochem. Z. 203 (1928)

370. 8 R. Willstatter and H.H. Escher, Hoppe-SeyZer's 2 . PhysioZ. Chem. 76 (1911) 214. 9 P. Karrer and G. Schwab, HeZv. Chim. Acta 23 (1940) 578. 10 R. Kuhn and E. Lederer, Hoppe-SeyZer's Z. PhysioZ. Chem. 200 (1931) 246. 11 R. Kuhn and H. Brockmann, Ber. 64 (1931) 1859. 12 R. Kuhn and E. Lederer, Naturwissenschaften 19 (1931) 306. 13 R. Kuhn and E. Lederer, Ber. 6 4 (1931) 1349. 14 R. Kuhn and E. Lederer, Hoppe-Seyler's Z. PhysioZ. Chem. 200 (1931) 108. 15 K. Egger, Pzanta 80 (1968) 65. 16 R. Kuhn and H. Brockmann, Hoppe-Seyler's Z. PhysioZ. Chem. 206 (1932) 41; Ber. 66 (1933) 407. 17 A . Winterstein and G. Stein, Hoppe-SeyZer's 2 . PhysioZ. Chem. 220 (1933) 247, 263. 18 A . Winterstein and K. Schon, Hoppe-SeyZer's Z. Physiol. Chem. 230 (1934) 139, 146, 158. 19 A . Winterstein and H. Vetter, Hoppe-SeyZer's Z. PhysioZ. Chem. 230 (1934) 169. 20 P. Karrer and K. Schopf, HeZv. Chim. Acta 15 (1932) 745. 21 L. Zechmeister and L. Cholnoky, Monatsh. Chem. 68 (1936) 68. 22 L. Zechmeister and L. Cholnoky, Die Chromatographische Adsorptionsmethode, Springer, Wien, 1937. 23 Nachr. Chem. Tech. 12 (1964) 286.

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247

MICHAEL LEDERER

MICHAEL LEDERER was born in Vienna, Austria, in 1924 and went to school there until 1938. At the age of 14 he emigrated to Australia and completed his secondary studies in an evening course offered by the Sydney Technical College. He then proceeded to the chemistry diploma course of the Sydney Technical College while working as an analyst first in a soap factory and then as a laboratory assistant with Bayer of Australia. On graduating he was appointed demonstrator at the Sydney Technical College and inscribed himself a t the Faculty of Science of Sydney University. In 1949 he obtained his B.Sc. from Sydney University and was appointed a lecturer at the Newcastle Technical College, Newcastle, New South Wales. In 1951 he first spent a short time at the Queensland Idstitute'of Medical Research and then left for Paris, to collaborate with his cousin Edgar Lederer on a book of chromatography. In 1954 he received the Docteur e's Sciences from the Sorbonne with a thesis on the paper chromatography of radioelements. In 1955 he was nominated Maftre de Recherches au Centre National de la Recherche Scientifique and received the Silver Medal of the C.N.R.S. In 1960 he was appointed the director of the Laboratorio di Cromatografia del Consiglio Nazionaledelle Ricerche, in Rome, Italy. His research interests are in the field of inorganic chemistry and the application of chromatographic and electrophoretic methods to the separation and study of inorganic complex compounds. Dr.M. Lederer has published several books (most in collaboration with Prof. E. Lederer) on chromatography and electrophoresis. He is founder and editor since its inception of the JownaZ of Chromatography and of Chromatographic Reviews.

248

The Sydney Technical College had the practice that a chemistry student, who had chosen physical chemistry as main subject, had to give a one-hour seminar on a topic chosen by the lecturer who was at that time the late professor Sir Ronald Nyholm. My friend, Peter Beckmann, received the assignment of "Chromatography". This meant in 1945 that one had to get hold of one of the rare copies of the book by Zechmeister and Cholnoky ( I ) and abstract a one-hour lecture from it. So far so good, but the lecture itself presented a problem as Beckmann's English, like my own, showed a strong middle-European bias. The only way to get his lecture into shape was to repeat it to me over-and-over again, every Sunday afternoon on our walks around Sydney. A t the end I also had to ask questions similar to those he could expect from the audience. Thus my introduction to chromatography: My own topic for the seminar was oxidation-reduction indicators. This I had chosen myself, as I was playing around with substituted diphenylamines at the time,

Fig. 27.1. Peter Beckmann, who had to lecture on chromatography, in the late 1940's.

A year later Sir Ronald appointed me as demonstrator in qualitative analysis. As my teaching load was only 21 hours, in the afternoons and evenings, this left me enough free time to study physiology and agricultural chemistry at Sydney University in the mornings. Now I had noted that the students in qualitative analysis all had trouble with one group of metals, namely As-Sb-Sn. Tin especially was hard to detect and As was often reported as present, when there existed only a precipitate of sulphur. I had seen and appreciated the article by A.J.P. Martin in Endeavour ( 2 ) showing some superb paper chromatograms of amino acids. I resolved then that such separation must also be possible with inorganic ions. My first paper dealing with a very simple separation of Sb(II1) from many metals was initially rejected by the Royal Society of New South Wales (as being not original!?!) and then accepted into one of the first issues of the newly founded AnaZytica Chimica Acta ( 3 ) . In the final year at the Sydney Technical College one also had to do a one-evening-per-week course on fire-assaying and I learned how to extract minute quantities of gold, silver and platinum metals

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from rather large samples by fusion methods. The sabsequent separations of these rare metals, once isolated, were very archaic and incomplete. I thus investigated how they would run on paper chromatograms and got an excellent separation of Au-Pt-Pd-Ag-Cu with butanol-HC1-water at the first try ( 4 ) . I then spent a happy time trying out all the interesting analytical separations I could think of (halides, organic acids, metals, acridine dyes) sometimes publishing a paper after only a few days work, much to the annoyance of my more serious colleagues. After graduating from Sydney University in 1949 I was appointed as lecturer in inorganic and physical chemistry at the Newcastle Technical College, Newcastle, N.S.W. Soon after starting there the paper by Durrum on the paper electrophoresis of proteins and peptides appeared (5). It took me about two weeks till I got the technique working also for inorganic ions and Mr. F.L. Ward, senior lecturer in physical chemistry joined me in the fun (6). Two years earlier my cousin Edgar Lederer had written a book on progress in chromatography (in French) ( 7 ) and had planned with the publishers that a companion volume on inorganic and theoretical chromatography would be written by Professor M. Haissinsky. However, just then Professor Haissinsky had worked with rather high doses of radioactivity and felt too tired to undertake the task. As I had just published a review on inorganic chromatography (8) my cousin invited me to write the companion volume and I gladly accepted. At the same time he looked around for an English publisher. He decided he would approach one whose books he liked. Looking through his library, what he liked mbst was the English edition of Karrer's Organic Chemistry and he thus wrote to Elsevier, Amsterdam, who accepted his offer. We soon realised that we could not make much headway with either manuscript if I did not join him in Paris. I thus left Australia for Paris late in 1951 and was soon accepted there as a research worker by Madame Joliot-Curie at the Institut du Radium. My Docteur 2s Sciences thesis was thus "La chromatographie sur papier des radioelements", in 1954. The second part of Recent Progress in Chromatography was published in French in 1952 (g), and the English version of the two books ( 1 0 ) and my Introduction to Paper Electrophoresis in 1955 ( 1 1 ) . At that time I was already busy working on a second edition of the chromatography book with my cousin and beginning to write a chapter on inorganic chromatography and electrophoresis for a handbook of microchemical methods ( 1 2 ) . The research work at the Institut du Radium was closely linked with the work of the physicists at the Institute. Their work required radioisotopes of high purity, for which paper chromatography is eminently suitable. Not only does it permit ready scanning of the chromatograms (if necessary by cutting up the paper in 1 cm strips) but it also permits preparative work with as much as 30-100 mg at a time. At one time we had several problems involving the separation of adjacent rare earths. After developing some not-very-efficient partition systems using butanol-acetylacetone-acetic acid-water we

250 felt that we had to resort to the classical work with ion-exchange columns as described by Ketelle and Boyd ( 1 3 ) . After building the set-up, involving rather long columns and heating jackets, we found that we had not the experience to get good separations and that the separate rare earths (when successful) were collected in a large volume of buffered citrate from which their isolation as "carrierfree" tracers was rather cumbersome if not impossible. In this work we used a collection of ion-exchange resins which had been bought by Dr. Bouchez when he separated some carrier-free alkalies some years earlier. One of the bottles contained Dowex 50 "colloidal aggregates", a product which was later withdrawn from commerce because the particles were too fine for the "usual" column chromatography. At the time we had worked with paper both in electrophoresis and chromatography and had "paper on the brain". Thus the obvious thing to do was to impregnate paper strips with a suspension of the colloidal aggregates of Dowex 50 which gave us excellent chromatograms of numerous rare earths as well as of other metal ions. We had thus described the first ion-exchange resin paper which was useful for chromatographic work in 1956 ( 1 4 ) . One aspect which we realised but did not emphasize in publishing the results was that

Fig. 2 7 . 2 . The editor of the Journal of Chromatography with the essentials of the trade: a type-writer, a lamp and Maru the tomcat sitting on the manuscripts.

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Fig. 27.3. ( l e f t ) . Miss M . K . Anton, the first desk editor of the J o u m l o f Chromatography. Fig. 27.4. ( r i g h t ) . Dr. Karel Macek and M. Lederer during a symposium in Liblice castle, in 1967.

complete separations for example of La and Y were possible with only very short developments, in other words that we had many more theoretical plates on our papers than were achieved in the very long columns then in use. By that time I had spent seven years doing research and writing on the average of ten pages a day for a book. Still in 1955 chromatography papers were rare in journals dealing with analytical chemistry. For example, the first papers on gas-liquid chromatography appeared in the Biochemical Journal, the fundamental work on ionexchange chromatography in the Journal o f the American Chemical Society, and most theoretical papers in the Journal o f t h e Chemical Society. It was evident to me that a journal was needed which dealt with this field. When I proposed this idea to Ur. P. Bergmans (then director of the Elsevier Publishing Co.) he was not enthusiastic. "Would this chromatography last?" "They already had a journal on analytical chemistry!" were the main arguments. A few years ago at

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the dinner celebrating the 100th volume of the Journal Of Chromatography he told me the real reason. The scientific publishing company was far in the red at the time with the holding company and all new ventures were to be reduced to a minimum. He evidently could not tell me this. However, a breakthrough came in 1957. Mr. Bergmas c m e to Paris and was asked by the International Council of Scientlfic Unions to publish their "ICSU Review": Very pleased with this Buccess, he rang m e up and we met at the Cafe Weber near the Madeleine. Seeing him in a good mood I again proposed a journal on chromatography. This time he felt he could risk it and agreed. The publishing details were thus entrusted to Dr. W. Gaade and much to our surprise the venture became a best-seller within two years and has not yet stopped growing. The first desk editor working with me was Miss M . K . Anton the most patient desk editor that ever existed, who, before retiring, trained her successor, Miss T. Sijpesteyn. I had come to Paris with the intention to help in the publication of a book and to return within a year to Australia and now I was already eight years in Paris without really meaning to stay there. Thus, when Professor V. Caglioti invited me to Rome to start the Laboratorio di Cromatografia del Consiglio Nazionale delle Ricerche, I gladly accepted, last but not least, because Northern European winters were not to our liking. The Laboratorio di Cromatografia was the first laboratory specifically concerned with chromatography at the time. There are now chromatography institutes in Germany, South Africa, Edinburgh and many others. Our main interest in the first years was in the mechanism of ion-exchange resin chromatography using ionexchange papers as an investigational method. Besides this we collaborated with the whole Istituto di Chimica Generale solving their chromatographic problems which lay within the field of coordination complexes. This led us to the phenomenon of outer-sphere complexing and its application in electrophoretic and chromatographic separations and to the study of hydrolysis polymers in aqueous solutions using gel filtration. The laboratory has also had lots of distinguished visitors: Professor A . Bevenue, Dr. Z. Deyl, Dr. J. Jankk, Dr. K. Macek, Professor A . S . Ritchie and Professor K.I. Sakodynskii, just to name a few. I first met Dr. Macek in Prague during the first symposium on flat-bed chromatography organised there in June 1961. He had published his bibliography of paper chromatography in Czech just a year earlier and was full of good ideas. The result of that meeting was the bibliography section of the Journal of Chromatography. In 1969 Dr. Macek spent a year in Rome, in order to organise the 4th Symposium on FlatBed Chromatography in Frascati. By then the Journal of Chromatography had grown to seven volumes per year and Dr. Macek accepted the post of associate editor dealing mainly with flat-bed chromatography and its pharmaceutical and medical applications. In 1974 the Journal of Chromatography had grown to 13 volumes a year. After working alone for 17 years (if we don't count the tomcat, an essential for good editing: he keeps me company but does not talk!) I was given a full-time editorial secretary (who alas stayed only for 24 years) to handle the correspondence and the checking of revised

253 papers. On this occasion we felt it was the right moment to split the journal into two. As we saw the most future in the biomedical applications of chromatography, we founded the Biomedical Applicat i o n s section with a separate editor (Dr. Karel Macek) and its own editorial board. The first volume appeared in 1977 and was so successful that two volumes had to be projected for 1978. REFERENCES 1 L, Zechmeister and L. Cholnoky, Die Chromatographische Adsorptionsmethode, Springer, Wien; 1st ed.: 1937, 2nd ed.: 1938. The English translation of the second edition: Principles and Practice o f Chromatography, Wiley, New York, 1941. 2 A.J.P. Martin, Endeavour 6 (1947) 21-28. 3 M. Lederer, Anal. Chim. Acta 2 (1948) 261-262. 4 M. Lederer, Nature (London) 162 (1948) 776-777. 5 E.L. Durrum, J . Amer. Chem. Soc. 7 2 (1950) 2943-2948. 6 M. Lederer and F.L. Ward, Aust. J . of Science 13 (1951) 114-115. 7 E. Lederer, Progre's re'cents de l a chromatographie. Premie're p a r t i e : Chimie organique e t biologique, Hermann & Cie., Paris, 1949. 8 M. Lederer, J . Proc. Roy. Australian Chem. I n s t . 17 (1950) 308326. 9 M. Lederer, Progre's ¢s de l a chromatographie. Deuxidme p a r t i e : Chimie mine'rale, Herman & Cie., Paris, 1952. 10 E. Lederer and M. Lederer, Chromatography - a Review o f Principles and Applications, Elsevier, Amsterdam, 1955. 11 M. Lederer, Introduction t o Paper Electrophoresis and Related Methods, Elsevier, Amsterdam, 1955. 12 M. Lederer, H. Michl, K. Schlogl and A. Siegel, "Anorganische Chromatographie und Electrophorese." In Handbuch der mikrochemischen Methoden, Vol. 111, F. Hecht and M.K. Zacherl, eds., Springer, Wien, 1961. 13 B.H. Ketelle and J.E. Boyd, J . Amer. Chem. Soc. 69 (1942) 28002812. 14 M. Lederer and S. Kertes, AnaZ. Chim. Acta 15 (1956) 226.

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ARNOLDO LIBERTI

ARNOLDO LIBERTI w a s born i n 1917, i n Rome, I t a l y . H e s t u d i e d a t t h e Un i v e r s i t y of Rome where he w a s awarded a D o c t o r a t e e m Zaude i n 1939. A f t e r s e r v i n g i n t h e army f o r seven y e a r s , he r e c e i v e d an I n t e r n a t i o n a l Education F e l l o w s h i p and j o i n e d t h e U n i v e r s i t y o f Minnesota where h e worked with P ro f e s s o r Kolthoff; he a l s o received an M.S. d e g r e e i n a n a l y t i c a l c h e m i s t r y from t h i s u n i v e r s i t y i n 1947. R e t u r n i n g home t o I t a l y he was nominated a s s i s t a n t p r o f e s s o r at t h e Universit y of Rome and, i n 1954, won a n a t i o n a l c o m p e t i t i o n t o become u n i v e r s i t y p r o f e s sor of a n a l y t i c a l c h e m i s t r y . H e t a u g h t a t t h e U n i v e r s i t i e s o f Messina (1954-1961) and Naples (1961-1969) and f i n a l l y r e t u r n e d t o t h e U n i v e r s i t y o f Rome a s head o f t h e A n a l y t i c a l Department. S i n c e 1968 he h a s a l s o been head o f t h e l a b o r a t o r y on A i r P o l l u t i o n ( L a b o r a t o r i o s u l l ' Inquinamento Atmosferico) of t h e C o n s i g l i o Nazionale d e l l e R i c e r c h e , t h e N a t i o n a l Research Council o f I t a l y . D r . L i b e r t y i s t h e a u t h o r and c o a u t h o r of o v e r 200 p a p e r s c o v e r i n g v a r i o u s a r e a s o f modern methods o f a n a l y t i c a l c h e m i s t r y such as p o l a r o graphy, s o l u t i o n e q u i l i b r i a , amperometric t i t r a t i o n s , coulometry, select i v e i o n e l e c t r o d e s , chromatography and environmental a n a l y s i s . I n each f i e l d h i s r e s e a r c h a c t i v i t y h a s r e c e i v e d wide r e c o g n i t i o n . D r . L i b e r t i was one o f t h e p i o n e e r s i n g a s chromatography where h i s c o n t r i b u t i o n s range from t h e development o f coulometry as a d e t e c t i o n d e v i c e t o column t e c h n o l o g y , i s o t o p e s e p a r a t ' l o n , aroma e v a l u a t i o n and environmental a n a l y s i s .

It used to be customary in Italian universities that when, as the result of a government competition, a young researcher received the title of professor, he had to move from his "own" university to a new one, usually in some remote area. This was also my case when, in 1954, I became professor of analytical chemistry and had to move from Rome, where I started my career, to the University of Messina, in Sicily. At that time, my scientific interest was mainly devoted to analytical electrochemistry and solution equilibria. When I joined the new university my aim was to establish a modern analytical school inspired by the good teachings of Isaac Kolthoff, with whom I had the good fortune to work at the University of Minnesota. As soon as I started to assign thesis work to my students I was quite surprised that almost all were asking for subjects related either to essential oils o r to agricultural juices. The reason for this was that at that time the only industrial activity in the Messina area was connected with agricultural production and manufacture of essential oils, juices and other products. It was quite frustrating for me, having devoted most of my research time to polarography and electrometric titrations, to find related subjects in the new area. Since I did not want to disappoint my students I started to familiarize myself with the procedures related to essential oil production. I was very much surprised, however, to see that in spite of the development of various modern analytical methods, the test which played a determining role in quality evaluation was the olfactoryappraisal, i.e., sniffing the oil sample. When a manufacturer wished to obtain a certification, after subjecting it to chemical tests, the oil was ultimately appraised by a special "smell" test by a qualified panel. Later on I learned that this was a "panel test". Quite different is the application of the human senses in scientific achievement. Some yield a response which cannot be quantified whereas for others, technical devices can yield quantitative information much more valuable than the ones supplied by a human sense. As an example, taste is by far a subjective evaluation whereas sight in terms of color evaluation can be replaced by applying the spectrum of electromagnetic radiations and specific detectors. When, in 1954, I had the good fortune to hear in Rome a lecture by A.J.P. Martin, who outlined the recent achievements in chromatography and showed how various mixtures might be separated in the gas phase, I definitely felt that gas-phase chromatography might seriously challenge the olfactory appraisal. I was so impressed by Martin's lecture that I asked him if I could spend a short time in his laboratory to learn how to build a gas density balance, (the only detector available at that time besides the katharometer) and have some practice in gas chromatography. In spite of the skepticism of Martin and James the first home-made gas chromatograph with a gas density balance as the detector, was soon built in Messina. I tried to find out how gas chromatography might replace the nose in essential oil evaluation. The first chromatogram of a bergamot oil obtained in 1956 showed the presence of only 6 components ( I ) and, from this result, I thought I

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F i g . 28.1. The f i r s t g a s chromatography b u i l d i n Messina i n 1955, u t i l i z i n g a gas d e n s i t y balance as the detector.

might be a b l e t o r e p r o d u c e t h e bergamot aroma, b u t I d e f i n i t e l y found I was wrong and q u i t e f a r from t h e g o a l According t o t h e column t e c h n o l o g y a v a i l a b l e a t t h a t t i m e , i t seemed t h a t t h e a n a l y s i s of a complex m i x t u r e c o n t a i n i n g compounds of w i d e l y d i f f e r e n t p o l a r i t y , might be c a r r i e d o u t o n l y by s e p a r a t i n g i t i n v a r i o u s c l a s s e s . I t a l s o appeared t o us t h a t i t would be a l o n g t i m e b e f o r e g a s chromatography c o u l d c o m p e t e w i t h t h e nose i n e v a l u a t i n g such a complex m i x t u r e a s an e s s e n t i a l o i l . A g a s d e n s i t y b a l a n c e w a s i n d e e d an o u t s t a n d i n g d e t e c t o r and one of i t s most a t t r a c t i v e f e a t u r e s w a s t h e p o s s i b i l i t y of d e t e r mining t h e m o l e c u l a r weight of an e l u t e d compound by u t i l i z i n g two c a r r i e r g a s e s of d i f f e r e n t m o l e c u l a r weight (2). T h i s i n t e r e s t i n g a s p e c t , however, h a s n o t found a wide a p p l i c a t i o n because o f t h e care required t o operate t h i s detector. The need f o r more s i m p l e and less s o p h i s t i c a t e d d e t e c t o r s induced m e t o d e v e l o p c o u l o m e t r i c p r o c e d u r e s a p p l i e d t o t h e q u a n t i t a t i v e d e t e r m i n a t i o n of e l u t e d compounds. The f i r s t a p p l i c a t i o n w a s i n t h e d e t e r m i n a t i o n of f r e e a c i d s , by r e p l a c i n g t h e p r e v i o u s l y used t i t r a t i o n p r o c e d u r e . The o p e r a t o r watched t h e e l e c t r i c s i g n a l of t h e i n d i c a t o r e l e c t r o d e and g e n e r a t e d c u r r e n t , which was r e c o r d e d t o b r i n g t h e s i g n a l t o z e r o ( 3 ) . The p r o c e d u r e was n o t a s t e d i o u s a s t h e one developed by M a r t i n and James, b u t d i d n o t a l l o w t h e o p e r a t o r t o r e l a x and smoke w h i l e o b s e r v i n g t h e development of a g a s chromatogram a s happens t o d a y . In o r d e r t o e x t e n d coulometry t o compounds which c a n n o t be t i t r a t e d d i r e c t l y , a g e n e r a l s e t - u p w a s d e v i s e d t o burn e a c h e l u t e d compound coming o u t o f a chromatographic column i n t o a c e l l where

...

258 the carbon dioxide set free was microtitrated by using an oxygen electrode set at pH 9.8 as the indicator. The set-up was entirely home-made and was an attempt at constructing a .&@s chromatograph which yielded a quantitative response for each compound through its combustion to carbon dioxide and its titration. The operator had to run the apparatus, which only in principle can be indicated as an automatic gas chromatograph.

Fig. 2 8 . 2 . Set-up for a gas chromatograph with coulometric titration of C O P obtained from the combustion of the sample components eluting from the column ( 4 ) . p=Manostat; r=flowmeter; q=cartidge for gas purification; L=chromatographic column; S=thermoregulator; b=furnace; B=titration cell; P A = microammeter; s=light source; F=photocell; C=coulometer; R=recorder. However, when we described this system in 1958 at the Amsterdam Symposium ( 4 ) , very little interest was shown by others. The introduction of ionization detectors and the development of instruments by the large manufacturing companies rendered my efforts almost obsolete. By good luck my young coworker D r . G.P. Cartoni, now a professor at the University of Rome, was awarded a fellowship at the University of Oxford and familiarized himself with the construction of the new detectors (hydrogen flame ionization and the argon ionization detector) and capillary columns. With the new experience previous equipment was set aside and work started with the "spaghetti kettle" gas chromatograph. This name was given to the experimental set-up because the external vessel, where the thermostatic fluid was

259

set, was a common aluminium kettle, which might be used to cook spaghetti. Onto the plate which covered the kettle, the detector was applied; flame ionization, argon ionization or electron capture detector, and the column was connected at the lower side of the plate.

Fig. 28.3. The "spaghetti kettle" gas chromatograph. The spaghetti kettle chromatograph was an excellent experimental tool which has been used extensively by myself and my co-workers Gian-Paolo Cartoni, Fabrizio Bruner and Antonio Di Corcia. We acquired our knowledge and experience in column technology by working on the spaghetti kettle. Fertile pioneering work was carried out with this experimental set-up and various areas of chromatography have been exploited. A number of these instruments have been built; some of them still work at various laboratories. Our aim was to be able to prepare high-resolution columns. After some trial and error, we were able to make glass capillary columns with high performance. We prepared both adsorption columns, by etching the glass capillary with sodium hydroxide, and partition columns, by coating them with stationary phases, utilizing various techniques ( 5 ) . Next, we set an ambitious goal: the separation of isotopic species by gas chromatography. A variety of systems have been studied and most organic compounds have been separated from their deuterated homologues. It was found that the gas chromatographic separation of an isotopic pair is affected by two counteracting isotope effects, the "normal" (i.e. the lighter species is eluted first) which is effective at very low temperatures and the

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"inverse" being predominant at high temperatures. Both effects are strongly affected by various absorbents and stationary phases (6-10).

Fig. 2 8 . 4 . (right-to-left) A . Liberti, A . Di Corcia, G.P. Cartoni and F . Bruner in front of one of the buildings of the University of Rome. The large amount of work carried out in isotopic species separation should have opened new roads in the technology of isotopes and probably those systems which might have some practical importance should have been further studied to evaluate the feasibility of isolation of isotopes of industrial importance. Although this aim has not been fulfilled, I wish to recall two achievements that in my opinion illustrate the hard work of our team in this field: the first is the quantitative separation of CH3D contained in natural methane achieved with a single injection in a capillary column at liquid nitrogen temperature ( 1 1 ) and the second is the significant enrichment (up to 5 0 % ) of CH3D from natural methane with help from a 120 m long packed column, which is very likely one of the longest packed columns ever constructed (12). The project is now over and it is difficult to establish-if it would be useful to carry on further work in this area and which advantages might be obtained f o r human society by a large investment in this area with the aim of obtaining isotopes of commercial interest by gas chromatography. It is however, a matter of satisfaction that two research groups, D r . F.S. Rowland's at Irvine, California, and Dr. J.A. Morrison's

at Hamilton, Canada, are using the column we developed. They are still the only tools available to obtain high purity deuterated compounds on a micropreparative scale. Besides the scientific and technical achievements a variety of applications have been developed by myself and my cowerkers such as checking the quality of food and the aroma of brandy, doping control etc. Gas chromatography became more popular (and us, as well) when it was found that the urine of the winner of the Giro d ' l t a l i a (Italy's most popular bicycle race) contained traces of an amphetamine-type compound. It was a great analytical success but sports fans did not share the same enthusiasm for gas chromatography, as the bicycle champion was disqualified: In the Istituto di Chimica Analitica of the Rome University there is a cupboard, which is almost a museum for gas chromatographic columns; etched columns ( 1 3 ) , carbonized columns ( 1 4 ) , thick-layer open-tubular columns ( 1 5 ) , sandwiched columns ( 1 6 ) etc. Each of them is related to ideas, which have been pursued with the aim of achieving better operating conditions in terms of resolution, efficiency and time of analysis. When gas chromatographs became commercially available and very refined gadgets and devices were developed to render more simple and comfortable the work of the operator, it was no longer necessary to construct home-made apparatus. There is however still a large area to be covered by the chemist: to work oncolumn technology. My coworkers and I continue to be active in this field. The column is the heart of a chromatograph and in spite of the progress there is still a lot of work to be done. When Professor Kiselev of Moscow visited my laboratory, commenting on the adsorption columns prepared by etching glass with sodium hydroxide he called my attention to the use of graphitized carbon black as an absorbing material. Many years of work have been spent by my group preparing, studying and evaluating these materials. Products of this kind are now available on the market and all researchers may enjoy the possibility of analyzing complex mixtures by combining the useful analytical properties of both gas-liquid and gas-solid chromatography, by working with columns representing the combined technique of gas-liquid-solid chromatography ( 1 7 , 18). By working with a very homogeneous material with a highly non-polar surface, almost completely free of specific active sites, the separation obtained is caused by the difference in geometric structure and polarizability. It has also been shown that by coating the surface of graphitized carbon black with different amounts of liquid phase, any type of compounds can be eluted with a linear isotherm while the amount of the liquid phase relative to adsorbent permits us to obtain a "tailor made" column. In this way, one of the main aims of modern chromatography seems to be reached: very short column and analysis time, with a high efficiency ( 1 9 ) . The problem of odours, outside the field of essential oils, brought a new interest with my nomination as the head of the Laboratory of Air Pollution of the Italian National Research Council. No longer was the rich fragranceof essential oils to be measured;

262 the problem nowinvolvedthe definition of the quality of air we breathe from our environment and in the factories where we work. The aim of gas chromatography was shifted to this new problem and columns to analyse sulphur-containing compounds ( Z O ) , polycyclic hydrocarbons ( 2 1 ) as well as dioxin (22) were studied. We have also developed adsorption tubes containing graphitized carbon black to trap organic pollutants which can then be heated directly and their adsorbed content injected directly into a gas chromatograph to yield a chromatographic pattern. Are we now at the end of the road I started 25 years ago to replace olfactory appraisal by a gas chromatographic analysis? The six components of the first gas chromatogram of bergamot oil have become about 350 if the determination is carried out by a sophisticated glass capillary column constructed according to the most refined technology (23). The combination of gas chromatography with mass spectroscopy also permits us to have information on most components which can be found in any complex mixtures and in the atmosphere and to identify them. The columns we have developed are sometimes able to solve problems and we are happy that our efforts represent an honest contribution to the development of chromatography. Although panel testing (using people with a special olfactory capacity) still exists, gas chromatography if applied correctly is now definitely yielding an objective response which challenges the one supplied by the nose. REFERENCES 1 A. Liberti and L. Conti, A t t i Convegno s u l l e essenze, Regg-Lo Ca t a b r i a , 956, pp. 1-8. 2 A . Liberti L. Conti and V. Crescenzi, A t t i Ace. Naz. L i n c e i 20 (1956) 623 3 A . Liberti, AnaZ. Chim. .4cta 17 (1957) 247-253. 4 A . Liberti and G.P. Cartoni, in Gas Chromatography 1 9 5 8 (Amsterdam Symposium), D.H. Desty, ed., Butterworths, London, 1958, Dp. 321-329. 5 F. Bruner, G.P. Cartoni and A . Liberti, Chim. I n d . (Mitan) 44 (1962) 999-1001. 6 A . Liberti, G.P. Cartoni and F. Bruner, J . Chromatogr. 1 2 (1963) 8-14. 7 A . Liberti, G.P. Cartoni and F. Bruner, in Gas Chromatography 1964 (Brighton Symposium), A . Goldup, ed., Inst. of Petroleum, London, 1965, pp. 301-312. 8 F. Bruner, G.P. Cartoni and A . Liberti, AnaZ. Chem. 38 (1966) 298-303. 9 G.P. Cartoni, A . Liberti and A . Pela, Anal. Chem. 39 (1967) 1618-1622. 10 A . Di Corcia and A . Liberti, Trans. Faraday SOC. 66 (1970) 967-975. 11 F. Bruner, C. Canulli, A . Di Corcia and A . Liberti, Naturt? (London) 231 (1971) 175-177. 12 F. Bruner, C. Canulli, A . Liberti and M. Marchetti, GGZ. Chim. ItGl. 103 (1973) 877-833. 13 A . Liberti, in Gas Chromatography 1966 (Rome Symposium), A.B. Littlewood, ed., Inst. of Petroleum, London, 1967, pp. 95-113.

263 14 A. Liberti and L. Zoccolillo, J . Chromatogr. 77 (1973) 69-73. 15 G. Goretti, A. Liberti and G. Nota, in Gas Chromatography 2968 (Copenhagen S y m p o ~ i ~ n )C.L.A. , Harbourn, ed., Inst. of Petroleum, London, 1969, pp. 22-31. 16 A. Liberti, G. Nota and G.C. Goretti, J . Chromatogr. 38 (1968) 282-286. 17 A. Di Corcia, A. Liberti and R. Samperi, Anal. Chem. 45 (1973) 1228-1235. 18 A. Di Corcia and A. Liberti, Advan. Chromatogr. 14 (1976) 305. 19 A. Di Corcia, A. Liberti and R. Samperi, J . Chromatogr. 267 (1978) 243. 20-F. Bruner, A. Liberti, M. Possanzini and I. Allegrini, Anal. Chem. 44 (1972) 2070-2074. 21 L. Zoccolillo, A. Liberti and D. Brocco, Atmospheric Environment 6 (1972) 715-720. 22 I. Allegrini, G. Bertoni, D. Brocco, A. Liberti and M. Possanzini. Chim. Ind. (Milan) 59 (1977) 541-544. 23 A . Liberti and G.C. Goretti, Essenze Deriv. Agrwn. 46 (1974) 197-208.

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S.R. LIPSKY

SEYMOUR ( ' S a n d y ' ) RICHARD LIPSKY was born i n 1924 i n San F r a n c i s c o , C a l i f o r n i a . H e o b t a i n e d h i s B.S. d e g r e e from t h e Univers i t y H e i g h t s C o l l e g e i n 1944, m a j o r i n g i n c h e m i s t r y and b i o l o g y . I n 1949 he r e c e i v e d h i s Medical Degree from t h e S t a t e U n i v e r s i t y o f New York's C o l l e g e o f Medicine. A f t e r completing h i s r e s i d e n c y t r a i n i n g i n Medic i n e a t t h e University of C a l i f o r n i a , i n 1952, he a c c e p t e d a p o s t d o c t o r a l r e s e a r c h f e l l o w s h i p w i t h P r o f e s s o r John P. P e t e r s i n t h e Department o f Medicine a t t h e Yale Univ e r s i t y School o f Medicine. S h o r t l y t h e r e a f t e r , he was a p p o i n t e d t o t h e Yale f a c u l t y , moving through t h e r a n k s o f i n s t r u c t o r , a s s i s t a n t and a s s o c i a t e p r o f e s s o r i n t h e Department o f Medicine. H e was c e r t i f i e d by t h e American Board o f I n t e r n a l Medicine i n 1954. I n 1966, because o f h i s i n t e r e s t i n t h e b a s i c s c i e n c e s , he was a p p o i n t e d t o t h e p o s t t h a t he p r e s e n t l y h o l d s a s p r o f e s s o r o f p h y s i c a l s c i e n c e s and d i r e c t o r of t h e S e c t i o n of P h y s i c a l S c i e n c e s a t t h e Medical School. D r . Lipsky i s t h e a u t h o r and c o a u t h o r o f o v e r 6 0 p a p e r s i n t h e a r e a s of l i p i d c h e m i s t r y , gas chromatography, l i q u i d chromatography and mass s p e c t r o m e t r y . H e has s e r v e d i n many d i f f e r e n t c a p a c i t i e s a s a c o n s u l t a n t t o i n d u s t r y a s w e l l a s t o d i f f e r e n t government a g e n c i e s such a s t h e N a t i o n a l I n s t i t u t e s of H e a l t h (NIH), I n s t i t u t e f o r Defense A n a l y s i s and t h e N a t i o n a l A e r o n a u t i c a l and Space A d m i n i s t r a t i o n (NASA). I n 1960, he was s e l e c t e d a s t h e P r i n c i p a l I n v e s t i g a t o r of NASA's Surveyor program f o r t h e d e t e c t i o n o f o r g a n i c compounds on t h e S u r f a c e o f t h e Moon by means of g a s chromatographic i n s t r u m e n t a t i o n . S e v e r a l y e a r s l a t e r , h i s p r o p o s a l f o r t h e development of a g a s chromatographic-mass s p e c t r o metric i n s t r u m e n t f o r a s i m i l a r t y p e of a n a l y s i s o f t h e s u r f a c e o f t h e p l a n e t Mars l e d t o h i s s e l e c t i o n a s P r i n c i p a l I n v e s t i g a t o r o f t h e NASA's Mariner program ( t h e f o r e r u n n e r of t h e r e c e n t Viking M i s s i o n ) . D r . L i p s k y ' s involvement i n gas chromatography s t a r t e d i n 1956. H e pioneered i n t h e a p p l i c a t i o n of t h e technique t o t h e a n a l y s i s of f a t t y a c i d s , t h e development of new i n s t r u m e n t a t i o n and t h e new t e c h n i q u e s o f g a s chromatography-mass s p e c t r o m e t r y and l i q u i d chromatography. A t p r e s e n t , h i s s c i e n t i f i c i n t e r e s t s i n v o l v e t h e a p p l i c a t i o n o f advanced chromatographic and s p e c t r o m e t r i c t e c h n i q u e s t o c e r t a i n a s p e c t s of t h e problem of chemical c a r c i n o g e n s i s .

In 1956, then being representative of the new emerging breed of physician scientist, I was deeply immersed in a comprehensive research project which involved a study of the rates of synthesis and transport of the specific fatty acids associated with the phospholipid, triglyceride, and cholesterol ester moieties found in the plasma of man. One of the complexities of this problem which had not been satisfactorily resolved at the time, was the development of a reliable, reproducible, sensitive, and accurate method for the separation and identification of the individual long straight chain saturated and unsaturated fatty acids as they occurred in biological materials. I had just spent an arduous and frustrating 9-12 months in attempting to adapt the reversed-phase liquid chromatographic procedures of Silk and Hahn and Howard and Martin (my first introduction to AJP!) to the problem at hand. My newly married technician and a wife, waiting at home, pregnant with our first child, did not fully appreciate the long and strenuous hours that were required to separate these infamous substances and to detect them by titration, often late at night, blurry eyed, in cramped laboratory quarters watching for an elusively distinct end point. In a rather hectic attempt to maintain domestic tranquillity in at least two different households I retreated to the library seeking a solution to my salvation. In one of those quirks of fate (in the past it seemingly took eons for me to find anything), I decided to walk over to the table that contained the recently arrived publications and for some unexplained reason I first picked up the latest issue of the Biochemical Journal and then quickly scanned through the table of contents. Ah - a familar name - my second encounter with Archer Martin! I quickly read the two articles published in this issue ( 1 , 2 ) and immediately experienced a sense of relief as my eyes became transfixed on a chromatogram showing the separation of the individual components of mixtures containing all of the fatty acids that I concerned myself with in my previous endeavor. Miraculously, it appeared that this new technique of gas chromatography possessed all the attributes I required to add a new dimension to my investigations. The speed was there - from C7 to C 1 8 in two hours, the reproducibility, the sensitivity, the accuracy - everything! I read and reread that article many times with an enormous sense of excitement. That evening my efforts were culminated with the formation of a very tidy list of materials and priorities that I considered essential for duplicating this new procedure of Martin and James that employed a gas as a vehicle for the first time. The only detail that provided me with some cause for concern was the construction of the detector - Martin’s ingenious gas density balance. Reflecting upon this, i t appeared that the wisest course of action was to communicate with Dr. Martin by letter requesting further details about the construction and operation of the detector as well as exploring the possibility of purchasing one from his source. The next morning a very polite and enthusiastic letter was on its way to England. During the next few weeks I relentlessly pursued my objectives on my list and soon much of the necessary equipment was constructed

267 or in hand. Included here was a dollar's worth of firebrick which one by one were placed in an old pillow case and promptly reduced to "brickdust" by our gleeful hammering during lunch breaks in a remote area of a nearby courtyard. During this time, the temperature-controlled cabinet, the columns, the systems for handling the gas flow and sample, took on an air of respectability. However, a response from Martin was not immediately forthcoming and having this elusive majestic gas density balance as part of the system seemed more remote than ever. Although somewhat disappointed, I was determined to explore alternative schemes for detecting the sample vapors as they emerged from the column. At this point, I enlisted the aid of two friends, Hal Henry and Maurice Godet, who at the time were graduate students in the DeparBment of Engineering at Yale. Together, we spent many evenings exploring ttgasIt detectors that could be applied to this new technique. In time, we quickly learned that the existing systems were either too insensitive or too expensive (the Sr 90 detector and associated electronics of Deal and Otvos (3)) or could not effectively operate at 20OoC. The elegance of the operating characteristics of Martin's detector were now appreciated more so than ever before. Finally, a decision was made to explore the possibilities of constructing a thermal conductivity device to meet these then rigorous specifications. One morning, some months later, a letter arrived from England. It was from Martin's younger colleague, Tony James. He was coming to New York and would be delighted to drop by in New Haven, to answer my questions and give a seminar at Yale on gas chromatography if I could make the appropriate arrangements. That old scientific "high" set in once again as I exhilaratingly reread his note. I quickly decided that the "millieux" in the Department of Medicine was not quite right for this event. With some trepidation, I approached the then Chairman of the Department of Biochemistry, a fine scientist but a rather taciturn and temperamental individual. After much discussion, the seminar was arranged. Tony James was magnificent! That Welshman had wit and style almost beyond belief. What oratory - the finest I encountered to that date - in my academic experience. The information flowed endlessly, in a brilliantly organized lecture. The seminar was an overwhemling success, Afterward, we enthusiastically talked for many hours, and amongst the overwhelming array of facts and figures I also learned that (a) Archer Martin rarely writes letters and (b) he meticously fabricates the gas density balance in his little work shop at his home in Abbottsbury - and had a long wait list. In fact Dr. James was in New York to deliver a long overdue gas density balance to "Pete" Ahrens at the Rockefeller Institute for his research studies on arteriosclerosis. In parting, I thanked Tony for a most memorable day and with mixed emotions,saw him to the train to New York. Shortly after this occasion, I received a call from Lloyd Guild of the Burrell Company in Pittsburgh who was responding to my written inquiry about my requirements for thermal conductivity detectors. Upon learning about my meeting with James and my existing knowledge of the field at the time, he suggested that he come to New Haven in order to explore the possibilities of collaborating in the development

268

Fig. 29.1. S . R . Lipsky (seated) and Maurice Godet in 1958, operating their gas chromatographic unit. of a high-temperature gas chromatographic instrument which would allow me to duplicate the analysis of mixtures of fatty acid methyl esters as performed by Martin and James. Several days later we met and exchanged information about the technique and the present status of thermal conductivity devices. As it turned out, Guild had a fair amount of experience with the analysis of gases and low-molecularweight compounds with a recently developed instrument utilizing a thermal conductivity sensing element, manufactured by Burrell. Not long after several additional meetings in Pittsburgh and New Haven, a Burrell instrument, the Kromo-Tog was installed in my laboratory with a thermal conductivity cell which could operate in the 200-250°C range. After much struggle with many of its eccentricicities (the overall instrument was not designed for high-temperature work), particularly with the detector filaments which seemed to burn out at the most inopportune times, my carefully prepared five foot "U" shaped column packed with Apiezon L coated on "brickdust" finally provided me with a separation of a mixture of fatty acids that was at least equivalent to that produced by Martin and James. Although exalted by these results, I was somewhat dismayed by the shortcomings of the instrument. Amongst other things, the poorly insulated glass columns heated by resistance wire controlled by Variacs gave rise to

269

erratic temperature control. This in turn, played havoc with the reproducibility of separation of certain of the C-18 fatty acids that I was particularly interested in. One of my goals was to split the column effluent before it entered the detector, trap out certain carbon-14 tagged acids and determine their specific activity. I promptly informed Guild about the problems with high-temperature operation. Although, personally very sympathetic, his management decided that they would like to see some return of their investment with their existing line of instruments before they made any additional major modifications. Once again we were on our own, but with a major difference - we got the whole thing to work - albeit not perfectly in all instances. In the ensuing weeks, Henry, Godet, and I concentrated on developing a new proportional temperature controller for our column compartment and further modifying thermal conductivity devices for high temperature operation. In short order, a respectable "home made" gas chromatograph emerged which provided me with the types of analyses of fatty acids that I learned to expect. Shortly thereafter, I had the opportunity to present some aspects of my work todate on the synthesis of fatty acids in the plasma of man at a regional biomedical meeting. During the talk, I showed some slides which depicted the types of separations of these substances that one could now obtain using the new English technique of gas chromatography. It was very well received and nothing unusual happened. I had no reason to expect otherwise at the time. Meanwhile, our further efforts in this area turned toward two specific areas: newer stationary phases and other more sensitive and stable detection systems. Reports began to crop up in the literature about the use of other thermally stable materials in this method. This intrigued me for several reasons. Efficiently packed columns coated with the nonpolar Apiezon grease did not provide any separation of two of the C-18 unsaturated fatty acids, linoleate (C-18:2) and linolenate (C-18:3) which I was particularly interested in. Moreover, the separation between the C-18 mono saturated acid, methyl oleate and lineolate was often incomplete. In addition, despite the presence of optimal operating conditions, the elution of the individual components of complex mixtures of methylesters of fatty acids of chain lengths up to 20 carbon atoms from the column frequently took three to four hours. With many samples at hand to analyze and with a temperamental detector that seemed to be coordinated with a particular phase of the moon, I accordingly began to test a wide variety of relatively high-molecular-weight materials as substitutes for the Apiezons ( 4 ) . One day, soon thereafter (in 1957) I received a phone call from a Dr. Evan Horning of the National Heart Institute of the National Institutes of Health in Bethesda, Maryland. He informed me that he was chairing a Committee composed of respresentatives from the food industry, government, and academia who were interested in the relationship of saturated and unsaturated fatty acids in the diet and the development of arteriosclerotic heart disease. Moreover, he stated that he had heard that I presented a paper recently in which I showed the results of the separation of fatty acids obtained by a "home made"

270 gas chromatographic unit, which were quite similar to that achieved by Martin and James. The long phone conversation ended with a request by Horning to bring his Committee to New Haven to view the technique in operation. I readily acceeded to his wishes and a meeting was arranged €or the beginning of the next week. The day before the delegation was to arrive, we tidied up our laboratory, a 12 by 15 foot room and - in a mood of anticipation decided to make our rather odd looking breadboard device respectible, by replacing several old components of the columns and detector systems with new ones. In retrospect this almost proved disastrous and something an old hand at the game would never have done. Late that day when the instrument was turned on - instant panic! Nothing worked. Although we had a collection of chromatograms that would illustrate the incredible resolving power of this tool, we felt that an actual demonstration of the equipment working was essential at this point. Over the next several hours, every component of the system which could be isolated and tested was found to be in good working order. The reassembled unit was turned on and the same difficulty prevailed. The recorder pen was wandering chaotically near the top of the chart and could not be brought down to baseline. Throughly exhausted and dejected, we tried to sleep for a few hours on the lab benches. We awoke very early that morning and much to our amazement the baseline had begun to stabilize. In hindsight, apparently the glass blower, in his anxiety to deliver our newly designed "U" shaped glass column in the shortest possible time did not "bake it out" in a high-temperature furnace overnight. What we had experienced was relatively large quantities of water vapor being eluted from the column into the detector - temporarily giving rise to enormous instability of the system. By 9:00 a.m. the instrument was churning away, elegantly separating one fatty acid from another as the Advisory Committee arrived. Amongst the visitors that day were Evan Horning, Charles Sweeley and Art Karmen (all of whom we developed very close ties with then and there - and each of these very outstanding individuals went on, independently, to make very significant contributions of their own to this rapidly emerging field). All went amazing well that day, almost beyond belief. Evan Horning obviously was enormously impressed with the technique and what we had done with it thus far. He immediately arranged for our laboratory to receive substantial financial support from the government to continue this work. His far-sightedness and enthusiasm at this particular point in time allowed all that followed to come into being. I have often reflected upon what the course of events would have been if we had not gotten our system to operate as well as it did on that memorable day. Besides these individuals, the intricate web that was beginning to form that day went on to involve me with Jim Lovelock, A1 Zlatkis, Robert Landowne, Csaba Horvfith, Ray Scott, Cliff Scott, Emmett Watson, and even Archer Martin, to name but a few. What would our lives have been like had things turned out differently. Food for thought, indeed! With adequate funding available to our laboratory, by early 1958 rapid and significant progress occurred in several different areas of

271 chromatography within a relatively short period of time. Soon after Orr and Callen announced the use of "Reoplex 400" as a polar phase for the analysis of the methyl esters of fatty acids ( 5 ) our group which included Robert Landowne at this time, introduced a series of polymers of ethylene glycol or diethylene glycol and dicarboxylic acids of decreasing carbon chain length (sebacate, adipate, gluterate, succinate) which made it possible for one to correlate the chemical nature of the stationary phase with the elution pattern of the saturated and unsaturated fatty acids. Under these circumstances, the rapid (up to C-20 in less than 60 minutes) and complete separation of all of the common fatty acid moieties was readily accomplished at temperatures less than 20OoC. Of all of the materials investigated at the time, the adipate and succinate polyesters soon became the most widely used stationary phase for this type of analysis (6). Along in here, as our work with different stationary phases intensified, our disenchantment with thermal conductivity detectors as sensing devices grew accordingly. Many of the polyester phases "bled" even after appropriate conditioning. This raised havoc with our filaments. Accordingly, we began to look again at different methods of detection. A letter to the Shell-Emeryville group (Deal, Otvos, Smith, and Zucco) brought a rapid and encouraging response along with an elegant set of blueprints of their "radiological detector for gas chromatography". Shortly thereafter the detector with small modifications and associated electronics was fabricated by our shop and the only ingredient missing was the Strontium 90 source which required an Atomic Energy Commission (AEC) license for its use. While this was pending, I contacted the U . S . Radium Company about its fabrication. Fortunately I happened to speak to a Ted Taylor of that organization. After much discussion about possible substitutions for Sr 90 I decided to visit him in Morristown, New Jersey. Taylor was most affable and informative. After learning that I did possess an AEC license for the use of soft beta emitters (in my work with carbon-14- and tritium-labelled fatty acids) he suggested the use of a tritium titanium foil for the Shell detector. Since this material would become available to us in short order we seized upon this opportunity even though it involved a slight redesign of the geometry of the cell. Upon trying the tritium source in the Shell system, two interesting facts emerged. When compared to our thermal conductivity device, the system was much more stable but the sensitivity was relatively low. Then another series of very fortuitous events occurred. I indirectly heard about Jim Lovelock's use of a Sr-90 detector at relatively high voltages employing argon in its metastable state as a carrier gas for very high excellent sensitivity. In short order, with the help of a borrowed high voltage supply and a cylinder of argon I tried the modified Shell detector with a tritium foil in this mode of operation. My results were again poor. Obviously since I could not repeat the findings of neither the Shell group nor Lovelock, something was inherently wrong with my approach. This time the answer was not long in coming. Jim Lovelock and I were to appear on the same program at the New York Academy of Sciences. He was speaking on his new detector (7) while I was to discuss new stationary phases for

272 the analysis of fatty acids (6). His talk followed mine. Near the conclusion of my presentation I showed a slide of the modified Shell detector with the tritium source and made mention of my difficulties with its performance in both modes of operation. At the end of the session Jim came over and introduced himself to me. His one question pinpointed the difficulty. What was the standing current of your source? "Something in the order of 5 x lo-'' amperes", I replied. "TOO low'', he responded. "You have to obtain a stronger source"! After much discussion, we found we had much in common. He too was involved in the analysis of fatty acids in his experiments on the freezing of red blood cells. By odd coincidence, whenever he sought to obtain results from samples he submitted to Tony James, there were difficulties with the gas density balance. Frustrated by these delays, he too developed his own detection system - a very unique one at that - with the highest sensitivity available at the time. Before the evening was over, I invited him to come to Yale for several months to work in collaborating with us on projects of mutual interest, Driving back to Connecticut that evening, the excitement which I dubbed "gas chromatography fever", a symptom common to many of us working in that field at the time, gripped me once again. The next morning I was on the phone to Ted Taylor explaining to him the urgency of developing higher activity tritium foils. Assured that he thought he could have several available to us within a few days, I went about making several small modifications in the detection system that were recommended by Lovelock. Within the week, one of the new tritium foils that arrived provided a standing current of about 3 x lo-' amperes and working precisely as Jim predicted. Obviously delighted with a stable and sensitive detector and new efficient stationary phases for the separation of the fatty acids, our need for additional instrumentation to explore other avenues with this incredible technique became obvious. About this time, a sales engineer by the name of Phil Hercz, who sold potentiometric recorders and temperature controllers for the Barber-Colman Company visited our laboratory frequently after we purchased two of his units. Being a bachelor at the time and recently arrived in the New Haven area, he would spend long evenings with us in the laboratory. He too was soon mesmerized by watching the rapidity of events as they occurred in the field. One day shortly thereafter, he invited me to join him at dinner that evening with his boss. Midway through the meal, the gentleman asked us if we could design a gas chromatograph for their company, since they already make certain of the components used in such systems. Sensing a good opportunity to update our "homemade apparatus" we agreed provided they could deliver the first two units to me within six weeks. The next day Henry, Godet, and I flew out to Rockford, Illinois, to meet with an instrument design team, who incidently did not know anything about gas chromatography but were quite competent in the areas of electrometers, voltage supplies, recorders and temperature controllers. Within short order we prepared specifications for a unique unit that had separate temperature controls for the injection port, column, and detector. The column compartment designed to accept a six foot "U" shaped glass

273 column which I found provided the best efficiencies was made from an aluminum block which could be cooled rekatively rapidly (in those days) by means of a circulating heat exchanger. The detector was an ionization chamber containing either a tritium titanium or a radium226 foil operated in the argon metastable state mode of Lovelock (arrangements were made to pay royalties to the British National Research Council). The Barber-Colman Model 10 was borne. Six weeks later when the first two units to be delivered to me were completed, I was invited to Rockford for a preview. I was shocked and delighted at the same time. The unit was enormous in size - encircled with heavy gauge steel - built for very rugged use in a factory instead of a laboratory. But it worked beautifully and was an instant success. Several years later it was replaced by the more elegant Model 5000. For the time being our instrument needs and those of many other investigators in the U.S.A. were fulfilled. When Jim Lovelock joined us at Yale, the International Symposium on Gas Chromatography at Amsterdam recently concluded. One of the highlights of the meeting was the presentation by Golay on the theory of capillary columns (8). Although his first attempts at practical results were relatively crude by todays standards, a significant increase in the resolution of several low-molecular-weight compounds was noted on a relatively short piece of narrow bore tubing using an insensitive thermal conductivity detector. It must have been obvious to many groups at the time that this was to be a giant step forward if capillary columns could be constructed to approach the theoretical efficiencies predicted by Golay. Again a favorable set of circumstances prevailed in our laboratory. With Jim's presence, we had a virtual monopoly at the time on the utilization of very sensitive ionization detectors for use with capillary columns which would only accept a small sample charge. Since we had a very adequate supply of radioactive foils that were easy to handle (i.e. tritium and -radium) plus an excellent machine shop, Jim's small dead-volume detectors were constructed and tested. Since the flow through capillary columns was in the order of 1-3 ml/min, Jim felt that the introduction of a "scavenger gas" would substantially reduce our dead volume and maintain the excellent resolution inherent in the capillary column. When he completed this modification of this micro version of the argon ionization detector, a simple sample splitter was constructed from a piece of narrow bore glass, silicone rubber column septa and a coil of stainless steel capillary tubing as a source of resistance for the split flow. A copper capillary tube coated with squalane made by Denis Desty - if I recall properly - and given to Jim prior to his departure for the U.S.A., completed the system. The sample came from a cigarette lighter filled with butane. Golay was proven right in practice. The results were astounding: Following this, we quickly decided to test the capabilities of the capillary column in resolving certain cis and trans fatty acid isomers (a separation never accomplished before by gas chromatography) by utilizing a 200 foot length of stainless steel capillary tubing x 0.010 inch internal diameter coated with a thin layer of Apiezon L.

274 We obtained efficiencies up to 200,000 theoretical plates and were able to separate methyl elaidate from methyl oleate in a relatively short period of time at 200°C ( 9 ) . Within days, word of these developments spread throughout the country. One of the first to call was a former Canadian who loved to play ice hockey in - of all places Houston, Texas. His name: A1 Zlatkis! It went something like this. "Could we come to Texas, tomorrow"? "No, we are terribly busy". More than an hour later, still on the phone: "OK, Jim has never been to Texas, he'll be there the day after next". This is the way it was for a long time to come! Shortly thereafter, we moved with dispatch to follow up on an early observation of Jim Lovelock: the development of negative peaks for certain substances as they were eluted from the chromatographic column when an ionization detector was operated at relatively low voltages. Again different versions of the micro-ionization detectors with the anode and cathode in different locations were fabricated in an attempt to formulate the optimal geometry for this particular application. One day, Jim poked a hole in one of the tritium foils we used for this application and the plane parallel detector was ready to test. A mixture of components containing different functional groups along with 2-3 hydrocarbons was prepared, injected onto a capillary column. Positive deflections were recorded for the hydrocarbons and a series of negative deflections for the ketones, aldehydes, alcohols and particularly the halogenated substances. The electron capture detector came into being with full force (10). Following Lovelock's departure from our laboratory, Landowne and I tested a variety of specific derivatives of steroids, sterols, and amino acids in a successful effort to enhance their detection by electron capture sensing system ( 1 1 - 1 3 ) . Still fascinated by the various ionization phenomena in gases, with Mike Shahin, we further explored the use of substances with high ionization potentials such as ultra pure helium, xenon, and krypton as carrier gases for the sensitive detection of permanent gases ( 1 4 ) . About this time, I became very much involved in NASA's interest in the detection of organic compounds ("life") on the surfaces of various planets that were scheduled to be visited by space probes. From previous experiences in our laboratory, in attempting to identify components of complex mixtures derived from biological materials by gas chromatography alone, I knew early on that this technique had to be augmented with an additional system for positive identification. In 1964, knowing of Ryhage's developments with the Becker separator, I thought that the then little known newly emerging combined techniques of gas chromatography-mass spectrometry could be ideally applied to this fascinating problem. Interestingly enough, although the concept of combined instrumentation was still "in utero" so to speak, NASA officialdom was so intrigued by the potential advantages of a miniaturized space-borne instrument of this type that it soon became an integral part of their planetary exploration program. Some twelve years later this device landed on the surface of Mars. From the impetus of this program, our interest in mass spectrometry grew. Soon, with the very able assistance of Walter McMurray

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275 and Brian N. Green, the first fast electrical scanning hi'gh-resolution mass spectrometer was developed in our laboratories. Obviously, the day after it was successfully completed, it was interfaced with a gas chromatograph (15). In 1966, some ten years after entering into the fascinating world of gas chromatography, some of the excitement began to wear off for me. The "public" entered the field in droves and an air of general contentment prevailed. Many of the basic problems had been satisfactorily resolved. We now were blessed with a wide selection of excellent detectors and columns assembled in attractive instruments by responsible commercial manufacturers. I felt it was time to move on. Accordingly, I decided to move into the field of liquid chromatography. Working in a Medical School, I was constantly reminded by my colleagues of all the biological substances (usually very polar or much higher molecular weight moieties) that couzd not be separated by gas chromatography. Fortunately at this time, an excellently trained chemical engineer, Csaba Horvhth joined our group. Horvhth, who as a graduate student with Halhsz at the University of Frankfurt, developed the support-coated open tubular columns, shared my views. Csaba, who worked in the dye industry in Frankfurt after leaving Hungary during the Uprising, had an excellent working knowledge of surface chemistry. Within a relatively short period of time, the technique of bonding a thin layer (pellicle) of an ion-exchange resin onto a surface-modified glass bead of uniform particle size, that would withstand rdatively high pressures (up to 3000 psi), was accomplished. These coated beads were meticulously packed into narrow-bore columns and were used for the rapid analysis of nucleosides, nucleotides, and bases at the picomole level. Emmett Watson developed a superb, sensitive small dead volume U V detector for us for this purpose ( 1 6 - 1 8 ) . High-performance liquid chromatography, as we dubbed this new version of an old technique, emerged onto the scene - soon to be aided by significant contributions by Huber, Kirkland, Knox, Majors, Scott, and others. This was an era chracterized by excitement, international good fellowship, rapid developments, and a fervor hard to describe - unless one was caught up in it. There are many fond memories in the laboratory and at the international symposia. I am sure that Art Karmen, the only other scientist who was also trained as a physician, enjoyed, as much as I did, the opportunity to be the "house physician" to our colleagues at these conferences. Why - we even "tidied" Archer Martin over his episode of acute thrombophlebitis at one of the sessions. What a small, wonderful world it was after all! In conclusion, I would like to pay tribute to the man whose genius was most responsible for it all, Professor A.J.P. Martin. He has twice made outstanding contributions to this field, in his discovery of partition chromatography and in his pioneering work on gas chromatography. He has thus altered for the better the lives of many of us. We, his scientific colleagues, thank him for allowing us to share with him this wonderful adventure.

276 REFERENCES

1 A . J . P . M a r t i n and A . T . James, Biochem. J . 63 (1956) 138. 2 A.T. James and A . J . P . M a r t i n , Biochem. J . 63 (1956) 144. 3 C . H . D e a l , J . W . O t v o s , V . N . Smith and P . S . Z u c c o , Anal. Cham. 28 (1956) 1958. 4 S.R. L i p s k y and R . A . Landowne, Biochim. Biophys. Acta 27 (1958) 666. 5 C . H . Orr and J . E . C a l l e n , J. Amer. Chem. Soc. 80 (1958) 249. 6 S.R. L i p s k y and R . A . Landowne, Ann. N . Y. Acad. Sci. 7 2 (1959) 559. 7 J . E . L o v e l o c k , A . T . James and E . A . P i p e r , Ann. N. Y. Acad. S c i . 72 (1959) 666. 8 M. J E. G o l a y , i n Gas Chromatography 1958 (Amsterdam Symposium), D . H . D e s t y , e d . , B u t t e r w o r t h s , London, 1958, p p . 36-55. 9 S . R L i p s k y , J . E . Lovelock and R . A . Landowne, ,J. Amer. Chem. Soc. 51 1959) 1010. 10 J . E Lovelock and S . R . L i p s k y , J . Amer. Chern. Soc. 8 2 (1960) 431. 11 S . R L i p s k y and R . A . Landowne, Anal. Chem. 33 (1961) 818. 12 R . A Landowne and S.R. L i p s k y , Anal. Chem. 3 4 (1962) 726. 13 R . A . Landowne a n d S . R . L i p s k y , Anal. Chem. 35 (1963) 532. 14 M.M. S h a h i n and S.R. L i p s k y , Anal. c"hem. 35 (1963) 467. 15 W . J . McMurray, B . N . G r e e n and S . R . L i p s k y , Anal. Chem. 3 8 (1966) 1194. 16 C . H o r v a t h , B. P r e i s s and S . R . L i p s k y , Anal. Chem. 39 (1967) 1422. 17 C . G . H o r v a t h and S . R . L i p s k y , J . Chromatogr. S c i . 7 (1969) 109. 18 C . G . H o r v a t h and S . R . L i p s k y , Anal. Chem. 41 (1969) 1227.

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277

JAMES E. LOVELOCK

JAMES EPHRAIM LOVELOCK w a s born i n 1919, i n Letchworth Garden C i t y , United Kingdom. H e graduated as a chemist from Manchester Univ e r s i t y i n 1941; i n 1948, he o b t a i n e d a Ph.D. i n Medicine a t t h e London School of Hygiene, and l a t e r , he was honored w i t h a D.Sc. i n Biophysics a t London U n i v e r s i t y . A f t e r f i n i s h i n g h i s u n i v e r s i t y s t u d i e s he worked f o r n e a r l y twenty y e a r s f o r t h e B r i t i s h Medical Research C o u n c i l , b o t h a t t h e Common Cold Research U n i t , i n S a l i s b u r y , and t h e N a t i o n a l I n s t i t u t e f o r Medical Res e a r c h , i n London. I n 1954, he was a t Harv a r d Medical School on a R o c k e f e l l e r t r a v e l i n g f e l l o w s h i p and i n t h e l a t e 1 9 5 0 ' s a s a v i s i t i n g s c i e n t i s t a t Yale U n i v e r s i t y Medical School. Between 1961 and 1964 he was p r o f e s s o r of Baylor C o l l e g e of Medicine and t h e U n i v e r s i t y of Houston, i n Houston, Texas. S i n c e 1964, he h a s been a f r e e - l a n c e s c i e n t i s t s e r v i n g a s a c o n s u l t a n t t o v a r i o u s companies and i n s t i t u t i o n s , w i t h a c o n t i n u i n g academic connection as a v i s i t i n g p r o f e s s o r t o t h e U n i v e r s i t y of Reading. D r . Lovelock i s t h e a u t h o r and coauthor of n e a r l y 200 s c i e n t i f i c p a p e r s d i s t r i b u t e d more o r less e q u a l l y among t o p i c s i n medicine and b i o l o g y , gas chromatography and atmospheric s c i e n c e . I n 1974 he was e l e c t e d a Fellow of t h e Royal S o c i e t y and i n 1975, he r e c e i v e d t h e M.S. T s w e t t Chromatography Medal. H i s c o o p e r a t i o n i n t h e space program w a s honored by NASA w i t h a C e r t i f i c a t e o f Recognition " f o r t h e c r e a t i v e development of a s c i e n t i f i c c o n t r i b u t i o n which h a s been determined t o be o f s i g n a l v a l u e i n t h e advancement o f t h e a e r o s p a c e technology program." D r . Lovelock's f i r s t i n t e r e s t i s i n t h e l i f e s c i e n c e s , o r i g i n a l l y i n medical r e s e a r c h b u t i n r e c e n t y e a r s i n t h e r o l e o f t h e Biosphere i n m a i n t a i n i n g t h e s u r f a c e c o n d i t i o n s of t h e E a r t h an o p t i m a l h a b i t a t f o r L i f e . H i s second i n t e r e s t , t h a t of i n s t r u m e n t d e s i g n and development, has o f t e n i n t e r a c t e d w i t h t h e f i r s t t o t h e i r mutual b e n e f i t . D r . Lovelock's involvement i n gas chromatography d a t e s back t o h i s days a t t h e National I n s t i t u t e f o r Medical Research where, i n 1956, he c o l l a b o r a t e d w i t h A.T. James on t h e gas chromatographic a n a l y s i s o f f a t t y a c i d s found i n l i p i d s from l i p o p r o t e i n s and blood c e l l s . I n t h e c o u r s e of h i s work, he developed t h e argon i o n i z a t i o n d e t e c t o r which proved t o be t h e o r i g i n of a family of d e t e c t o r s . The most important member of t h i s family w a s t h e e l e c t r o n c a p t u r e d e t e c t o r which revolut i o n i z e d environmental a n a l y s i s .

278 It was in the spring of 1951 when I first heard those fateful words "Gas Chromatography" which were to transform my life and that of my family. At the time I was working at an out-station of the National Institute for Medical Research, the Common Cold Research Unit, near Salisbury in Wiltshire. My career as a staff scientist seemed assured with years ahead to disentangle such elementary medical problems as the common cold and blood coagulation. The message was brought to me by a colleague, Dr. Keith Dumbell, who had returned from a visit to our parent Institute in London, with an account of an Open Day there. Among the laboratories of the Institute which demonstrated their work was that of A.J.P. Martin. He and A.T. James demonstrated the separation of fatty acid methyl esters with their new technique ( 1 ) . As is my custom, I did not listen carefully to Dr. Dumbell's account and instead recreated in my own mind what seemed to be a gas chromatography column. It was composed of froth, stationary bubbles of a gas, with liquid somehow percolating past them. This inverted notion stayed with me until in October of that year when I moved back to London. Here I was assigned a new medical problem, that of finding the nature of the damage to living cells by freezing and how to prevent it. In the exciting scientific environment of the National Institute, I soon discovered, as were most things discovered, by conversation in the cafeteria, just what a gas chromatograph really was and was taken to see that most elegant of detectors, the gas density balance. It was not until 1956, however, that I was able to participate actively in the development of gas chromatography, until then I was wholly concerned with problems of cryobiology. In 1956, A.J.P. Martin told me that some of the more interesting possible further developments of gas chromatography required a much more sensitive detector and he asked if I had any ideas which might lead to its development. It so happened that in the late 1940's I had had the task of developing a sensitive anemometer. Its purpose was to measure the small airmovementswhich determine whether an indoor environment is comfortable or not. It needed to be both extremely sensitive and omnidirectional in response and yet could be carried in one hand for field use. My first attempt was an accoustic anemometer constructed from war surplus radar equipment. It worked quite well but the source of sound it used, a 20 kHz aluminium resonator behaved like a laser. Its beam was so powerful that it would ignite cotton wool held in it, it could suspend coins in the air and no doubt would have deafened the neighbourhood bats. Needless to say this apparatus was neither portable nor acceptable to my sponsors and there seemed little chance then of making it so. Another approach was needed and it came to me that might be possible to label the air with ions and observe air motion through their ability to convey an electrical charge (2). I therefore coustructed a simple device consisting of a brass ball 1 cm in diameter mounted on a 2 cm long stalk. The ball was coated with about 15 pCi of radium which was extracted from the luminous dials of discarded aircraft instruments. The ball was surrounded by three symmetrically disposed wire circles

to form an open sphere. The ions produced by the alpha radiation from the radium were collected on the outer rings by polarising the space between them and the ball. Any air movement through the cage carried away ions which might otherwise have been collected. It was extraordinarily sensitive, and could even detect air movements as low as 1 ft./min. Its drawback was that it was also very sensitive to the presence of a wide range of chemical substances. Particularly those associated with smoke, such as cigarette smoke. For this reason it never succeeded as an anemometer but it is the basis of ionisation smoke and fire detectors now in common use. This entertaining but apparently barren quest between 1946 and 1948 was to bear fruit ten years later. In 1957 it seemed to me that the disadvantages of the anemometer might be turned to advantage as the basis of a gas chromatography detector. The notion of ionisation detectors was not new in gas chromatography, for Boer ( 3 ) had developed the ionisation cross-section detector of Otvos and Stephenson ( 4 ) to be a gas chromatography detector. This device was and still is a reliable detection method with characteristics very similar to those of the thermal conductivity detector. It was never a serious rival to this detector because of its requirement for a radioactive source. Indeed all detectors requiring radioactivity, no matter how small, have been dogged by formidable bureaucratic hindrance masquerading under the false colours of safety regulations. Only those such as the electron capture detector which, so far, are indispensable have survived this handicap. Back in 1956 my brief from Archer Martin was to invent something more sensitive than the gas density balance. The specifications of the ionisation cross-section detector clearly showed that it was not a likely candidate for this role and there seemed to be little chance of greatly improving it. I now recalled my earlier experiences with the anemometer and also those of physicists in earlier days who had experienced great difficulties in purifiying the gases they used for their experiments on ionisation phenomena. It seemed that the greatest disturbance of gaseous ionisation was when substances were present which attached electrons. So the first ionisation detector I tried was an electron capture detector (ECD). It consisted of a simple ion chamber containing a strontium-90 source, very similar in dimensions and design to the cross-section detector used by Boer, but operated with nitrogen as a carrier gas, instead of hydrogen or helium, and with an applied potential of only a few volts, instead of several hundred volts as with the cross-section detector. The National Institute was well equipped with mechanical and electronic workshops and possessed a loan pool of test equipment. Even so back in those days the yearly allowance of a staff scientist for apparatus and equipment was about f100. In present terms this had the purchasing power of about US$1500. Althoughnot anunreasonable sum forthose times, it meant that new ventures such as the making of speculative gas chromatography detectors grew from stony ground. The electrometer amplifiers I used were home built and they were connected to simple inexpensive galvanometric pen recorders.

I am deeply grateful to A.T. James who at that time unstintingly taught me the arts of chromatography. I hope that he in return gained from me something of biology and biochemistry. He gave me my first gas chromatgraph column, it was a straight 4 ft. long 5 mm diameter glass tube. It contained Celite coated with Apiezon L. I mounted it inside a long metal bar which was heated electrically and the temperature maintained more or less constant by manual adjustment of a variable transformer. The detector was joined at one end of this column through a short length of silicone rubber tubing. The whole assembly was insulated with glass wool and mounted vertically. This was so that the National Institute procedure of applying samples by opening the column head and pipetting on a 1-pl sample could be performed. The system was first tried with nitrogen carrier gas using it as a cross-section detector, it worked just as Boer had reported but confirmed that the cross-section detector was less sensitive'than the gas density balance. I then reduced the applied potential and reversed the connections to the recorder so as to observe electron attachment if it took place. I tried the chromatography of mixtures of common laboratory solvents and it seemed to be extraordinarily sensitive but also very erratic in its behaviour. After injecting a mixture containing carbon tetrachloride it ceased to work and nothing I did would restore its function. It took me a week to discover that enough of the CCl4 was still desorbing from the silicone rubber tubing connecting the column to the detector to saturate the latter. I now realised that we had something very sensitive indeed and reassembled the apparatus in as clean a form as I could and cautiously applied a 0.1-ul mixture of the fatty acid methyl esters which Martin and James wished to analyse. The result was impressive, a set of off-scale peaks marched across the resultant chromatogram, I called Tony James who brought with him a sample of allegedly pure methyl octanoate. We applied this to the column and were amazed to see peak after peak appearing, sad to say none of them had the retention time of methyl octanoate. We now know of course that the ECD is not sensitive to fatty acid methyl esters and that what we were seeing were probably traces of halogenated compounds, impurities in the sample. The ECD clearly did not meet the requirements of gas chromatography in 1956 and so rather reluctantly, for it was a fascinating and challenging device, I put it aside. I then moved on to see if other ionisation processess could be exploited for detection. Whatever the process the key seemed to be to ionise the solute molecules whilst leaving the carrier gas unchanged. There is a large difference between the ionisation potential of most carrier gases and that of most organic chemicals. Nitrogen, for example, ionises at 15 eV whereas nearly all organics at less than 12 eV. Could conditions be arranged in an atmospheric pressure ion chamber so that the mean electron energy was greater than 12 but less than 15 eV? At this point I had a very lucky break. When I ordered a new cylinder of nitrogen from the Institute stores they were temporarily out of stock and the only gas available was argon. Now the ionization potential of argon is closely similar to that of

281 nitrogen and therefore this gas seemed worth trying. I therefore set up a column with argon flowlng and connected to the same ion chamber which had served earlier as the ECD. This time, however, I applied a fairly high potential, 700 volts, which was insufficient to cause field-intensified ionisation of the argon but which I thought might lead to ionisation of the solute molecules, The first trial of this system using a 1-1.11 mixture of fatty acid methyl esters gave a splendid chromatogram with large peaks and a straight noise-free baseline. Repeated trials with different conditions of operation showed that this detector was consistent in its behaviour and might well form the basis of the one that Martin and James sought. Eventually the argon was used up and I returned to nitrogen as the carrier gas and was amazed to find that no similar sensitive response was available. Indeed with nitrogen the device was no more sensitive than the ionisation cross-section detector which in fact it was. A few separate experiments showed that detection was by the Penning effect. In the argon detector ( 5 ) ionisation is by collision with argon metastable atoms rather than by direct electron impact. My first encounter with other chromatographers of the U.K. was at a meeting of the Gas Chromatography Discussion Group at Oxford in 1957 where I presented a paper describing both the electron capture and the argon detector. The argon detector met with an enthusiastic response and enjoyed a brief year or two of wide-spread use. Its great sensitivity and almost universal range of response made possible experimentswith high pressure and capillary columns which otherwise were difficult or impossible with the less sensitive detectors then available. It also opened up the general application of gas chromatography in the biochemical field. By 1959 however, its rival the flame ionisation detector with its better accuracy and reliability and freedom from radioactive sources was taking over and soon replaced it in all but the most specialised activities. During the heyday of the argon detector R.P.W. Scott and D.H. Desty were frequent visitors and our newly discovered chromatographic arts were freely exchanged with them for theirs. A greatly valued gift of this kind was a 100 ft. squalane-coated copper capillary column from Desty. This I used in experiments to develop an argon detector with a small sensing volume. This column worked superbly well until destroyed by overheating but as is often the way with the first tries at a new technique it recoated successfully. Tony James took news of the argon detector to the U . S . A . on a visit in early 1958 and as a consequence I was invited there in April of that year to read a paper at a meeting of the New York Academy of Sciences. I had been to Boston for a year in 1954 to work at Harvard Medical School so America itself was not unfamiliar. On this second visit, however, I was astonished by the interest and enthusiasm stirred by the possibilities of the argon detector. In the U.K. of those days science came second in prestige to the humanities and applied science-and the development of hardware tended to be regarded as an affair for technicians or amateur enthusiasts. Indeed at the National Institute, the time I had available for detector development was a small proportion of that devoted to my

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Fig. 30.1. An early example of the use of a capillary column (100 ft. x 0.01 in. cupro-nickel coated with Apiezon L) and two detectors in parallel. The upper chromatogram is with an argon detector and the lower one with an electron capture detector. Sample volume was 1 ug; the sample consisted of a mixture of cyclohexane (solvent) (1), fluorobenzene (2), chlorobenzene ( 3 ) , m/p-dichlorobenzene (4), o-dichlorobenzene (5), 1,3,5-trichlorobenzene (6), 1,2,4-trichlorobenzene ( 7 ) and 1,2,3,-trichlorobenzene ( 8 ) . Column temperature: 125OC; carrier gas: argon.

main task of lipid biochemistry. Strangely, in spite of these stringencies the output of the National Institute was impressive. It seems that the innovation like grass does better when cut and trodden on than when overfed. At the New York meeting I met S.R. Lipsky of Yale University who was kind enough to invite me to his laboratory in New Haven for a visit of six months or more in the coming autumn. Immediately after the meeting and before returning to London I responded to an invitation from Keene Dimmick to spend a few days with him at his home in Walnut Creek. He and his wife Adele were just starting in their home what was to become a sizeable segment of the gas chromatography industry. I shall always remember their kind hospitality and the sight of the Dimmick children filling gas chromatographic columns. Perhaps

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Fig. 30.2. J.E. Lovelock in 1961, at the University of Houston, Houston, Texas.

I should mention that the conditions of my service for the U.K. Medical Research Council forbad any consulting arrangements and the patents of inventions were held by them for the common good. This had some pecuniary disadvantages but we were well paid and in the long term it enabled me to make many friends among the scientists and engineers employed in the instrument industry without a feeling of constraint that an exclusive tie to one or other of them would have engendered. At Sandy Lipsky's laboratory later that year I was able to reduce to practice an accumulation of ideas and partially completed projects. Most important of these was the electron capture detector (6) which through its capacity to discover pesticides in just about everything everywhere set the scene for Rachel Carson and the environmental movement. In November 1958 I made my first visit to that warm and humid climate of Houston and there first met A1 Zlatkis. I took with me a recoated edition of Desty's capillary column and an argon detector specifically made for use with it. This powerful combination resolved "platformates" and "reformates" and other products of the Houston oil industry as never before. It was a great pleasure to be able to share the professional satisfaction of those chemists who with Marcel Golay's splendid invention the capillary column (7) and a sensitive ionisation detector were able to make exquisitely perfect chromatograms. Such were their quality 'that when Zlatkis and I tried to publish one ( 8 ) , the referee complained that they were to good to be real and must have been faked. Fortunately we suceed in convincing him otherwise. In my last years at the National Institute in London I was able to devote most of my time to detector research. The developments of

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this period up and till October 1961 included the photoionisation detector among others and are described in a review paper ( 9 ) . During this period also Peter Simmonds came to work with me and thus began a collaboration and friendship which has lasted until the present day. Everything that has happened to me since mostly as an independent scientist working from my home in Wiltshire was made possible by Archer Martin's invention. If you have the hardware with which to solve an otherwise intractable problem it is possible to cross all of the scientific frontiers from astronomy to zoology and indeed to travel the Earth itself. REFERENCES 1 A . T . James and A.J.P. Martin, Biochem. J . 50 (1952) 679. 2 J.E. Lovelock and E.M. Wasiliwska, J . S c i . Instrum. 26 (1949) 367. 3 H. Boer, in Yapour Phase Chromatography ( 1 9 5 6 London Symposim), D.H. Desty, ed., Butterworths, London, 1957, pp. 169-184. 4 J.W. Otvos and D.P.S. Stevenson, J . Amer. Chem. Soc. 7 8 (1956) 546. 5 J.E. Lovelock, J. Chromatogr. 1 (1958) 35. 6 J.E. Lovelock and S . R . Lipsky, J . Amer. Chem. Soc. 82 (1960) 431. 7 M. J. E. Golay, in Gas Chronatography 1958 (Amsterdam Symposium), D.H. Desty, ed., Butterworths, London, 1958, pp. 36-55. 8 A . Zlatkis and J.E. Lovelock, Anal. Chem. 31 (1959) 620. 9 J.E. Lovelock, Anal. Chem. 33 (1961) 162.

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A.J.P. MARTIN

ARCHER JOHN PORTER MARTIN w a s born i n 1910, i n London, England. H e s t u d i e d a t Cambridge U n i v e r s i t y and r e c e i v e d h i s Ph.D. i n 1936. Between 1932 and 1938, h e was a s s o c i a t e d w i t h t h e Dunn N u t r i t i o n a l L a b o r a t o r y o f Cambridge U n i v e r s i t y and t h e n , between 1938 and 1946, w i t h t h e Wool I n d u s t r i e s Research A s s o c i a t i o n . I t was i n t h i s labor a t o r y i n 1941, t o g e t h e r w i t h R.L.M. Synge, t h a t h e developed p a r t i t i o n chromatography, and i n 1944, t o g e t h e r w i t h R . Consden and A.H. Gordon, p a p e r chromatography. I n 1948, a f t e r a two-year s t a y a t a p h a r m a c e u t i c a l company he j o i n e d t h e L i s t e r I n s t i t u t e o f t h e Medical Research Council and, i n 1950, t h e N a t i o n a l I n s t i t u t e f o r Medical Research a t M i l l H i l l , London. Here, t o g e t h e r w i t h A.T. James, he developed g a s - l i q u i d p a r t i t i o n chromatography. S i n c e 1956 h e h a s served as a c o n s u l t a n t t o v a r i o u s l a b o r a t o r i e s and i n s t i t u t i o n s . Between 1964 and 1974, h e w a s an e x t r a o r d i n a r y p r o f e s s o r a t t h e Technische Hogeschool ( T e c h n o l o g i c a l U n i v e r s i t y ) i n Eindhoven, The N e t h e r l a n d s , and i n 1973-1974 p r o f e s s o r i a l f e l l o w a t t h e U n i v e r s i t y o f S u s s e x , B r i g h t o n , England. I n 1974, he was a p p o i n t e d Robert A. Welch P r o f e s s o r of Chemistry a t t h e U n i v e r s i t y of Houston, Houston, Texas. D r . Martin - t o g e t h e r w i t h R.L.M. Synge - r e c e i v e d t h e Nobel P r i z e i n Chemistry i n 1952 f o r t h e i n v e n t i o n of p a r t i t i o n chromatography. H e h a s h o n o r a r y d o c t o r a t e s from t h e U n i v e r s i t i e s o f Leeds and Glasgow, i s a f e l l o w o f t h e Royal S o c i e t y , a honorary member of a number of s o c i e t i e s and t h e r e c i p i e n t o f s e v e r a l awards and medals o f v a r i o u s s c i e n t i f i c s o c i e t i e s , among them t h e M.S. Tswett Chromatography Medal and t h e American Chemical S o c i e t y Award i n Chromatography. H e i s a Command e r o f t h e B r i t i s h Empire and a r e c i p i e n t of t h e J a p a n e s e Order o f t h e R i s i n g Sun.

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When I was aschoolboy, I was exceedingly interested in chemistry and read my elder sister's university text books. I do not remember learning any chemistry at all at Bedford School, since I was always well ahead of what I was supposed to be learning in physics and chemistry. I took great interest in distillation and was particularly impressed by distillation columns. By the time I went to Cambridge University, in 1929, I had found a number of books describing the chemical engineering side of distillation columns and noted that at that time industrial research on the preparation of good columns was much in advance of laboratory research. Eighty-plate columns turning out many tons of alcohol or petroleum were available in the industry, but distillation systems in laboratories contained, at best, only a few tens of plates. At Cambridge University, I became interested in countercurrent separations and plate theory. After graduation in 1932 and spending one year in the Physical Chemistry Laboratory, I joined the Dunn Nutritional Laboratory, at Cambridge. I was interested in vitamins, and decided to look for vitamin E , unknown at that time. Various members of the laboratory were concerned with the carotenes and in 1933 Dr. A . Winterstein from Richard Kuhn's laboratory in Heidelberg visited us and demonstrated a chromatogram of a crude carotene solution on a chalk column; the carotene separated appropriately into bands of various colours. I was fascinated to see the relationship between the chromatogram and distillation columns and to realize that the processes involved in the separation of the carotenes and of volatile substances by distillation column were similar; there was relative movement of the two phases and it was their interaction at many points that gave rise to good separations. I continued to work with vitamin E and Dr. T. Moore and I started separating carotene by distribution between two solvents using separating funnels. I was sufficiently mathematically inclined to work out the extent of separation that can be obtained in this way, and was appelled to find how small this was with a single extraction. So, I set up chains of separating funnels, moving upper and lower layers countercurrently,but found that even when one has such a small number as, say, six funnels, just shaking and separating the layers becomes a full-time job. I had always been interested in engineering processes (after all, my original intention was to become a chemical engineer) and so, I started to devise machines to do the countercurrent extractions. The first machine was designed for the first stage in the separation of vitamin E ; vegetable oils were saponified and the soaps extracted with ether. After various modifications, I could achieve about eight theoretical plates and obtained very efficient extraction in this way. This method was satisfactory for extracting a particular substance from one liquid to another but much more was needed to separate two or more substances of closely similar partition coefficients. It was clear to me that it was not worthwhile trying to make an apparatus with less than about two hundred theoretical plates and so, I amplified the type of apparatus I had used for the ether extraction of soaps.

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F i g . 31.1. A.J.P. Martin a t t h e Dunn N u t r i t i o n a l L a b o r a t o r y of Cambridge U n i v e r s i t y , i n t h e mid-1930's. I n t h e f i n a l d e s i g n , 45 h a l f - i n c h t u b e s , each about f i v e f o o t l o n g , were s t a c k e d v e r t i c a l l y i n a r a c k . A p a i r o f narrow t u b e s r a n up between t h e t o p of one t u b e and t h e bottom of t h e s u c c e e d i n g t u b e and t h i s w a s r e p e a t e d f o r t h e whole s e r i e s . When a p u l s e of l i q u i d was pushed t h r o u g h t h i s a p p a r a t u s t h e h e a v i e s t l i q u i d , which had c o l l e c t e d a t t h e bottom of t h e t u b e , was f o r c e d t o t h e t o p of t h e n e x t t u b e i n t h e series, and a b a l l v a l v e p r e v e n t e d t h e l i q u i d from d r o p p i n g back. S i m i l a r l y t h e l i g h t e s t l i q u i d , which had c o l l e c t e d a t t h e t o p of t h e t u b e , w a s pumped down t o t h e bottom of t h e - n e x t t u b e (on t h e r e v e r s e s t r o k e ) and so on. These s u c c e s s i v e s t r o k e s produced a c i r c u l a t i o n between t h e bottom of one t u b e and t h e t o p of t h e n e x t and made a v a i l a b l e , i n e f f e c t , a c o n t i n u o u s 200-foot column f o r s e p a r a t i o n p u r p o s e s . The 90 b a l l v a l v e s r a t t l i n g on t h e i r seats made a n o i s e l i k e t h e s e a on s h i n g l e ! I t w a s a c o n s i d e r a b l e e f f o r t t o make t h i s machine and when i t was f i n i s h e d i t was a month b e f o r e I c o u l d summon t h e courage t o t r y i t . But a p a r t from some f i r e s and d i f f i c u l t i e s when t u b e s b r o k e ,

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the machine did ultimately work. I was able to Separate vitamin E into several obviously different and distinct fractions, for the first time. I have always had the difficulty in writing up my results and I never published this work (although it does appear in my Ph.D. thesis) or pursued it further, but it left me with a profound interest in countercurrent problems. In 1937 mychief,Sir Charles Martin, introduced me to R.L.M. Synge who was working on a scholarship from the International Wool Secretariat at the Biochemical Laboratory at Cambridge. Synge was trying to improve the methods for the analysis of proteins. He had measured the partition coefficients of acetylamino acids between chloroform and water and thought that a separation method could be based on this sort of method. But his technique with separating funnels wasnot good enough. Sir Charles suggested that my apparatus be used. Since chloroform and water were not suitable phases for it, we designed another completely different machine in which these two compounds could be used. This was made in Cambridge for Synge to my design, but it didn't work, and I took it and Synge to the Wool Industries Research Association, in Leeds, where I had moved in 1938. We were eventually successful in using this machine to separate, and measure fairly accurately, the monoamino, monocarboxylic acids in wool ( 1 ) . It was a fiendish piece of apparatus, we had to sit by it for a week for one separation; it had 39 theoretical plates and filled the room with chloroform vapour. We used to watch it in $-hour shifts. We had constantly to adjust small silver baffles to keep the apparatus working properly. One of the effects of four hours of chloroform intoxication was that when our partner arrived to take the next shift he was invariably sworn at by the one who had been watching the machine. Another curious effect of the chloroform was that when I went into the fresh air, it smelt particular. This was my first experience of the interesting phenomenon of negative smell and may have been partly responsible for my current interest in the physiology of the sensation of smell. I continued designing new machines that I hoped would be more satisfactory, but although I worked out some dozens of ideas none of them produced a machine that was sufficiently cheap and easy to seem worth making. In 1940 it occurred to me that the crux of the problem was that we were trying to move two liquids in opposite directions simultaneously. Equilibrium had to be established rapidly or the experiment took far too long, but this meant converting the liquids to very fine droplets and if the droplets were too small they would not settle out or move into the required direction within any reasonable period of time. This meant that the machine was bound to be a compromise unless I could either introduce centrifugal force to speed up the movement of droplets or think of a completely different system. Then I suddenly realized that it was not necessary to move both liquids; if I just moved one of them the required conditions were fulfilled. I was able to device a suitable apparatus the very next day, and a modification of this eventually became the partition chromatograph with which we are now familiar. Synge and I took silica gel intended as a drying agent from a balance case,

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290 ground i t u p , s i e v e d i t and added w a t e r t o i t . W e found t h a t w e c o u l d add a l m o s t i t s own weight o f w a t e r t o t h e g e l b e f o r e i t become n o t i c e a b l y w e t . W e p u t t h i s m i x t u r e o f s i l i c a g e l and w a t e r i n t o a column, p u t t h e a c e t y l a m i n o a c i d s on t o t h e t o p and poured chloroform down t h e column. W e wondered how w e s h o u l d know where t h e amino a c i d s w e r e i n t h e column and when t o e x p e c t them t o emerge a t t h e bottom o f t h e t u b e . By t h e end of t h e f i r s t day t h e r e was no s i g n of them. To f i n d o u t what was happening i n t h e column w e added methyl o r a n g e t o

F i g . 31.3. A page of t h e notebook of A . J . P . M a r t i n and R.L.M. Synge d i s c u s s i n g t h e experiment on t h e s e p a r a t i o n of a c e t y l l e u c i n e and a c e t y l p r o l i n e .

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the liquid on the silica gel and thus were able to see the acetylamino acids passing down the column as a red band. One foot of tubing in this apparatus,could do substantially better separations than all the machinery we had constructed until then. Normal chloroform contains about 1% of ethyl alcohol as a stabilizer. The first experiment we did, with chloroform straight out of a bottle in the laboratory, gave the results we expected, and we separated acetylproline and acetylleucine. We next used carefully distilled chloroform and were surprised to find that the amino acids did not move from the top of the column. The reason for this, of course, was increased absorption due to the absence of ethyl alcohol. When we added alcohol to the chloroform our bands could move down the column again. But it was difficult to produce a satisfactory colour change; we needed large amounts of acids with our original silica gel-methyl orange system. So we experimented with different ways of making precipitated silica, and eventually developed a process of stirring hydrochloric acid into sodium silicate. This process reliably produced material that behaved as we wished in the columns. But this work was more magic than science; we never understood in detail what we were doing. Later we changed the indicator; at one time we used pelargonin which we extracted ourselves from various flowers.

R

I

z $

a-

Rs VALUES IN COLLlDlNE ' 0.2 0.4 0.6 0.8 1.0

0

Fig. 31.4. Diagram showing the expected positions of amino acids on a two-dimensional paper chromatogram, prepared with phenol-ammonia (0.3%) and collidine solvents. Al=alanine, Ar=arginine, As= aspartic acid; Cy=cystine; Glu=glutamic acid; Gly=glycine; H=histidine; HP=hydroxyproline; IL=isoleucine; La=lanthionine, L=leucine; Ly= lysine; M=methionine; NL=norleucine; NV=norvaline; Or=ornithine; dAl=phenylalanine; P= proline; Se=serine; Th=threonine; Tr=tryptophan; Ty=tyrosine; V=valine. From the paper by Consden, Gordon and Martin (3).

This work was eventually published in 1941 ( 2 ) . In this paper we noted that the mobile phase could just as well be a gas as a liquid, We also predicted that with such a system, very refined separations of various kinds of compounds would be possible. Although this paper was widely read by chemists in different fields, no one thought this prediction worth testing experimentally. In spite of all our efforts we could separate only the monoamino, monocarboxylic acids. We could not make the system work for the dicarboxylic or basic amino acids. So we looked for materials other than silica to hold the water and our first choice was paper. I had seen paper chromatograms of dyes and was familiar with the uptake of water by cellulose, so paper was an abvious choice. Dr. A.H. Gordon, who was now working with us at Leeds, looked through BeiZstein's Fiandbuch der Organischen Chemie to find a colour reaction that would reveal our amino acids on the paper; he found ninhydrin which proved admirable for our purpose. Our first paper chromatograms were circles of paper in a Petri dish containing water and water-saturated butanol fed by capillarity to the centre by a tail on which a drop of amino acid solution had been placed. When the butanol reached the edge, the paper was dried and sprayed with ninhydrin in dry butanol. Later, we used strips of paper in test-tubes and more suitable containers boxes in which the air was kept saturated with water - with troughs containing the mobile solvent into which the tops of the strips could dip. Several boxes were needed since it was characteristic of the method that though it was not particularly quick, very little work was needed to run many strips simultaneously. An important step was running the chromatogram in two dimensions. The first solvent spread the amino acids in a line near one end of the paper from a spot near the corner; then, after drying, we turned the paper through a right angle and spread the line of spots into a two-dimensional pattern by using a different solvent (3). I would like to mention here that the first two-directional separation that I did was with electrophoresis in one direction in an acetate buffer and paper chromatography in the other. The tract for the electrophoresis was isolated from the rest of the column by saturating the paper with paraffin wax on either side. But chromatography turned out to be more satisfactory than electrophoresis at that time, so I did not work with electrophoresis again until 1944. Our next problem was to deal with the curious fact that in some, but not other, solvents the purple amino acid spots had a pink "beard" underneath them; and as they ran further down the paper the purple colour showed less and the pink more. The purple-coloured spots of leucine and phenylalanine, for example, had almost vanished before they got to the bottom of the paper, leaving only a faint pink blob. This unsatisfactory colour change was particularly marked with papers on which the chromatogram had been run in two directions: in phenol in one direction and in collidine in the other. This two-directional system was convenient because we could distinguish between the acidic, basic and neutral compounds. However, when the separation was run in an atmosphere of ammonia so as to increase the pH, a further problem was observed: the paper became covered with black spots.

293 E v e n t u a l l y w e found t h e c a u s e o f t h e s e problems. We i d e n t i f i e d t h e c a u s e of t h e b l a c k s p o t s on t h e p a p e r a s copper from t h e f a n s used f o r d r y i n g t h e p a p e r s i n t h e l a b o r a t o r y ; t h e s e f a n s had a b a d l y s p a r k i n g commutator which f i l l e d t h e room w i t h c o p p e r . The l a r g e amounts of copper i n t h e Leeds atmosphere a l s o c o n t r i b u t e d t o t h e copper on o u r p a p e r s . The b l a c k c o l o u r a t i o n was due t o t h e c a t a l y t i c o x i d a t i o n of phenol by copper i n t h e p r e s e n c e of ammonia, and t h e p i n k b e a r d s were caused by a copper complex of t h e amino a c i d t h a t formed on t h e p a p e r . The f o r m a t i o n o f t h e s e complexes c o u l d be supp r e s s e d by i n c l u d i n g a complexing a g e n t f o r copper cyanide f o r example - i n t h e c o a l g a s w e p u t i n t o t h e atmosphere i n t h e box. W e were p l e a s e d t o f i n d t h a t o u r s y s t e m worked e q u a l l y w e l l f o r p e p t i d e s and s u p r i s e d t o f i n d t h a t i t c o u l d s e p a r a t e almost carbohydrates(4) e v e r y o t h e r group o f compounds i t was t r i e d o n and f l o w e r p e t a l a n t h o c y a n i n (5, 6 ) a r e examples. F . H . P o l l a r d ( a t B r i s t o l ) worked w i t h m e t a l s and found t h a t t h e y a l s o c o u l d be s e p a r a t e d by p a p e r chromatography. Synge and I were busy w i t h t h e amino a c i d s and p e p t i d e s , b u t w e were v i s i t e d by many s c i e n t i s t s who were i n t e r e s t e d i n o u r t e c h n i q u e . They came and looked a t t h e p a p e r chromatograms and t h e n went away and used p a p e r chromatography f o r t h e i r own s e p a r a t i o n s . Paper chromatography was amazingly a p p l i c a b l e t o t h e s e p a r a t i o n o f widely d i f f e r e n t groups o f c h e m i c a l s . Synge and I , i n our f i r s t p a p e r on p a r t i t i o n chromatography ( 2 ) had e v o l v e d a t h e o r y r e l a t i n g t h e speed of t h e zones t o t h e p a r t i t i o n c o e f f i c i e n t . F u r t h e r , by i n t r o d u c i n g t h e concept of t h e t h e o r e t i c a l p l a t e f o r chromatograms, a p r e d i c t i o n c o u l d be made about t h e s h a p e of t h e zones and t h e i r r a t e of b r o a d e n i n g . L a t e r , a f t e r work w i t h p e p t i d e p a p e r chromatograms, I found i t p o s s i b l e , by assuming t h a t t h e f r e e e n e r g y o f t r a n s f e r o f a compound from one phase t o a n o t h e r was an a d d i t i v e f u n c t i o n of t h e f r e e e n e r g i e s o f i n d i v i d u a l atoms o r groups o f atoms, t o f o r e c a s t w i t h r e a s o n a b l e accuracy t h e p a r t i t i o n c o e f f i c i e n t and chromatographic b e h a v i o u r o f p e p t i d e s and many o t h e r s u b s t a n c e s . I n 1948 I moved f o r a s h o r t t i m e t o t h e L i s t e r I n s t i t u t e of P r e v e n t i v e Medicine i n London and t h e n t o t h e N a t i o n a l I n s t i t u t e f o r Medical R e s e a r c h , M i l l H i l l , where, i n 1950, Tony James j o i n e d m e ( h e had p r e v i o u s l y been working w i t h Synge). James and I t r i e d t o s e p a r a t e o u r m a t e r i a l s u s i n g c r y s t a l l i z a t i o n on a column - what i s now known a s z o n e - r e f i n i n g . T h i s p r o j e c t looked h o p e l e s s f o r a f e w months, and James became more and more d i s c o u r a g e d ; w e c o u l d do much b e t t e r w i t h a c o u p l e of b e a k e r s t h a n w i t h a l l t h e c o m p l i c a t e d a p p a r a t u s w e had c o n s t r u c t e d . So ( t o improve James' morale) I s u g g e s t e d t h a t w e go back t o t h e p r e d i c t i o n mentioned i n t h e f i r s t p a p e r Synge and I wrote on p a r t i t i o n chromatography (21, t o u s e a g a s i n s t e a d of a l i q u i d a s t h e mobile p h a s e ; I was s u r e t h i s would work. P r o f e s s o r J . Popjak had asked m e f o r a more r e f i n e d method t h a n p a p e r chromatography f o r s e p a r a t i n g f a t t y a c i d s and I t h o u g h t t h a t g a s chromatography might be a b l e t o do t h i s . So w e s p e n t o u r f i r s t week w a i t i n g f o r t h e bands t o come o u t o f a g a s chromatograph; i n f a c t , t h e y had a l l come o u t i n t h e f i r s t few s e c o n d s . We used q u a r t e r - i n c h - b o r e g l a s s t u b i n g , about 15 i n c h e s l o n g , packed w i t h C e l i t e (which had been found

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294 to be the most convenient material to use with liquid-liquid columns). We passed nitrogen in at one end of the column, the other end of which was provided with a capillary that dipped into a test tube containing indicator solution. A small conical flask, instead of a burette, held the titrant. The flask had a doubly bored stopper, one hole carrying a tube that passed from the bottom of the flask to a jet just above the level of the liquid in the test tube, while the other hole had a piece of valve rubber attached that could be milked between finger and thumb to express a drop of titrant from the jet. James sat with a stop watch and a piece of graph paper and timed and plotted the drops while I watched the test tube and put in a drop of titrant whenever the colour of the indicatorchanged. Plotting the number of drops against time yielded a series of steps. The height of the steps denoted the quantity of acid emerging, and their position on the time axis showed the retention time. We first separated the methylamines, since they would run at room temperature. Later, using a steam jacket for the column, we separated the first members of the fatty acid series. Initially we used a fatty, oily material, but this gave very distorted bands. I had enough experience with chromatography by this time to realize that non-linear absorption creates tailing. But on these plots we had the reverse of tailing - a long front and a sharp tail - which we eventually realized was due to the association of the fatty acid to dimers; in other words, dimerization was a considerable problem to us for six months or more. By adding a soluble acid (such as stearic acid) in excess to the stationary-phase liquid, we were able to sort out this difficulty and obtain reasonably shaped bands. This technique worked very well for fats and oils and equally well for mines. We obtained our first useful results six weeks after starting the experiment. This was the beginning of gas chromatography for us. The details still had to be worked out, but it is really astonishing how closely similar this first column was to many columns still in use today. We wanted to illustrate the technique by using it to separate some natural mixtures, so we tried to identify the amine responsible for the fishy smell of stinking goose-foot (Chenopodiwn v u l v a r i m ) . We found trimethylamine in this plant and were able to separate the three methylamines and ammonia quite readily from it. We next made an automatic titrating machine. This was a rather Heath-Robinson arrangement. The titration was recorded by using the eye to detect colour changes in the indicator and the whole thing was operated by manual drive. This was a most demanding machine; if one looked away for a second a kink in the curve appeared! As a next step, we incorporated a motor with a photocell, to drive the machine automatically. In 1951, we summarized our results in a paper presented at the Oxford Congress for Analytical Chemistry (7) and then, in a detailed manuscript submitted on June 5 , 1951 to the Biochemical JournaZ (8). In this, we discussed the results on the analysis of fatty acids, described the automatic titration machine and developed the theory of gas-liquid partition chromatography, based on the original paper I published in 1941 with Synge (2) but modifying it to allow for the

compressibility of the mobile phase. This was followed by another paper coauthored by James, G.H. Smith and myself of the analysis of methylamines ( 9 ) , and then by several papers in the next years.

w

a

0

TIME, MINUTES

Fig. 31.5. The separation of acetic, propionic, isobutyric and n-butyric acids by gas chromatography. Column length, 4ft.; liquid phase, stearic acid (10% w/w) in DC-550 silicone; nitrogen pressure, 480 torr ; flow-rate , 33 cm3/min ; column temperature, 100 OC. A, experimental curve; B, differential of experimental curve. From the paper by James and Martin (8). Soon after the publication of these papers, we started to receive many visitors who were interested in the new technique. One of the firsts was N.H. Ray from the Research Laboratories of Imperial Chemical Industries, Ltd; he actually visited us before the publication of our paper. He wanted to examine compounds which were not detectable by titration and we suggested the use of a thermal conductivity detector cell because Claesson at Uppsala University had already described the use of such a detector in gas adsorption chromatography (10). In 1954 he published his results (11, 12) but his method was not completely satisfactory for us. I tried to device a better method and developed the gas density balance ( 1 3 ) , a good detector of its day. This is the summary of my contributions to chromatography; since 1955, I have worked mainly in other areas. Since my involvement, parti: tion chromatography has developed much further than Synge and I originally expected, perhaps most surprisingly in connection with the quantity of material needed for analysis. Accepted methods of amino acid analysis before we began our work required half a kilogramme of protein and about six months’ work for a monoamino, monocarboxylic acid analysis. The silica partition columns require a few milligrammes and paper chromatograms only a few microgrammes of protein. Now gas

296

chromatographs with modern detectors can work with nanogramme quantities and the new selective detectors can even detect picogramme quantities. Thus, within a thirty-five year period, the quantity of sample needed has been reduced by a factor of 10l2. I am hopeful that methods I hope to work on, either myself or through others, will reduce this quantity by a further 103-106 without loss of accuracy. ACKNOWLEDGEMENT This contribution is based on a presentation during a CIBA Foundation Symposium held in 1969 ( 1 4 ) . We are grateful to the CIBA Foundation for the permission to utilize this text. Copies of the pages of our original notebooks were kindly provided by Dr. R.L.M. Synge. REFERENCES 1 A.J.P. Martin and R.L.M. Synge, Biochem. J . 35 (1941) 91-121. 2 A.J.P. Martin and R.L.M. Synge, Biockem. J . 35 (1941) 1358-1368. 3 R. Consden, A.H. Gordon and A.J.P. Martin, Biochem. J . 38 (1944) 224-232. 4 S.M. Partridge, Nature (London) 158 (1946) 270-271. 5 E.C. Bate-Smith, Nature (London), 161 (1948) 153. 6 E.C. Bate-Smith, Nature (London) 161 (1948) 835-836. 7 A.T. James and A.J.P. Martin, Biochem. J . 48 (1951) vii. 8 A.T. James and A.J.P. Martin, Biochem. J . 50 (1952) 679-690. 9 A.T. James, A.J.P. Martin and G.H. Smith, Biochem. J . 52 (1952) 10 11 12 13 14

238-242. S. Claesson, A r k . Kemi Miner. Geol. 23A (1952) 1. N.H. Ray, J . AppZ. Chem. 4 (1954) 21. N . H . Ray, J . AppZ. Chem. 4 (1954) 82-85. A.J.P. Martin and A.T. James, Biockem. J . 6 3 (1956) 138-143. A. J.P. Martin, “Historical Background.” In Gas Chromatography

i n Biology and Medicine (CIBA Foundation Symposiwn, London, February 5-6, 1969), R. Porter, ed., J. & A. Churchill Ltd., London, 1969, pp. 2-10.

297

STANFORD MOORE and W.H. STEIN

STANFORD MOORE was born i n 1913, i n Chicago, I l l i n o i s and grew up i n N a s h v i l l e , Tennessee where he r e c e i v e d h i s B.A. d e g r e e from V a n d e r b i l t U n i v e r s i t y , i n 1935. H i s gradua t e t r a i n i n g was a t t h e U n i v e r s i t y o f Wisconsin, i n Madison, where he r e c e i v e d h i s Ph.D. i n 1938; h i s t h e s i s was a p r o j e c t i n b i o c h e m i s t r y under t h e d i r e c t i o n o f Karl P a u l Link. The s u b j e c t s of h i s i n i t i a l res e a r c h e s was t h e c h a r a c t e r i z a t i o n o f carboh y d r a t e s a s benzimidazole d e r i v a t i v e s . Through L i n k ' s f r i e n d s h i p w i t h Max Bergmann, D r . Moore was l e d t o j o i n t h e l a t t e r group a t t h e R o c k e f e l l e r I n s t i t u t e f o r Med i c a l Research i n New York C i t y , i n 1939. After three years of apprenticeship i n p r o t e i n c h e m i s t r y as a member o f t h e s t a f f o f t h e Bergmann L a b o r a t o r y , which t h e n i n c l u d e d W.H. S t e i n , D r . Moore was away f o r t h r e e y e a r s a s a j u n i o r adm i n i s t r a t i v e o f f i c e r w i t h wartime governmental a g e n c i e s . Return t o t h e R o c k e f e l l e r I n s t i t u t e i n 1945 marked t h e b e g i n n i n g of t h e c o l l a b o r a t i o n w i t h D r . S t e i n . D r . Moore became a member o f R o c k e f e l l e r I n s t i t u t e and when t h e I n s t i t u t e became a u n i v e r s i t y , he was a p p o i n t e d Prof e s s o r o f Biochemistry. I n 1950, D r . Moore occupied t h e Francqui C h a i r of t h e U n i v e r s i t y o f B r u s s e l s ; i n 1951, he s p e n t a h a l f y e a r a s a v i s i t i n g i n v e s t i g a t o r i n Cambridge, England and i n 1968, a semester a s a v i s i t i n g p r o f e s s o r i n h e a l t h s c i e n c e s a t V a n d e r b i l t U n i v e r s i t y School o f Medicine. D r . Moore i s a member o f t h e U.S. N a t i o n a l Academy of S c i e n c e s . H e h a s been t h e t r e a s u r e r (1956-1959) and p r e s i d e n t ( 1966) o f t h e American S o c i e t y o f B i o l o g i c a l Chemists and a member of t h e S o c i e t y ' s e d i t o r i a l board (1950-1960); he s e r v e d a s t h e chairman o f t h e Panel on P r o t e i n s o f t h e Committee on Growth of t h e N a t i o n a l Research Council (1947-1949), chairman o f t h e O r g a n i z i n g Committee f o r t h e S i x t h I n t e r n a t i o n a l Congress o f Biochemistry (1964) and p r e s i d e n t of t h e Feder a t i o n o f American S o c i e t i e s f o r Experimental Biology (1970). H e h a s honorary d o c t o r a t e s from t h e F a c u l t y of Medicine o f t h e U n i v e r s i t y o f B r u s s e l s , t h e U n i v e r s i t y o f - P a r i s , and t h e U n i v e r s i t y of Wisconsin. H e r e c e i v e d t o g e t h e r w i t h D r . S t e i n t h e American Chemical S o c i e t y Award on Chromatography and E l e c t r o p h o r e s i s and t h e R i c h a r d s Medal, and from t h e C a r l s b e r g L a b o r a t o r y i n Copenhagen t h e Lindstrfim-Lang Medal. I n

1972, D r . Moore, t o g e t h e r w i t h D r . S t e i n and D r . C h r i s t i a n B. Anfinsen, was awarded t h e Nobel P r i z e i n Chemistry. D r . Moore and D r . S t e i n have c a r r i e d o u t fundamental r e s e a r c h work i n p r o t e i n chemistry and t h e u t i l i z a t i o n of chromatographic methods f o r t h e d e t e r m i n a t i o n of amino a c i d s .

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WILLIAM HOWARD STEIN was born i n 1911, i n N e w York C i t y . H e f i r s t s t u d i e d a t Harvard C o l l e g e , where he r e c e i v e d h i s B.S. degree i n 1933. H e r e c e i v e d h i s Ph.D. i n biochemist r y i n 1938 from t h e Department o f Biochemist r y o f t h e College o f P h y s i c i a n s and Surgeow o f Columbia U n i v e r s i t y , N e w York C i t y . H e t h e n j o i n e d t h e Department o f D r . Max Bergmann a t t h e R o c k e f e l l e r I n s t i t u t e a s an a s s i s t a n t . A f t e r D r . Bergmann's d e a t h he became a s s o c i a t e member (1949) and member of t h e I n s t i t u t e i n 1952. H e was appointed P r o f e s s o r of Biochemistry i n 1955. H e was a v i s i t i n g l e c t u r e r a t t h e U n i v e r s i t y of Chicago i n 1961, a t Haverford College i n 1962, and a t Harvard U n i v e r s i t y i n 1964. D r . S t e i n i s a member of t h e U.S. Nationa l Academy of S c i e n c e s and t h e American Academy of A r t s and S c i e n c e s . H e served a s a member of t h e Council of t h e I n s t i t u t e of Neurological Diseases and (1961-1966), of t h e Medical Advisory Board of Blindness of t h e N . I . H . Hebrew U n i v e r s i t y -Hadassah Medical School (1957-1970), as a t r u s t e e of Montefiore H o s p i t a l i n N e w York C i t y (1949-1974) and a s t h e chairman of t h e U.S. N a t i o n a l Committee f o r Biochemistry (1968-1969) and o f t h e E d i t o r i a l Committee o f t h e American S o c i e t y o f B i o l o g i c a l Chemists (1958-1961). H e was a member of t h e E d i t o r i a l Board of t h e Journal of BioZogicaZ Chemistry (1962-1964) and became i n 1964 an a s s o c i a t e e d i t o r and i n 1968 e d i t o r o f t h e j o u r n a l . . B e c a u s e o f i l l n e s s he r e s i g n e d t h i s post i n 1971. D r . S t e i n has honorary d o c t o r a t e s from Columbia Universit y and t h e A l b e r t E i n s t e i n College o f Medicine of Yeshiva U n i v e r s i t y ; he a l s o r e c e i v e d t h e Award o f Excellence from Columbia U n i v e r s i t y Grad u a t e F a c u l t y and A l u m n i A s s o c i a t i o n . Together with D r . Moore, D r . S t e i n r e c e i v e d t h e American Chemical S o c i e t y Award i n Chromatography and E l e c t r o p h o r e s i s and t h e Richards Medal. From t h e C a r l s b e r g Laboratory i n Copenhagen t h e y r e c e i v e d t h e Linderstgim-Lang Award. I n 1972, D r s . Moore and S t e i n were awarded t h e Nobel P r i z e i n Chemistry j o i n t l y w i t h D r . C h r i s t i a n B. Anfinsen. D r . S t e i n and D r . Moore have c a r r i e d o u t fundamental r e s e a r c h work i n p r o t e i n chemistry and t h e u t i l i z a t i o n of chromatographic methods f o r t h e d e t e r m i n a t i o n of amino a c i d s .

299

Our story begins in the late 1930's when we both joined the laboratory of Max Bergmann at the Rockefeller Institute for Medical Research in New York City. From his apprenticeship with Emil Fischer in Berlin and his own researches in Dresden, Bergmann came to the Rockefeller Institute in 1934 with a dedication to the task of understanding protein molecules in organic chemical terms. In that tradition, one of the first steps would be the expression of the empirical formula of a polypeptide chain in terms of the numbers of each of the constituent amino acid residues. Fractional distillation of amino acid esters,-.specificprecipitation methods, colorimetric analyses, and microbiological assays had all been applied, with varying degrees of success, to the analysis of complicated mixtures of amino acids. We devoted our first years with Bergmann to the extension of a solubility product method ( I ) which grew from work on the gravimetric determination of amino acids and represented an attempt to widen the range of this type of analytical approach (2). The proline and glycine contents of gelatin and collagen ( I ) , the leucine content of egg albumin (3), and the glycine content of silk fibroin (3) were determined by a procedure that was successful but (we would be the first to say) was slow and tedious. The research on this subject was interrupted by the war years, which took us both to other tasks. During this period, illness terminated Bergmann's careerprematurely in 1944. But the spirit which he had helped to convey to these two young assistants was still in the forefront of our minds when we returned to basic research in 1946. In the meantime, two elegant ideas had been generated, one in England and one down the hall from our laboratory in New York, both of which offered new hope to the analytical biochemists. Lyman Craig, at the Rockefeller Institute, had invented his procedure for countercurrent distribution in 1943 ( 4 ) , and had demonstrated (5) the potential resolving power of well-instrumented, stepwise liquid-liquid extraction. He suggested that we undertake to develop the technique for the separation of the amino acids in a protein hydrolysate. In the course of pondering his suggestion, we also reflected on the applicability of the new technique of liquid-liquid partition chromatography announced by Martin and Synge in 1941 (6) and which, in the form of paper chromatography ( 7 ) , was already revolutionizing many aspects of biochemical experimentation. We decided to investigate whether we could obtain quantitative results by partition chromatography in column form. Martin and Synge (6) had suggested that if quantitative amino acid analyses could be achieved, the chromatographic method would offer advantages in speed and simplicity over the use of a liquid-liquid extraction train for routine use.

Chromatographic experiments Martin and Synge (6) had first studied the partition chromatography of N-acetyl amino acids on silica gel columns, but for quantitative work we preferred to chromatograph amino acids as such in order to avoid any errors that might be introduced by variations

300 in yields in the derivatization. Elsden and Synge ( 8 ) and Synge ( 9 ) in 1944 had obtained qualitative separation of free amino acids by partition chromatography on starch columns. Our first experiments ( 1 0 ) with an n-butanol-water solvent system to separate phenylalanine, leucine, and isoleucine on a column of potato starch gave us irregular peaks and variable recoveries. A step toward successful chromatography came from giving the column a preliminary wash with 8-hydroxyquinoline to remove metal ions; with paper chromatography the chelation of cupric ions had proved to be advantageous (7). As soon as we had succeeded in obtaining symmetrical and well-resolved peaks with our test system of three amino acids, we were sufficiently encouraged to undertake the design of an automatic fraction collector ( 1 1 ) and a quantitative micro method for analysis of the effluent fractions ( 1 2 ) in order to facilitate a systematic investigation of the variables that might extend the method to the seFaration of all of the common amino acids of protein hydrolysates. We wished to divide the effluent into small fractions of known and constant volume (1 or 2 ml) so that analysis thereof would permit the construction of effluent concentration curves which would reveal the full resolving power of the system. The design that we developed used a photoelectric eye to count drops and an automatic re-set counter to activate the turning of a circular rack to move a new tube under the column after a given number of drops ( 2 1 ) . The next step was the quantitative analysis of the numerous fractions thus collected. The color reaction between ninhydrin and amino acids had been studied extensively since the introduction of the reagent by Ruhemann ( 1 3 ) in 1911:

0

II

Na citrate HI R - CI - W H + 2 @>C/OH/ 'OH buffer pH5 *

100" c.

mz

a

i

Ninhydpin

E

>C-N=C
'i

ONa d-NH, acid

0

+COz

1 + R-C=C

II

Diketohydpind lidene- Carbon Aldehydt diket&yclPin&rnine dioxide

-Yet the yield of blue color at 570 nm (from diketohydrindylidinediketohydrindamine) had not been a linear function of amino acid concentration. By carrying out the reaction in evacuated tubes, we showed that the yield of color at low amino acid concentrations was markedly improved and we attributed this result to a reduction in the amount of dissolved oxygen. The addition of stannous chloride as an internal reducing agent in the reagent solution made it possible to obtain linear and reproducible results ( 1 2 ) ; later versions of the method used hydrindantin (the reduced form of ninhydrin) as the reducing agent ( 2 4 ) and substituted dimethyl sulfoxide ( 1 5 ) for the monomethyl ether of ethylene glycol initially used as the organic solvent in the reagent solution. Proline gave acceptable yields of color at 440 nm.

301 Thus equipped with fraction collectors and an accurate and sensitive method for the analysis of hundreds of tubes per day on a routine basis, we undertook to work out a system with starch columns which would include the seventeen most common constituents of an acid hydrolysate of a protein (11). Several hundred chromatograms later ( 2 6 ) we had a system which would accomplish the task. The recoveries were 100 k 3% with loads corresponding to 2.5 mg of protein. We applied the method to the determination of the amino acid composition of B-lactoglobulin and bovine serum albumin ( 2 7 ) .

Effluent cc.

g

+ 2.0-Leucine Isaleuclne

B ie + c acid Lysine

"I

I

149

163

Fig. 32.1. Separation of amino acids from a synthetic mixture by chromatography on starch columns (1948-1949). A , Solvent: n-butanol-benzyl alcoholwater (1:1:0.288). Column: 1.9 x 30 cm. Load: ca. 20 mg of amino acids. Elapsed time: 3 days ( 1 1 ) . B, Solvents: n-butyl alcohol-n-propyl alcohol0.1 N HC1 (1:2:1), followed by 2:l n-propyl alcohol-0.5 N HC1 (2:l). Column: 0.9 x 30 cm. Load: ca. 3 mg of amino acids. Elapsed time: 5 days. The alanine-glutamic acid overlap was resolvable with a third column ( 1 6 ) .

179

302 Thus, by 1950 a quantitative chromatographic method for amino acid analysis was available and was utilized in studies of the time on purified peptides and proteins. The first investigators to set up the procedure outside of our laboratory were Pierce and du Vigneaud, our neighbors at Cornell Medical College, in order to determine the amino acid composition of oxytocin (18). The starch columns worked, but they were not rapid. It took two weeks to complete a full analysis. In thinking about column packings that might be amenable to more rapid flow-rates, our attention was drawn to the experiments published in 1949-1950 by Cohn (19, 2 0 ) on the chromatographic separation of purines, pyrimidines, and nucleotides on the acid form of sulfonated polystyrene (Dowex 50) or on a quaternary ammonium base (Dowex 1 or 2). The availability of synthetic organic ion exchange resins in small bead or finely powdered form was stimulating the development of a number of new chromatographic

Fig. 32.2. Separation of a synthetic mixture of amino acids on a 0.9 x 150 cm column of the sodium form of Dowex 50-X4 (1954). Load: ca 0.1 mg per amino acid. Flow-rate: ca. 8 ml/h (23).

- Gradually increasing pH 02-

01-

and @a+](0.2N pH 31

-

1.4~ pH 51)

no------+

303 procedures in analytical chemistry and we decided to see whether sufficient resolution of amino acids could be obtained. Partridge (21) was studying the possibilities of Tiselius and Claesson's displacement development method for the separation of amino acids on ion exchange resins on a preparative scale. When we tested the resolution obtainable by elution analysis with Dowex 50, the results were encouraging (22). If charge were the only factor operating, it would be difficult to separate the neutral amino acids from one another, but the polystyrene matrix supplies a non-polar component to the stationary phase. The rate of elution of an amino acid from such a resin is thus a result of the affinity of the column packing for both the ionic and the non-ionic portions of the molecule. A single column system was developed (22), using buffered Dowex 50 (ca. 8% cross-linked) which gave an analysis of a protein hydrolysate in half the time required with starch. With a column of 4% crosslinked Dowex 50, the resolution on a 150 cm column was increased to cover about fifty amino acids and related compounds (23). The method, at this stage of its development, was used by us and by others in the 1950's to obtain quantitative amino acid analyses of hydrolysates of purified proteins and foods and to determine the free amino acids in physiological fluids ( 2 4 , 2 5 ) and mammalian tissues (26). In cooperation with Werner Hirs, who was the first post-doctoral fellow to join us in these endeavors, the research was extended to the isolation of amino acids on a preparative scale by elution from ion exchange columns with volatile buffers (27) or volatile acids (28). As experience showed the utility of the approach, it seemed worthwhile to invest the effort necessary to make the analysis more rapid and more automatic. In cooperation with Darrel Spackman, who joined us in 1954, the time of a manual analysis was reduced to 48 hours ( 2 9 ) and concurrently an automatic recording instrument was designed and built for the performance of such analyses ( 3 0 ) ; the effluent was analyzed continuously. The 1958 model of the apparatus gave a complete analysis of an acid hydrolysate of a protein in an overnight run, physiological fluids were analyzed in two days. The sample required was in the range from 0.1 to 1.0 micromole per amino acid. In the subsequent twenty years, extensive academic and industrial researches on the instrumentation and on improved sulfonated polystyrene beads have reduced the time of analysis to about 1 hour and lowered the load to a few nanomoles. The recent introduction of reagents which yield fluorescent products from amino acids is extending the sensitivity to the picomole range. The standard methods of amino acid analysis do not provide separation of the D- and L-isomers. In cooperation with James Manning ( 3 1 ) , the technique was extended to the determination of the optical antipodes by derivatization of DL-amino acid with the N-carboxyanhydride of L-leucine or L-glutamic acid and chromatographic separation of the resulting diastereoisomeric L-D and L-L dipeptides. The resolution is sufficient to permit one part of D-amino acid to be detected in the presence of 1000 parts of the L-is omer

.

304

Fig. 32.3. Chromatographic analysis of a mixture of amino acids automatically recorded in 22 hours (1958; (30)). Column: 0.9 x 150 cm, Amberlite IR-120. Load: 1 micromole per amino acid. Since our principal aim was the study of protein structure, the extension of the chromatographic method to the purification of proteins was one of our early goals. The resolving power of chromatography was appealing as a possible means of obtaining a single molecular species for structural study. In 1950 Palbus and Neilands (32) obtained encouraging results on the use of a column of polymethacrylic acid resin (Amberlite IRC-50) for the purification of cytochrome c. In cooperation with Werner Hirs (33, 3 4 ) , in 1951, we were able to obtain finite distribution coefficients with bovine pancreatic ribonuclease using IRC-50 and chromatographic behavior comparable to that of a simple amino acid on an ion exchanger. Concurrently, Martin and Porter ( 3 5 ) chromatographed ribonuclease with a novel partition system. Boardman and Partridge (36) studied in detail the elution of cytochrome c from IRC-50 and obtained efficient elution analyses. Tiselius ( 3 7 ) , in the course of studying adsorbents f o r proteins, systematically explored hydrophilic gels of calcium phosphate in 1954 and suggested that hydrophilic matrices might be preferable to hydrophobic ones for many protein separations. Sober and Peterson ( 3 8 ) , in the same year, provided the key idea that opened ion-exchange chromatography to proteins in general when they introduced exchangers with a hydrophilic matrix such as cellulose. Protein structure work also presented the problem of how to separate peptides obtained by enzymic hydrolyses of polypeptide chains. Dowex 50 with the cross-linkage reduced to 2% provided a

305

Fig. 3 2 . 4 . Moore and Stein at the bench model of the automatic amino acid analyzer (1965). sufficiently porous matrix for effective quantitative chromatography of peptides containing up to 2 2 residues (Hirs et al. ( 3 9 ) ) . This technique was a key one in our structural work on ribonuclease ( 4 0 ) ; today the separation of peptides depends heavily upon the technique of gel filtration introduced by Porath and Flodin ( 4 1 ) and upon the use of cellulose- or dextran-based ion exchangers.

HistoricaZ Perspective In the course of the utilization of the chromatographic method in the solution of practical problems in protein chemistry, our thoughts ( 4 2 ) have often turned to the history of the technique. When we were graduate students, the method introduced by Tswett at the turn of the century was experiencing a renaissance in the field of organic chemistry and was being applied for the separation of carotenoids by Kuhn and Lederer, Karrer, Zechmeister and Cholnoky, and Strain. Progress in sterol chemistry was being stimulated by the technique. To the biochemist, who was working primarily with water-soluble compounds, chromatography was not a method which he then thought of as a likely laboratory tool. Martin and Synge opened a new chapter on the subject by making partition chromatography a

306 practical procedure for a host of the water-soluble substances of biochemical concern. The commercial development of synthetic ionexchange resins, originally prepared to remove dissolved salts from water and applied analytically during the war years for the fractionation of the fission-products produced in nuclear reactors, provided a new adsorptive mechanism for the separation of solutes from aqueous solution. Since so many of the constituents of living matter are ionic, the scope of ion-exchange chromatography was quickly appreciated. Yet whatever the mechanism of the partition or adsorption, the basic concept of Tswett remains at the heart of the process. The multi-plate nature of the column-eluent system gives the method a potential resolving power which he appreciated qualitatively and quantitatively. It has been our privilege, over the years, to discuss the principles and the practice of the chromatographic method with E. Lederer, Zechmeister, Craig, Martin, Synge, Tiselius, Partridge, Porath, Cohn, and Sober. The recent decades have seen a re-birth of Tswett's method that speaks for the elegance of the original idea. Also a major contribution to the progress of the science has come from cooperation between scientists in academic research and in commercial instrument design. The skill and the enterprise that have placed on the market automatic instruments for many types of chromatography has had a key role in the biochemical progress of our times . REFERENCES 1 M. Bergmann and W . H . Stein, A new principle for the determination of amino acids and its application to collagen and gelatin, J . BioZ. Chem. 228 (1939) 217-232. 2 S . Moore, W.H. Stein and M. Bergmann, Protein constituent analysis by the solubility product method, Chem. Rev. 30 (1942) 423-432. 3 S. Moore and W.H. Stein, Determination of amino acids by the solubility product method, J . Biol. Chem. 150 (1943) 113-130. 4 L.C. Craig, Identification of small amounts of organic compounds by distribution studies. Application to atabrine, J . BioZ. Chem. 150 (1943) 33-45. 5 L.C. Craig, Identification of small amounts of organic compounds by distribution studies. Separation by counter-current distribution, J . BioZ. Chem. 155 (1944) 519-534. 6 A.J.P. Martin and R.L.M. Synge, A new form of chromatogram employing two liquid phases. 1. A theory of chromatography. 2. Application to the micro-determination of the higher monoamino-acids in proteins, Biochem. J . 35 (1941) 1358-1368. 7 R. Consden, A.H. Gordon and A.J.P. Martin, Qualitative analysis of proteins. A partition chromatographic method using paper, Biochem. J . 38 (1944) 224-232. 8 S.R. Elsden and R.L.M. Synge, Starch as a medium for partition chromatography, Biochem. J . 38 (1944) ix. 9 R.L.M. Synge, Analysis of partial hydrolysate of gramicidin by partition chromatography with starch, Biochem. J . 38 (1944) 285-294.

10 S. Moore and W.H. S t e i n , P a r t i t i o n chromatography o f amino a c i d s on s t a r c h , Ann. N.Y. Acad. S c i . 49 (1948) 265-278. 11 W.H. S t e i n and S. Moore, Chromatography o f amino a c i d s on s t a r c h columns. S e p a r a t i o n o f p h e n y l a l a n i n e , l e u c i n e , i s o l e u c i n e , m e t h i o n i n e , t y r o s i n e , and v a l i n e , J. Biol. Chem. 176 (1948) 337-365. 12,s. Moore and W.H. S t e i n , P h o t o m e t r i c ninhydron method f o r t h e u s e i n t h e chromatography of amino a c i d s , J. BioZ. Chem. 176 (1948) 367-388. 13 S. Ruhemann, T r i k e t o h y d r i n d e n e h y d r a t e , J. Chem. Soc. 99 (1911) 792800 , 1486-1492. 14 S. Moore and W.H. S t e i n , A modified n i n h y d r i n r e a g e n t f o r t h e photometric d e t e r m i n a t i o n of amino a c i d s and r e l a t e d compounds, J. Biol. Chem. 211 (1954) 907-913. 15 S. Moore, Amino a c i d a n a l y s i s : aqueous d i m e t h y l s u l f o x i d e as s o l v e n t f o r t h e n i n h y d r i n r e a c t i o n , J. BioZ. Chem. 243 (1968) 6281-6283. 16 S. Moore and W.H. S t e i n , Chromatography o f amino a c i d s on s t a r c h columns. S o l v e n t m i x t u r e s f o r t h e f r a c t i o n a t i o n o f p r o t e i n hydrolys a t e s , J. BioZ. Chem. 178 (1949) 53-77. 17 W.H. S t e i n and S. Moore, Amino a c i d composition o f 8 - l a c t o g l o b u l i n and bovine serum albumin, J. BioZ. Chem. 1 7 8 (1949) 79-91. 18 J.G. P i e r c e and V. du Vigneaud, S t u d i e s on h i g h potency o x y t o c i c m a t e r i a l f r o m beef p o s t e r i o r p i t u i t a r y l o b e s , J. Biol. Chem. 186 (1950) 77-84. 19 W.E. Cohn, The s e p a r a t i o n o f p u r i n e and p y r i m i d i n e b a s e s and nucleot i d e s by i o n exchange, Science 1 0 9 (1949) 377-378. 20 W.E. Cohn, The anion-exchange s e p a r a t i o n o f r i b o n u c l e o t i d e s , J. Amer. Chem. Soc. 7 2 (1950) 1471-1478. 21 S.M. P a r t r i d g e , Displacement chromatography on s y n t h e t i c ion-exchange r e s i n s . 3. F r a c t i o n a t i o n o f a p r o t e i n h y d r o l y s a t e , Biochem. J . 4 4 (1949) 521-527. 22 S. Moore and W.H. S t e i n , Chromatography of amino a c i d s on s u l f o n a t e d p o l y s t y r e n e r e s i n s , J. BioZ. Chem. 192 (1951) 663-681. 23 S. Moore and W.H. S t e i n , P r o c e d u r e s f o r t h e chromatographic d e t e r m i n a t i o n o f amino a c i d s on f o u r p e r c e n t c r o s s - l i n k e d s u l f o n a t e d p o l y s t y r e n e r e s i n s , J. BioZ. Chem. 211 (1954) 893-906. 24 W.H. S t e i n , A chxomatographic i n v e s t i g a t i o n o f t h e amino a c i d c o n s t i t u e n t s o f normal u r i n e , J. Biol. Chem. 201 (1953) 45-58. 25 W.H. S t e i n and S. Moore, The f r e e amino a c i d s o f human blood plasma, J. BioZ. Chem. 211 (1954) 915-926. 26 H.H. T a l l a n , S. Moore and W.H. S t e i n , S t u d i e s on t h e f r e e amino a c i d s and r e l a t e d compounds on t h e t i s s u e s o f t h e c a t , J. Biol. Chem. 211 (1954) 927-939. 27 C.H.W. H i r s , S. Moore and W.H. S t e i n , I s o l a t i o n o f amino a c i d s by chromatography on i o n exchange columns; U s e o f v o l a t i l e b u f f e r s , J. BioZ. Chem. 195 (1952) 669-683. 28 C.H.W. H i r s , S. Moore and W.H. S t e i n , The chromatography o f amino a c i d s on i o n exchange r e s i n s . Use of v o l a t i l e a c i d s f o r e l u t i o n , J. Amer. Soc. 76 (1954) 6063-6065. 29 S. Moore, D.H. Spackman and W.H. S t e i n , Chromatography o f amino a c i d s on s u l f o n a t e d p o l y s t y r e n e r e s i n s . An improved system, Anal. Chem. 30 (1958) 1185-1190.

308 30 D.H. Spackman, W.H. S t e i n and S . Moore, Automated r e c o r d i n g a p p a r a t u s for use i n t h e chromatography o f amino a c i d s , Anal. Chem. 30 (1958) 1190-1206. 31 J . M . Manning and S. Moore, D e t e r m i n a t i o n of D- and L-amino a c i d s by i o n exchange chromatography a s L-D and L-L d i p e p t i d e s , J. B i o Z . Chem. 243 (1968) 5591-5597. 32 S. P a l b u s and J . B . N e i l a n d s , P r e p a r a t i o n o f cytochrome c w i t h t h e a i d of i o n exchange r e s i n , Acta Chem. Scand. 4 (1950) 1024-1030. 33 C.H.W. H i r s , W.H. S t e i n and S. Moore, Chromatography of p r o t e i n s . R i b o n u c l e a s e , J. Amer. Scc. 7 3 (1951) 1893. 34 C.H.W. H i r s , S. Moore and W.H. S t e i n , A c h r o m a t o g r a p h i c i n v e s t i g a t i o n of p a n c r e a t i c r i b o n u c l e a s e , J. B i o l . Chem. 200 (1953) 493-506. 35 A.J.P. M a r t i n and R.R. P o r t e r , The chromatographic f r a c t i o n a t i o n of r i b o n u c l e a s e , Biochem. J. 49 (1951) 215-218. 36 N.K. Boardman and S.M. P a r t r i d g e , S e p a r a t i o n of n e u t r a l p r o t e i n s on ion-exchange r e s i n s , Biochem. J. 59 (1955) 543-552. 37 A . T i s e l i u s , Chromatography o f p r o t e i n s on c a l c i u m phosphate columns, Ark. Kemi 7 (1954) 443-449. 38 H.A. Sober and E . A . P e t e r s o n , Chromatography o f p r o t e i n s on c e l l u l o s e i o n - e x c h a n g e r s , J. Amer. Chem. Soc. 76 (1954) 1711-1712. 39 C.H.W. H i r s , S. Moore and W.H. S t e i n , P e p t i d e s o b t a i n e d by t r y p t i c h y d r o l y s i s o f p e r f o r m i c a c i d - o x i d i z e d r i b o n u c l e a s e , J. B i o l . Chem. 219 (1956) 623-642. 40 S. Moore and W.H. S t e i n , Chemical s t r u c t u r e o f p a n c r e a t i c r i b o n u c l e a s e and d e o x y r i b o n u c l e a s e , Science 180 (1973) 458-464. 41 J . P o r a t h and P. F l o d i n , G e l f i l t r a t i o n ; a method f o r d e s a l t i n g and group s e p a r a t i o n , Nature 1 8 3 (1959) 1657-1659. 42 W.H. S t e i n and S. Moore, Chromatography, S c i e n t i f i c Amer. 184 (1951) 35-41.

309

H.W. PATTON

HUGH WILSON PATTON was born in 1921, in Lebanon, Tennessee. He obtained a B . S . in Chemistry from Middle Tennessee State University, Murfreesboro, in 1945, and a Ph.D. in Physical Chemistry from Vanderbilt University, Nashville, Tennessee, in 1952. From 1950 to 1953 he was professor of chemistry at Arkansas State Teachers College. In 1953, he joined the Tennessee Eastman Company as a research chemist. He served in various other assignments within the Tennessee Eastman Laboratories including assistant director of research between 1970 and 1973. Since 1973 he has been a vice president of Eastman Chemical Products, Inc. , and is at present responsible for technical servicq and new products. Dr. Patton has published a number of papers on various subjects. He wrote two chapters in the book Principles and Practice of Gas Chromatography edited by R.L. Pecsok (J. Wiley & Sons, New York, 1959) which represented one of the first books on the subject. Dr. Patton's involvement in gas chromatography started in July 1953. In September 1954, he presented the first American paper on the instrumentation and application of the technique at a nationwide meeting.

310 I first heard of gas chromatography in early July, 1953, from W.I. Kaye who was my supervisor at that time. I had been working as a research chemist in the Tennessee EastmanResearCh Laboratories for about three months. Another employee of these laboratories (J.E. Guillet, now at the University of Toronto) was on educational leave in England. He become aware of the early work of A.J.P. Martin and his associates in 1952, and wrote to our director of research to suggest that Martin's technique should be studied in our laboratories. In my first conversation with W.I. Kaye on the subject of gas chromatography, I was asked to study the available literature, and do preliminary experiments to determine the applicability of this technique to analytical problems of interest to us. At this point we did not have Martin's papers available, but did have a letter from our friend in England describing the method with some comments on equipment he had observed while visiting in the laboratory of N.H. Ray. I did a review of the literature and found a number of papers dealing with separation of gases and vapors using columns of adsorbents of various kinds and using a variety of means to move sample components along the column. About three months after I started this project, J.S. Lewis joined me and we worked together for many years. From the beginning it seemed to us that a proper detector and recording system was a necessary key to rapid evaluation of separation techniques. My first experiments used an infrared spectrometer to detect hydrocarbons gases as they were eluted from a charcoal column with nitrogen carrier gas. The separation took hours, it was inefficient and infrared detection was cumbersome and insensitive. A part of the information we received from Guillet was a diagram and description of a thermal conductivity cell. It was made of glass, had a single, straight platinum filament and allowed the effluent gases to pass directly through the cell. We had a cell of this kind made in our glass shop. It worked, but it was rather insensitive and quite susceptible to variations in temperature and flow-rate. In the course of seeking for improvements and alternatives we found in our laboratories commercial thermal conductivity cells which were used for gas analysis; e.g., hydrogen in hydrocarbons. In each cell there were four compact, helically wound tungsten filaments within a cubical brass block. The mechanical and electrical design compensated effectively for temperature and flow variations. Four our purpose, the response time of these cells was much too long. It was obvious that a change in the internal geometry to provide more nearly direct passage of effluent gases over the filaments would improve response time, but our colleagues who had experience with the cells were apprehensive about the effect of such changes on baseline stability. We tried a number of different arrangements (drilled or plugged holes in the brass block) until we found an acceptable compromise between response time and sensitivity to changes in flow rate. It turned out that the disturbance caused by passing carrier gas through

311 the filament cavities was less severethan we expected. In addition, we found that it was not necessary to put the effluent gases straight through the cavity to have adequate respoase time. Our compromise geometrycausedthe gases to pass through one end of the filament cavity. The internal geometry is shown in our original paper ( I ) . This design was made available to the Gow-Mac Instrument Co., the manufacturer of the commercial cell which we had modified. Commercial cells of this design were available by the time our paper appeared in print. Ready availability of a good thermal conductivity cell made it easy for analysts to construct their own chromatographs. Having developed a satisfactory detector, we began to recognize the remarkable scope and power of the method. Our attention was focussed early on means of analyzing mixtures of light hydrocarbons because of important projects which required such analyses and for which no satisfactory method was available. Thus we tried a variety of adsorbents, carrier gases and operating conditions for this purpose. Gas-liquid methods were given second priority because, at the time, gas-adsorptian chromatography seemed better suited for our purposes. The equipment used for our earliest work with thermal conductivity cells was assembled from components which were available around the laboratory. These parts were simply attached to an ordinary distillation rack.

Fig. 33.1. Photographof one of the first gas chromatographs constructed at Tennessee Eastman Company.

312 Except for the early information from our friend in England, and the published literature, we had no contact with other groups working on gas chromatography until after our first paper was presented. We wrote up our early work on gas adsorption chromatography of hydrocarbons in the spring of 1954 and presented a paper at the Symposium on Advances in Separation of Hydrocarbons and Related Compounds before the Division of Petroleum Chemistry at the 126th National Meeting of the American Chemical Society in September (1954) ( I ) . I was astounded at the interest shown in this work. I remember standing outside the meeting room answering questions from a group of interested people for more than an hour. Breakfast and luncheon discussions were arranged for the next two days. Invitations to speak on gas chromatography came during the remainder of the ACS meeting and in the weeks that followed. It was obvious that there was intense interest in gas chromatography, especially among instrument comoanies. Many chemical and petroleum companies were already aware of the work of Martin and others in England, and some of them hadtheirown programs well under way. It was a time of great excitement and expectation. Everyone who was involved in these early discussions could see that gas chromatography had an unprecedented potential as an analytical method. Even s o , I think we all underestimated that potential by a large factor. Although our initial work at Tennessee Eastman Company gave priority to gas-adsorption chromatography, we had a parallel program on gas-liquid chromatography and our first paper on this subject was presented by J . S . Lewis at the Southeastern Regional Meeting of the American Chemical Society in November, 1955 (2). One of the early invitations for a paper came from the program committee of the Third National Air Pollution Symposium. We did a project in which we collected exhaust gases from my automobile and analyzed them by means of gas chromatography. We also used mass spectrometry, and infrared and ultraviolet spectroscopy on the separated fractions. This small exploratory project demonstrated the power of gas chromatography, especially in combination with other methods, to analyze complex mixtures such as those encountered in air pollution studies (3). Another interesting early application involved gas chromatographic analysis of cigarette smoke. The more volatile components of this exceedingly complex mixture were separated and analyzed to a degree that was previously impossible ( 4 ) . Although the thermal conductivity cell described earlier was satisfactory for most purposes, we discovered that the difference in geometry of the sample and reference channels sometimes resulted in spurious signals from abrupt ambient pressure changes. This kind of disturbance became especially bothersome during a project to determine trace components in hydrocarbons. Lewis and I spent an interesting evening in an otherwise abandoned laboratory testing various electrical and mechanical sources of noise. We discovered that the main cause of the noise was opening of the external doors to the building, quite some distance from our laboratory. Cells with identical paths for sample and reference gas, were found to be much less susceptible to such effects. In addition, such cells enabled use of two columns (one at a time) with one detector ( 5 ) .

313 The need for standardization and storage and retrieval of gas chromatographic data was recognized early in the Tennessee Eastman Research Laboratories. Over a period of years, J . S . Lewis was primarily responsible for development and refinement of a system which was later adopted by ASTM and made generally available (6). In the early 1960's it was possible to plot the number of papers published on gas chromatography vs. time and get a good fit to a logarithmic function. Extrapolation of this plot led to the ludicrous prediction that all scientific journals would be completely filled with such papers within a few years. Even though this projection must have failed sometime during the years, it is probably fair to say that gas chromatography has become the most widely useful analytical method yet devised. REFERENCES 1 H.W. Patton, J . S . Lewis and W.I. Kaye, 126th National American Chemical Society Meeting, New York, N. Y . , September 12-1 7 , 1954; Anal. Chem. 27 (1955) 170. 2 J . S . Lewis, H.W. Patton and W.I. Kaye, Southeastern Regional American Chemical Society Meeting, Columbia, S . C., November 3-5, 1955; Anal. Chem. 28 (1956) 1370. 3 H.W. Patton and J . S . Lewis,Fractionation and Analysis on a Micro Scale by Gas Chromatography, Third National A i r Pollution Symposiwn, Pasadena, C a l i f . , A p r i l 18-20, 1955. 4 H.W. Patton and G.P. Touey, Anal. Chem. 28 (1956) 1685. 5 J . S . Lewis and H.W. Patton, in Gas Chromatography (1957 Lansing Symposium), V.J. Coates, H . J . Noebels and I.S. Fagerson, eds., Academic Press, New York, 1958; pp. 145-153. 6 J . S . Lewis, G. McCloud and W. Schirmer, J . Chromatogr. 5 (1961) 541.

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315

C.S.G. PHILLIPS

COURTENAY STANLEY GOSS PHILLIPS was born in 1924 in Newport, Monmouthshire, England. He was educated at Haileybury and at Merton College, Oxford University, receiving his B.A. degree in 1949, M.A. in 1949 and D.Sc. in 1964. In 1948 he was appointed Fellow and tutor in Chemistry at Merton College, and later university lecturer in the Inorganic Chemistry Laboratory, Oxford University. In 1965, he was visiting professor at the University of California, Riverside. Most of D r . Phillips' published work has been in or related to gas chromatography, including the first book on Gas Chromatography published by Butterworths in 1956. He has also written a two-volume text on Inorganic Chemistry with R.J.P. Williams published by Oxford University Press in 1965 and 1966. Dr. Phillips is one of the founder members of the (Gas) Chromatography Discussion Group and has served twice as its chairman. Honoring his wideranging achievements he was awarded the M.S. Tswett Chromatography Medal in 1975. Dr. Phillips' involvement in gas chromatography began in 1946, when following the classic studies of Claesson, in Sweden, he further developed the various adsorption techniques. He was one of the first who realized the importance of the work of A.T. James and A.J.P. Martin in gas-liquid partition chromatography. Since then he has been involved in many aspects of gas chromatography such as the study of inorganic volatiles, the investigation of heterogeneous catalysis and various adsorbents.

316 I have been led to believe that intelligent scientists pick their research topics by a process of logic: many of mine seem to have arrived by accident. Others have arisen because I have been unusually fortunate in having over the years a steady stream of able research students; alas, in what follows it will not be possible to refer by name to more than a few of them. I began my scientific research in September 1945 in a dusty corner of a room in the same department in which I work today. One of my earliest encounters was with the great Professor Soddy (of isotope fame) who as a result of the news of the atomic bomb decided to return to sort out his old radioactive specimens which had been stored for many years in cupboards beside the apparatus I had just inherited. Now we have, in Oxford, a truly admirable feature of the undergraduate chemistry course, the Part 1 1 , in which all students do one year of unadulterated ”research”; not everyone discovers a great deal new about the natural world in his or her year, but all seem to learn a great deal about themselves and probably for the first time in their lives do some real science rather than indulge in mere book learning or craft training. My Part I1 supervisor, and incidentally my predecessor as Fellow and Tutor at Merton College, was Dr. B. Lambert. He had become famous and, somewhat reluctantly, financially secure as a result of his work on the charcoal gas mask in World War I. The Fellows of Merton then decided that, despite his being a mere chemist, he must have some intellkgence and duly elected him to a fellowship. He was a beautiful experimentalist and in particular an outstanding amateur glassblower; his technique was quite distinct from the professional and involved taking great care in setting up the work in clamps so that a fine result could be obtained even by those with little real glassblowing skill. He certainly taught me something of the art of making experimental life easier by careful planning and design of experiments. In many ways his own experimental work was too good, his measurements often being so detailed that it was difficult to explain them: his contemporary C.N. Hinshelwood did better science with less convincing experimental expertise. My Part I1 project involved very precise gas density measurements with a quartz buoyancy microbalance. Fortunately, most of the constructional and preparative work had already been done before I arrived, but I still had to learn the hard way to make allowance for every tiny source of possible error and to take extreme care with each measurement. It was a little like playing ”snakes and ladders” in that one slip would take one back down a snake to the position one was in some six or eight weeks before. Though I learned a lot it was not really my scene, and ever since I have found myself attracted to those experiments which can be set up to give one an answer in a matter of days. We finished the work in little over a year, incidentally determining the atomic weights of carbon and nitrogen relative to oxygen with a precision and reliability close to that which can be achieved quite simply with a mass spectrometer ( 1 ) . We then (1946) had to find a real D. Phil. project. Lambert pointed out that it would be decidedly useful for my professional advancement to have some bigger guns behind me, and so we jointly approached

317 Hinshelwood to act as what one might call consultant supervisor. He was then interested in the gas kinetics of hydrocarbons and was seeking a better analytical method. His own experience in World War I1 with the differential percolation of gases through gas-mask charcoals suggested that this might form the basis of a satisfactory technique. And s o , Lambert and I set about building in glass what was to be essentially a gas-solid elution gas chromatographic system using a thermal-conductivity detector and with the temperatures controlled by means of vapour baths. Our original intentions were deflected when an observant colleague pointed out a tiny snippet in an article by Ralph Muller in Analytical Chemistry. He wrote about the work at Uppsala of Stig Claesson who had successfully used displacement gas chromatography for the separation of hydrocarbons on charcoal (2). We communicated with Claesson who was very helpful and as his results had proved so promising we modified our own design to be able to use his displacement method. This indeed worked very well, and my first gas chromatographic results appeared in print in 1949 (3). In the meantime (December 1947) I had, much to my surprise, been elected to a tenure fellowship and tutorship at Merton. I had also been told to go abroad for eight months to "grow up" or, as the College euphemistically expressed it, gain a wider scientific experience. By a stroke of good fortune Linus Pauling was then the visiting Eastman Professor at Oxford, and he was immensely generous with letters of introduction and with advice on what to do. Among other things he pointed out that the U.S.A. was obviously the country to go to, that California in addition to its intrinsic attractions was conveniently placed to justify many visits both going to it and returning, and that as he would be in Oxford perhaps Berkeley would be more exciting than California Institute of Technology for an extended stay. Wisely and happily I followed his advice. For me the abiding scientific result of this American experience was the strengthening of my faith in not being dissuaded from doing something merely because it had not been done before. I am sure that it can often be a most fruitful exercise to ask oneself precisely why something has not been done. When I returned in October 1948 I had to take on a good deal of teaching, including a mass of material which I thought I had learned as an undergraduate. I think I learned more chemistry in one year of teaching than in three years of undergraduate learning, and I have since found that one of the best ways to learn something is to undertake to give a lecture course on the subject. I also believe that this can sometimes be as interesting to the student as receiving the accepted word from an established authority. Anyway, as a result of this theory I have written books on Inorganic Chemistry and on Group Theory and have become something of an expert on security matters. In those days, I was also the only science tutor at Merton, which meant that apart from having to keep up with all branches of chemistry and acting as "moral" guide to physicists and engineers, I was frequently approached by elderly dons who felt that I should be able as a chemist to deal with all their personal medical problems! I now had to begin supervising a number of research students

318 which meant looking around for some other fields. We did then some work on the separation of high polymers by liquid chromatography, and I became interested in a variety of electrochemical phenomena (the cracked-tube, the glass-rod and the rotating platinum polarographs, undervoltage and chemical electrostatics) as well as continuing with my interest in displacement gas chromatography, where among other things we began to play with a variety of possible detectors. It is perhaps worth mentioning that at this time the head of my department felt that gas chromatography would have no future and that I was able to proceed largely because two visitors from I.C.I. were generous enough to put the opposite view to him. In 1951, I became aware of the work of James and Martin on gas-liquid elution chromatography, and it soon became all too apparent that this was going to be the superior analytical gas chromatographic technique. In hindsight, of course, this should have been obvious to me when I first started in gas chromatography, but I had just not analysed the problem clearly enough to see it. Curiously I believe I would have stumbled by accident into gas-liquid elution chromatography if I had not heard of Claesson's work, for our initial experiments had shown some years before that better elution results were obtained when we had set out to heavily "poison" the charcoal adsorbent with an involatile liquid. Perhaps there is a lesson to be learned here about not reading too much in the literature before one starts experimenting in a new direction. On the other hand, the basic idea of gas-liquid chromatography had been clearly set out in Martin and Synge's original paper on liquid-liquid partition chronatography which I had read but had not registered properly. On reflection I believe that one often does the most original and productive work with only a brief initial look at the literature, followed at a later date by a more thorough reading when one's own experimental experience makes one more alive to the finer points. At this time, therefore, my research group joined the bandwagon of gas-liquid chromatography. It was a happy period for not only was there so much waiting to be done, but there was a remarkable spirit of cooperation among all those who were engaged in developing and applying the technique. This was particularly marked in the activities of the (Gas) Chromatography Discussion Group which over the years has done so much to disseminate ideas and to brihg together chromatographers from all over the world. It has also, to my mind, through the dedicated labour and inspiration of C.E.H. Knapman, produced the model abstracting service. Our own experimental contributions were fairly obvious ones. Thus, for example, we did the first work on temperature-programming which I saw merely as the analogue in gas chromatography of gradient elution in liquid chromatography ( 4 ) . We also devised a number of detectors, e.g. flowimpedance, specific-heat, surface-potential, and dielectric-constant which are now probably only of historical interest. As was perhaps natural for one of the few academic laboratories working then in gas chromatography, we became interested in its non-analytical applications. The determination of adsorption isotherms and the thermodynamic study of adsorption processes had been a feature of our dis-

319 placement work ( 5 ) ; the thermodynamic study of solutions by gas-liquid chromatography followed logically under the impetus of A.B. Littlewood (6). In those days we also felt that it would be possible to set up standard sets of chromatographic data which would be readily and accurately reproduced from one laboratory to another. Subsequent experience has shown this to be much harder than we had imagined, particularly because of oxidative and other degradation of stationary phases in normal operation. A not unhappy act of fate had placed me i n the Inorganic Chemistry Laboratory at Oxford University, and here I have remained over 30 years despite the fact that my experimental interests have mainly been in the application of what is basically a physical technique to the study of hydrocarbons. However, quite early on (1954) we recognised that gas chromatography could have important applications in inorganic chemistry, and indeed because superior separations are carried out with small sample quantities in an inert atmosphere, the technique is in many ways ideally suited to the discovery and investigation of new inorganic materials many of which are readily attacked by air. The first examples we looked at were the silanes, of which only the first three had been identified and these only with great difficulty. They can be made easily, e.g. by the hydrolysis of magnesium silicide, but are spontaneously inflammable on contact with air. We soon found that there were in fact a whole range of silanes (we investigated up t o Si9H20) which were the exact analogues of the straight and branched-chain paraffin hydrocarbons and which were readily separated by gas-liquid chromatography (7). Furthermore, much of the identification could be accomplished using variations of the gas chromatographic procedures. Thus the retention data of the silanes (and similarly later of the germanium and mixed silicongermanium hydrides such as Si2Ge3H12) followed beautifully the pattern established by the hydrocarbons with the n-isomers lying on a straight line in the plot of log(retention time) against number of silicon atoms, their molecular weights could be determined at the microgram level using a combination of thermal-conductivity (or PV measurement) and density-balance detectors, their formulae could be established by conversion to and chromatographic analysis of the chlorides (e.g. Si2Ge3H12 to 2 Sic14 + 3 GeCl,, + 12 HCl), and the straight-chain isomers removed by 52 molecular sieve. This work was extended to the silicon and germanium alkyls, boron hydrides, and the borazines (derivatives of the aromatic ring B3N3H6). In the last case we were able readily to distinguish the number of free NH groups in the molecule by comparison of retention data using a hydrogen-bonding (oxygen-acceptor) and non-hydrogen-bonding stationary phase ( 8 , 9 ) . The preparation and identification of new compounds in this manner can be remarkably easy, and I have always felt that the potential of gas chromatography and now HPLC has not been fully realised by those who work on the preparation of new inorganic compounds, particularly in the organometallic field. Another inorganic application arose from the work of Bradford, Harvey and Chalkley with columns containing silver nitrate. We developed a whole series of gas-liquid columns containing metal atoms

320 (e.g. the metal stearates, and the dodecyl-salicyl-aldimines of Ni, Dd, Pt and Cu ( 1 0 ) ) with which very specific separations could be achieved because of the sample acting as a ligand in complexing to the metal atoms. Conversely measurements of retention data were shown to provide very subtle information about metal-ligand equilibria. These measurements neatly complemented the more traditional studies in aqueous solution in that they were measured against an organic background, could involve only very small energy changes, and could be made extremely rapidly (in principle down to something like one per second). Again this is an area where an enormous amount of work could be done relatively easily by those interested in metal-ligand interactions. The method can also be applied to other complex equilibria as we showed by work with tri-o-thymotide (inclusion compounds) and desoxycholic acid (as a model for biological processes) ( 1 2 ) . In 1962, another curious twist of fate resurrected my interest in gas-solid chromatography. C.G. Scott had been working for many years in the laboratories of the Lobitos Oil Company and had become internationally known for his work on elution gas-solid chromatography. He had, however, entered industry directly from school, bypassing the normal university route. Through R.P.W. Scott (no relation) I discovered to my great and pleasant surprise that C.G. would like to work for a degree with me in Oxford. Now the University of Oxford is a remarkable institution in that, if the cause is good enough, nearly anything can be done. So with the assistance of letters of support from two Nobel prize winners (Martin and Hinshelwood) C.G. Scott came to Oxford for two years to continue and to develop his work for the D. Phil. degree. He naturally rekindled my enthusiasm for adsorbents, so that a number of my research students continued the theme which he had begun of modifying alumina (later Si02, Ti02 etc.) surfaces with various salts to produce highly-satisfactory stationary phases for gas chromatography. One of the peculiarities of these materials is that significant changes can be made by substituting one alkali halide for another, while very profound changes result from the use of ions such as Ag+, Cu+, or Cd2+. The displacement method also came back into its own and we were able to show that this could be used for relatively large-scale (up to 50 g) separations and for the concentration (e.g. by 10' for alkenes in alkanes) of trace impurities ( 1 2 ) . This work of Scott has also led on to our present-day activities, The first of these arose by accident. One of my research students was investigating the properties of a KCl/A1203 column and obtained a peculiar chromatogram which had clearly arisen from the partial reaction of his sample (an alkyl halide) to give olefins by elimination of HC1. It seemed to me that we might get more information about the reaction by stopping the gas flow for a few minutes so as to allow the reaction to proceed while the chromatography was interrupted. Much to my initial surprise this worked much better than I had thought, for the stopped-flow peaks thus generated proved to be extremely sharp as proper reflection shows they must be, From this rather modest beginning we have since developed and adapted a whole series of gas chromatographic methods ( 1 3 , 2 4 ) which now enable us to study (particularly heterogeneous) catalytic processes

321 using modules constructed from conventional gas chromatographs. These methods make possible a remarkably detailed study of the kinetics of a whole variety of catalytic reactions with ease and rapidity, and with a much greater degree of control and insight than is possible by more conventional methods. As an example, we have recently been investigating the hydrogenolysis of hydrocarbons on Ni/SiOz, the initial work on which has formed the D. Phil. thesis of K.F. Scott, who is the son of R.P.W. Scott ( 1 5 ) . These studies have also forced us to develop a number of ancillary techniques, including methods for the removal of oxygen down to at most 1 part in (16). Another current interest has arisen from the displacement work of C.G. Scott and indeed goes in some ways right back to my own beginnings in gas chromatography. He had shown that the displacement method can be used for highly selective preparative separations and for trace concentration, but large amounts of pure displacer material were required, and after use the column had to be cleared of this by heating. It seemed to me that one could get over these two difficulties by use of a moving heater (not in itself a novel idea in gas chromatography) and so we embarked on what we call heater-displacement chromatography. This has proved to be an excellent method for specific separations (e.g. the hexane isomers at 99.93 to 99.99% purity at the rate of 1 g/min, from a 1 m x 1 cm diameter column) and can in principle be operated on a continuous basis and be scaled up considerably with larger diameter columns. We have also combined our catalytic experience with this preparative work. Thus, for example, using a chloride-modified Pt/A1203 as stationary phase, n-hexane is isomerised to a mixture of the hexane isomers in the heater zone, and these isomers are then separated on the column ahead of the heater. Furthermore, the less-branched isomers, which are more strongly adsorbed, continuously come back into the heater zone where they are reisomerised so that the final result is almost complete conversion to the least-adsorbed isomer, 2,2-dimethylbutane ( 1 7 ) . Thirty or so years is a long time to have been associated with one technique: yet I have no regrets. Gas chromatography has been a continuing challenge and has taken me into a wide variety of chemical problems. As an academic it has brought me into closer con'tact with industry than many of my colleagues. This has been a most enjoyable and refreshing experience, a tremendous asset to one who has had to advise many able young men and women in their choice of career, and at times not without financial reward. As a research supervisor it has provided my research pupils (1 or 3 year) with a technique with which they can obtain results sufficiently quickly and simply for them all to be able to start planning their own work at quite an early stage. It seems to me that the acquisition of established expertise is all too often the major feature in the early years of a student's research as well as during his undergraduate career. It is said that as one grows older one notices that policemen grow younger: perhaps in the same vein I notice that my younger colleagues seem to find it harder to find new research fields for themselves. It may, however, be that, like oil, simple and worthwhile

322 research is something of a wasting asset, an asset of which I have been allowed more than my own fair share. REFERENCES 1 B. Lambert and C.S.G. Phillips, P h i l , Trans. Roy. Soc. A 2 4 2 (1950) 415. 2 s. Claesson, A r k . Kern; Min. Geol. A 23 (1946) 1. 3 C.S.G. Phillips, Disc. Faraday Soc. 7 (1949) 241. 4 J.H. Griffiths, D.H. James and C.S.G. Phillips, Analyst (London) 77 (1952) 897. 5 D.H. James and C.S.G. Phillips, J . Chem. Soc. (1954) 1066. 6 A.B. Littlewood, C.S.G. Phillips and D.T. Price, J . Chem. Soc. (1955) 1480. 7 K. Borer and C.S.G. Phillips, Proc. Chem. Soc. (1959) 189. 8 C.S.G. Phillips and P.L. Timms, Anal. Chem. 35 (1963) 505. 9 C.S.G. Phillips, P. Powell and J.A. Semlyen, J . Chem. Soc. (1963) 1202. 10 G.P. Cartoni, R.S. Lowrie, C.S.G. Phillips and L.M. Venanzi, in Gas Chromatography 1960 (Edinburgh Symposium), R.P.W. Scott, ed., Butterworths, London, 1960, p. 273. 11 A.O.S. Maczek and C.S.G. Phillips, J . Chromatogr. 29 (1967) 7. 12 C.G. Scott and C.S.G. Phillips, in Gas Chromatography 1964, (Brighton Symposium), A. Goldup, ed., Inst. of Petroleum, London, 1965, p. 266. 13 R.M. Lane, B.C. Lane and C.S.G. Phillips, J . Catalysis 18 (1970) 281. 14 C.S.G. Phillips and C.R. McIlwrick, Anal. Chem. 4 5 (1973) 782. 15 K.F. Scott and C.S.G. Phillips, J . Chromatogr. 112 (1975) 61. 16 C.R. McIlwrick and C.S.G. Phillips, J . Physics E 6 (1973) 1208. 17 C.M.A. Badger, J.A. Harris, K.F. Scott, M.J. Walker and C.S.G. Phillips, J . Chromatogr. 126 (1976) 11.

323

J.O. PORATH

JERKER OLOF PORATH was born i n 1921, i n S a l a , Sweden. H e e n t e r e d t h e U n i v e r s i t y o f Uppsala i n 1942, p a s s e d h i s " f i l o s o f i e kandidatexamen" (B.S.) i n 1946, and a f t e r s t u d i e s a t Stockholm's Hogskola, t h e " f i l o s o f i e magisterexamen" (M.S.). H e o b t a i n e d t h e l i c e n t i a t e d e g r e e (Ph.D.) a t t h e U n i v e r s i t y of Uppsala, i n 1950. H e also s t u d i e d a t t h e I n s t i t u t f u r Krebsforschung, i n H e i d e l b e r g (1950), and a t t h e Hormone Research L a b o r a t o r y , U n i v e r s i t y o f C a l i f o r n i a a t Berkeley (1951-1952). I n 1957, he r e c e i v e d t h e Swedish d o c t o r ' s d e g r e e (D.Sc). D r . P o r a t h was a p p o i n t e d a s s i s t a n t prof e s s o r a t Uppsala U n i v e r s i t y i n 1957, a r e s e a r c h f e l l o w a t t h e Swedish N a t u r a l S c i e n c e Research Council i n 1960 and obt a i n e d a p e r s o n a l p r o f e s s o r s h i p i n 1964. H e succeeded Arne T i s e l i u s a s J a c o b s o n i a n P r o f e s s o r i n Biochemistry i n 1968. D r . P o r a t h i s t h e a u t h o r of a l a r g e number o f p a p e r s i n b i o c h e m i s t r y and chromatography. H e h a s been v i s i t i n g p r o f e s s o r a t San F r a n c i s c o Medical School and a t t h e Canadian Medical Research C o u n c i l , i n Mont r 6 a l . H e is member o f a number o f academies and s c i e n t i f i c s o c i e t i e s among them t h e Royal Swedish S o c i e t y o f S c i e n c e and t h e Royal Swedish Academy of S c i e n c e . D r . P o r a t h was awarded t h e S i x t e n Heyman's P r i z e , t h e A r r h e n i u s P l a q u e t t e ( t o g e t h e r w i t h P. F l o d i n ) , t h e Gold Medal of t h e Royal Swedish Academy o f E n g i n e e r i n g , t h e Honorary P l a q u e t t e o f t h e French S o c i e t y of B i o c h e m i s t r y , t h e B j o r k e n ' s P r i z e and t h e 1978 P r i z e Biochemical A n a l y s i s of t h e Deutsche G e s e l l s c h a f t f u r K l i n i s c h e Chemie. I n 1971, he r e c e i v e d a honorary M.D. from t h e U n i v e r s i t y o f Uppsal a . D r . P o r a t h is a p i o n e e r i n t h e a p p l i c a t i o n o f chromatography i n b i o c h e m i s t r y and p a r t i c u l a r l y i n t h e u s e of s i z e - e x c l u s i o n ( g e l ) chromatography and a f f i n i t y chromatography.

I was introduced to the earlier concepts of chromatography for the first time after reading Harry Willstaedt's booklet ( 1 ) published in 1938. The first experiments according to the Tswett-type chromatography were made in my home on very simple sugar and alumina columns. I enjoyed the separation of chlorophyll and carotenoids from maple leaves and separation of some mushroom pigments. It took, however, about 10 years of university studies before I began doing serious chromatographic work. In the meantime, I was engaged with synthetic work in organic chemistry. This was time-consuming since chemicals were scarce during this period of World War I1 and even simple compounds had to be synthesized. Much time was also spent on the purification of the raw products. After some time, however, I realized that my work in the field of organic chemistry had no promising prospects unless and until I gained a deeper insight into the purification processes that I needed. It was at this stage that I ventured to work for Arne Tiselius since there were "corridor" rumours that he and Stig Claesson had invented ingenious separation methods. I was hoping that I would further my career in organic chemistry if I only acquired their new techniques, When I joined the Institute of Biochemistry I had passed examinations for The Svedberg (I was probably his last student in physical chemistry) and had a reasonably good background in organic chemistry corresponding to a Ph.D. I had also introduced, for the first time in Sweden, courses in organic qualitative analysis of students in organic chemistry. My background was thus of great help for my future research endeavours in chromatography. Tiselius accepted me as a research student and advised me to isolate biologically active peptides such as bacitracin and pituitary hormones. The transfer to the new Institute brought me into immediate international contacts. The atmosphere in the overcrowded laboratory was most stimulating. Various chromatographic techniques, including gradient elution, were in their developmental stage at that time. However, the methods were not as effective as I had hoped and I myself became involved in improving chromatography and this turned out to be my fate: I never went back to classical organic chemistry work. My earlier contributions were rather modest. Maybe the introduction of "stepgraded" columns of saturated charcoal ( 2 ) - now revived by Shaltiel in hydrophobic interaction chromatography (3) - and some volatile buffer systems, e.g., triethylamine acetate (now frequently used for nucleotides) were the only ones which still seem to have some applicability (4). Following Sober and Peterson's introduction of cellulose ion exchangers, I synthesized and tested cellulosesulfonic acid exchangers (5), a work that, in historical retrospect, may be considered as the forerunner of the sulfonic exchangers based on Sephadex, agarose and other hydrophilic gels. I still consider that much of the work with saturated charcoal, displacement and gradient elution chromatography was a necessary step in the right direction but such work did not yield satisfactory solutions to the separation problems in protein and peptide chemistry. The clean-cut separations obtained by linear adsorption or partition

325 chromatography were desirable but the recurring problem was to find suitable adsorbents and operation conditions to achieve such goals. In the search for adsorbents that made ,linear chromatography possible it was accidently observed that thiamine phosphates were partially separated on starch in an electrophoretic experiment in which the current was by neglect not switched on. I then did some preliminary studies to explain this observation and found that amino acids, peptides and proteins were separated by molecular sieving while many dyes were retarded by adsorption. Some of the latter also migrated as compact zones. These observations made me rather optimistic regarding the possibility of finding adsorbents for linear chromatography and molecular sieves suitable for peptides and proteins. The discovery of the first useful molecular sieve, which has been described elsewhere ( 6 , 7 ) , came some years later. After Per Flodin had left the Institute for an employment at Pharmacia I used to continue our discussions on further improvements in column electrophoresis. In particular, wewere concerned about the drawbacks of the available supporting media and were thus motivated to look for other suitable supports. It was at this time that Per Flodin informed me that he had seen a sample of cross-linked dextran on one of the shelves at Pharmacia which one can test as a bed support. This specimen had been synthesized by Bjorn Ingelman some years earlier but nobody had any suggestion as to what it could be used for. Some months later I had the material in my hand. The first experiments that were done with it to test for zone spreading convinced me that the same kind of phenomena were operating in the column of crosslinked dextran as was found earlier with the starch beds. An idea then occurred to me that this gel material would perhaps serve as a more suitable molecular sieve than starch. Flodin and I (8) then persued this idea and embarked on a program to explore cross-linked dextran, cellulose, starch and polyvinylalcohol. Cross-linked dextran was found to be the best suitable support. The cross-linked dextrans were synthesized at Pharmacia. The tests were chiefly made in my laboratory but also by Flodin at Pharmacia. Together with Walter Bjork I made the first fractionation of proteins on the new gel ( 9 ) . In a subsequent report, my collaborators and I described, as early as 1959, the use of gel filtration for studies of complex association and dissociation phenomena, exemplified with protein-bound oxytocin and vasopressin ( 1 0 ) . Pharmacia later marketed the gel under the trade name of Sephadex. By changing the degree of cross-linking, Sephadex of suitable permeability for different molecular size ranges could be produced. Flodin's systematic studies were of decisive importance in this respect and for the further development of the Sephadex products. He published his own contributions in a doctoral thesis ( 2 1 ) . I will never forget the scepticism that met our attempts to introduce the molecular sieves. Almost nobody except ourselves believed in cross-linked dextran as a separation medium. One very frequently raised objection was concerned with the limited range of operational R values: less than 0.4-1.0: The underlying mechanism was also anotffl er topic of controversy. I was convinced, however, that

326 size separations were based on differences in volumes of the gel matrix available to the separable solutes. Flodin shared by views. Tiselius was not convinced in the beginning. He believed that separation was caused by differences in diffusion rates. To convince my colleagues that differential volume accessibility alone could explain molecular sieving, I described at the Florence Conference in 1962 a very simple model ( 2 2 ) . Laurent and Killander (2.3) later formulated a more elaborate theory and with the advance of "gel-permeation chromatography" (GPC), the mechanism of separations on gel supports has been treated in more detail by Giddings and others. It disturbed me, however, that the "dynamic" hypothesis presented by Ackers and others was widely accepted and was even quoted in textbooks of biochemistry as the only explanation for molecular sieving. The development of GPC on polystyrene resins also has its origins in Uppsala since one of the representatives of Waters Associates tried in our laboratory and at Pharmacia to adapt their refractometer to the gel filtration procedure. As a result the potentialities of molecular sieving for technical polymers and petroleum products were realized by him and the knowledge transferred to J.C. Moore and his associates. In this context I think it is appropriate to give credit to B . Cortis-Jones ( 2 4 ) who first tried to apply Sephadex to polymers dissolved in organic solvents. After our first publication of the method of gel filtration on cross-linked dextrans, there was a delay of about 3-4 years before Sephadex gel chromatography was generally adopted. The main reason was probably the fact that Flodin and I had discussed at length the possible drawbacks and limitations in this new method rather than concentrating primarily on its advantages. This fact was often pointed out to me by Tiselius who thought that I had a pessimistic view of the practical applications of the newly invented technique. Moreover, and for the same reason, there have been delays in the publication of some newly discovered methods which had repeatedly placed my collaborators and me behind in the race for recognition. This is as such of minor importance but is of decisive value as far as raising funds for new research projects is concerned. In retrospect, however, I still hold the opinion that a comprehensive criticism of one's own discoveries - irrespective of its potential wide applicability is justifiable and in the end rewarding. The availability of Sephadex and agarose, the latter introduced by HjertCn as a molecular sieve ( 1 5 ) , prompted us to use them as starting materials for the synthesis of specific adsorbents by coupling suitable ligands onto them to serve as adsorption centers. Initially a number of special problems were encountered which were solved after extensive studies. As a result a number of coupling methods have been devised to meet the demands for the immobilization of proteins or low-molecular-weight solutes containing NH2-, SH-groups etc. A break-through in our efforts in this direction came with the incidental discovery of the "cyanogen bromide" coupling procedure (26) which is described elsewhere (27).

327 For several years we were engaged in the development and study of immobilized enzymes and biospecific adsorbents based on cellulose, Sephadex and agarose. Our contributions in this field were, however, overshadowed by the work of Cuatrecasas and associates in spite of the fact that they also used our techniques. My collaborators Lars Sundberg and Kare Aspberg and I were confident that we would obtain a patent for enzyme inhibitors coupled to agarose as well as a general patent for lectins coupled to agarose ( 1 8 ) . Unfortunately, however, K.O. Lloyd published a paper (which we were not aware of) on the use of concanavalin-Sepharose (using again the cyanogen bromide method) just two weeks before we submitted our application for a patent. Consequently, we had to withdraw our claims - a fact that seriously hampered our possibilities of obtaining financial support for further research on lectins. In spite of this, however, we are still persistently following up the program aimed at finding highly selective lectin-based adsorbents for glycoproteins and carbohydrates. The divinyl sulfone (DVS) coupling method ( 1 9 ) has been extremely useful for coupling of carbohydrates - a prerequisite for isolating lectins from plant extracts by "affinity chromatography". This method has also been used with great success for the synthesis of immunosorbents. The recent advancements in "affinity chromatography", gas and high-pressure liquid chromatography and not least the remarkable achievements in gel electrophoresis in various forms are most impressive. Much effort is at present directed towards the production of apparatus and bed materials as well as the formulation of elaborate theories in order to increase the plate numbers in chromatographic beds rather than the search for newer principles of separation. The number of commercially available but more or less similar types of apparatus for GC and HPLC is bewildering. One is thus inclined to ask whether chromatography had not reached the stage of diminishing returns. It would thus appear that we already have a sufficiently effective arsenal of techniques to cope with all possible fractionation problems which can be encountered in biochemistry and the chemistry of natural products. In my opinion, however, this is not the case for several reasons. The most important is perhaps the lack of effective preparation methods for fractionation in non-aqueous or mixed organic-aqueous solvents. More efficient systems for watersoluble mixtures are also desirable in order to shorten the time required for fractionation. Rather than increase the efficiency in terms fo plate height to the extreme I believe that it is more important to develop different techniques based on new separation parameters. With this objective in mind, we have recently synthesized new kinds of adsorbents by means of bisoxirane (19,20) and other coupling agents. Recently, my efforts have been directed to problems in an almost "virgin" field in chromatography, viz. charge-transfer adsorption in aqueous systems. After synthesizing and studying some amphiphilic adsorbents (21) we found it more reasonable to direct our efforts in charge-transfer adsorption since Hjerth and others simultaneously and independently were investigating hydrophobic interaction chromatography. In an early phase of our research on molecular sieves,

328 we had observed that Sephadex exhibits a weak affinity for aromatic compounds (22,23) - an affinity that can occasionally be used for solving practical separation problems. It occurred to me then that "aromatic adsorption" might be enhanced by introducing certain ligands into the gel matrix. Such studies were initiated in the mid-sixties. Nitrophenylethers of Sephadex and agarose were tried but the adsorbents produced initially were too weak for practical applications. However, after 8-9 years of occasional trials the events took a turn for the better and we came upon 2,4-dinitrophenylthioether derivatives of Sephadex ( 2 4 ) . These adsorbents have an affinity for aromatics strong enough to warrant intensified research in the field. We were also able to attach chlorodicyanobenzoquinone to Sephadex and agarose in concentrations of up to about 200 pmoles per gramme dry gel. Such quinone gels showed the expected strong electron acceptor properties. Some electron donor gels have also been synthesized and shown to be promising adsorbents for nucleotides. Although quantum nechanical calculations afford a sound theoretical basis for evaluation of the chromatographic behaviour and also serve as valuable guides for selecting effective charge-transfer ligands, serious deviations from the expected results are often encountered. We have interpreted the occurrence of these deviations as caused by solvation effects and contributions of other forces. Hydrogen bonding, ion-dipole and dipole-dipole interactions are likely to play a role in some cases, e.g., in the quinone gels. Hydrophobic interactions cannot be the dominant or sole factors responsible for adsorption in cases where the adsorption is strengthened as the temperature is decreased. This is in fact always the case for the strong acceptor or donor ligands hitherto studied. As mentioned above, we have obtained many unexpected results in connection with different kinds of gel chromatography. The observed phenomena cannot be explained solely in terms of molecular sieving. Hydrogen bonding and dipole-dipole interactions are probably also involved. This apparently seems to be the case for the results obtained on gels consisting of an interwoven network of agarose and polyacrylamide (e.g . , Sephacryl) (25). The metal chelate gels comprise a special kind of electron acceptor adsorbent of great promise (26). The separations obtained are due to differences in affinities of solutes for specific transition metal ions (e.g., Cu2+, Zn2+, Fe3+) which are strongly bound to the gel matrix via chelating ligands. Further systematic studies are necessary before metal chelate adsorption methods can be applied for the separation of peptides and proteins. At the Institute of Biochemistry in Uppsala we are still following traditions from the time of The Svedberg and Arne Tiselius (27). At times we have asked ourselves if we have become so fascinated with separation problems that we are engaged primarily in the art for the art's sake. I do not seriously believe this to be true. At the same time, we have also been engaged in research in other areas of biochemistry. I have thus had the luck of initiating many other types of research projects that have been completed successfully thanks

329 0

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Hours

n=4

40-

m

60-

I

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Fig. 35.1. Fractionation of mixtures of oligoethylene glycols representing an early illustration of "high-performance liquid chromatographyll ( 2 9 ) . A 0 . 4 g sample of oligoethylene glycol monophenylethers was chromatographed on three columns C 1 , C2 and C3 (110, 50 and 110 cm respectively) with Sephadex LH-20 in 96% ethanol. The effluents from the columns were recorded separately with three Uvicords (n=O refers to phenol). In water solution at least 16 components could be completely separated. When the tetramer had left column C1 all the columns were connected in series and the run continued. The separation is due to a combined effect of molecular sieving and a weak chargetransfer and hydrophobic adsorption.

330 to my skillful collaborators. For the immediate future, my intentions are to concentrate my efforts mostly on the study of brain chemistry. A declaration of my future intentions here may seem unwise and contrary to what my teacher, Arne Fredga, used to say. According to him, our illustrious chemist Jons Jacob Berzelius was of the opinion that a good chemist should never reveal in advance his intentions regarding forthcoming research projects or make announcements about possible outcomes of future research. Be that as it may, I am sure that my work in brain chemistry will undoubtedly force me to improve classical fractionation methods further and will also justify a continuation of our attempts to find new adsorbents and new chromatographic systems. Every scientist with some decades of experience in research probably has his own "cabinet of curiosities" with small inventions and discoveries which are unpublished or forgotten. In my cabinet I have "zone precipitation" (28), fractionation of mixtures of oligoethylene glycols (29) (in efficiency approaching HPLC), and "hydroxylated" gels. Some day I would like to bring out these objects from the cabinet and develop them into useful tools and techniques. As a matter of fact, some of them have already been taken out and transformed into more useful inventions by others. A case in point is the method of concentrating protein solutions with dry Sephadex which was published long ago by Flodin, Gelotte and myself (30). This method is now adopted to heal wounds using cross-linked dextran under the trade name Debrisan ( 3 1 ) . Immunoassay techniques were also expanded following the advent of Sephadex and suitable coupling methods (32). These examples serve to illustrate that developmental work in chromatography has significantly contributed to progress in other areas of interest such as in medicine and in the many disciplines of the natural sciences. REFERENCES 1 H. Willstaedt, Om naturfiirglimnen och deras biologiska betydelse, Studentforeningen Verdandis smiskrifter, No. 4 0 , Stockholm,l938 (in Swedish). 2 J . Porath, 4 r k . Kemi 7 (1954) No. 57. 3 s. Shaltiel, Methods Enzymol. 34 B (1974) 126. 4 J. Porath, Nature (London) 775 (1955) 478. 5 J. Porath, Ark. Kemi 11 (1959) No. 11. 6 J. Porath, Lab. &act. 16 (1967) 834. 7 S. Hjert6n and J . Porath, UppsaZa Univ. 500 Years: 9 Chemistry, Acta Univ. Upsaliensis, 1976, pp. 27-66. 8 J. Porath and P. Flodin, Nature (London) 183 (1959) 1657. 9 I. Bjork and J. Porath, Acta Chem. Scand. 13 (1959) 1256. 10 E.B. Lindner, A. Elmqvist and J. Porath, Nature (London) 184 (1959) 1565. 11 P . Flodin, Dextran Gels and Their Application i n Gel FiZtration, Ph. D. Thesis, Uppsala, 1962. 12 J . Porath, J . Pure AppZ. Chem. 6 (1963) 233. 13 T.C. Laurent and J. Killander, J . Chromatogr. 14 (1964) 317. 14 B. Cortis-Jones, Nature (London) 191 (1961) 272.

331 15 S. HjertBn, Arch. Biochem. Biophys. 99 (1962) 466. 16 R. Axen, J . P o r a t h and S. Ernback, Nature (London) 214 (1962) 1302. 17 J . P o r a t h , Nature (London) 218 (1968) 834. 18 K. Aspberg and J . P o r a t h , Acta Chem. S c a d . 24 (1970) 1839. 19 J . P o r a t h and R. A x h , Methods Enzymol. 44, 11 B (1976) 19. 20 L. Sundberg and J . P o r a t h , J. Chromatogr. 90 (1974) 87. 2 1 J. P o r a t h , L. Sundberg, N. F o r n s t e d t and I . Olsson, Nature (London) 245 (1973) 465. 22 .J. P o r a t h , Biochim. Biophys. Acta 39 (1960) 193. 23 G . G e l o t t e , J. Chromatogr. 3 (1960) 330. 24 J . P o r a t h and K. Dahlgren Caldwell, J . Chromatogr. 133 (1977) 180. Janson, J . Chromatogr. 25 M. B e l e w , J . P o r a t h , J . Fohlman and J.-C. 1 4 7 (1978) 205. 26 J. P o r a t h , J . Carlsson, I . Olsson and G . B e l f r a g e , Nature (London) 258 (1975) 598. 27 J . P o r a t h , S e p a r a t i o n Methods and Arne T i s e l i u s , i n Hormonal Prot e i n s and Peptides, Vol. 1 5 , C.H. L i , e d . , Academic P r e s s , N e w York, 1978, pp. 159-185. 28 J . P o r a t h , Nature (London) 196 (1962) 47. 29 J . P o r a t h , Adsorption and Molecular S i e v i n g i n Sephadex and Sephadex D e r i v a t i v e s , i n Gas Chromatography 1968 (Copenhagen Symposium) , C.L.A Harbourn, e d . , I n s t . of Petroleum, London, 1969, pp. 212-216. 30 P. F l o d i n , B. G e l o t t e and J . P o r a t h , Platme (London) 188 (1960) 493. 31 U. Rothman, T h e o r e t i c a l Viewpoints on Chromatography of Wounds, i n Sven. K i r . 31 (1974) 1. 32 L. Wide and J . P o r a t h , Biochim. Biophys. Acta 130 (1966) 257.

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333

VICTOR PRETORIUS

VICTOR PRETORIUS was born i n 1928, i n R a n d f o n t e i n , T r a n s v a a l , South A f r i c a . H e s t u d i e d a t t h e University of P r e t o r i a r e c e i v i n g h i s B.S. and M.S. d e g r e e s i n 1949 and 1951 r e s p e c t i v e l y . In 1952 he w a s awarded a Rhodes S c h o l a r s h i p t o Oxford U n i v e r s i t y , England, where he worked under t h e Nobel L a u r e a t e S i r C y r i l Hinshelwood. He finished h i s studies there receiving the Ph.D.(Oxon.) i n 1955. R e t u r n i n g t o t h e Univ e r s i t y of P r e t o r i a , D r . P r e t o r i u s was a p p o i n t e d a s e n i o r l e c t u r e r i n 1957 and p r o f e s s o r and head o f t h e Department o f P h y s i c a l Chemistry i n 1960. I n 1966 he became d i r e c t o r of t h e Research Unit f o r Chromatography and i n 1973 t h e d i r e c t o r o f t h e I n s t i t u t e f o r Chromatography o f t h e U n i v e r s i t y of P r e t o r i a . D r . P r e t o r i u s i s t h e a u t h o r of o v e r 90 s c i e n t i f i c p a p e r s ; he c o e d i t e d w i t h A . Z l a t k i s a monograph on p r e p a r a t i v e g a s chromatography which has a l s o been t r a n s l a t e d i n t o R u s s i a n . D r . P r e t o r i u s is a member o f v a r i o u s o f f i c i a l committees i n t h e Republic o f South A f r i c a . H e h a s s e r v e d a s p r e s i d e n t o f t h e South A f r i c a n Chemi c a l I n s t i t u t e (1966-1967), t h e J o i n t Council of South A f r i c a n Scient i f i c S o c i e t i e s (1969-1970), t h e Chemistry S e c t i o n o f t h e S u i d - A f r i k a a n s e Akademie v i r Wetenskap e n Kuns (1971-1972), t h e A s s o c i a t e d S c i e n t i f i c and T e c h n i c a l S o c i e t i e s o f South A f r i c a (1969-1970 and 19741 9 7 5 ) , t h e N a t i o n a l Advisory Committee of S c i e n t i f i c P u b l i c a t i o n s (19681976) and t h e S e l e c t i o n Committee f o r Rhodes S c h o l a r s h i p (1965-1970). C u r r e n t l y , h e i s a member of t h e N a t i o n a l Committee o f t h e I.U.P.A.C. and t h e Board of C o n t r o l of t h e Bureau f o r S c i e n t i f i c P u b l i c a t i o n s . D r . P r e t o r i u s r e c e i v e d t h e Rhodes S c h o l a r s h i p i n 1952, and t h e C a r n e g i e F e l l o w s h i p i n 1964; he was awarded t h e AE&CI g o l d medal, t h e Havenga P r i z e i n Chemistry of t h e Suid-Afrikaanse Akademie v i r Wetenskap e n Tegnologie, t h e g o l d medal o f t h e South A f r i c a n Chemistry Ins t i t u t e and t h e M.S. T s w e t t Chromatography Medal. H e is a f u l l member o f t h e Suid-Afrikaanse Akademie v i r Wetenskap e n Kuns, and s e r v e d a s t h e chairman o f i t s Chemistry S e c t i o n i n 1971-1972. D r . P r e t o r i u s ' r e s e a r c h i n t e r e s t s have c e n t e r e d almost e x c l u s i v e l y around chromatography. In 1958, he p i o n e e r e d i n t h e development o f t h e flame i o n i z a t i o n d e t e c t o r . Besides t h i s , h i s c o n t r i b u t i o n s range from t h e t h e o r e t i c a l a s p e c t s of b o t h gas and l i q u i d chromatography t o p r e p a r a t i v e chromatography.

334 My a s s o c i a t i o n w i t h chromatography began i n 1953 when I was s t u d y i n g f o r my d o c t o r a t e under S i r C y r i l Hinshelwood a t Oxford U n i v e r s i t y . My problem w a s t o u s e a mass s p e c t r o m e t e r t o a t t e m p t t o u n r a v e l t h e wide range of p r o d u c t s formed d u r i n g ' t h e g a s phase p o l y m e r i z a t i o n of o l e f i n s - a f o r m i d a b l e t a s k , as i t t u r n e d o u t . Consequently t h e news t h a t Ray ( I ) a t I . C . I . had s u c c e s s f u l l y used t h e new t e c h n i q u e o f g a s - l i q u i d chromatography t o s e p a r a t e and d e t e c t a wide v a r i e t y of chemical compounds w a s r e c e i v e d by u s w i t h g r e a t i n t e r e s t and a f t e r a v i s i t t o Ray w e immediately set about b u i l d i n g a g a s chromatograph w i t h a k a t h a r o m e t e r d e t e c t o r . As I r e c o l l e c t t h e a p p a r a t u s gave more t r o u b l e t h a n r e s u l t s a n d , when I l e f t t o r e t u r n t o South A f r i c a a t t h e end o f 1954, i t had n o t r e a l l y f u l l f i l l e d i t s promise. However I was s u f f i c i e n t l y i n t r i g u e d t o c o n t i n u e w i t h t h e work i n South A f r i c a , I n f a c t I w a s f o r c e d t o look f o r an a l t e r n a t i v e t o a mass s p e c t r o m e t e r f o r my r e s e a r c h on g a s k i n e t i c s s i n c e my budget w a s less t h a n US$lOO! During t h e p e r i o d 1955-1960, a f t e r I r e v i s i t e d t h e United Kingdom and Europe, I worked i n v i r t u a l l y complete i s o l a t i o n i n South A f r i c a . Because of t h i s , many of my e a r l y i d e a s , which subs e q u e n t e v e n t s proved t o have been w o r t h w h i l e , were p r e m a t u r e l y abandoned. Lack of f u n d i n g t u r n e d e x p e r i m e n t a l p r o c e d u r e s , which appeared s i m p l e enough i n England, i n t o i n s u r m o u n t a b l e o b s t a c l e s . As an example i t t o o k m e t w o y e a r s t o a c q u i r e a c r u d e , second-hand recording potentiometer! The Oxford v e n t u r e i n t o g a s chromatography l e a d m e , e a r l y i n 1955, t o a t t e m p t t o d e v e l o p e a d e t e c t o r which would be b e t t e r t h a n a k a t h a r o m e t e r . My background i n mass s p e c t r o m e t r y i n f l u e n c e d m e t o b e l i e v e t h a t t h e most p r o f i t a b l e d i r e c t i o n t o e x p l o r e would n o t

I

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i n v o l v e t h e measurement of a b u l k p r o p e r t y b u t would invoke an ioni z a t i o n p r o c e s s and t h e subsequent measurement of an i o n c u r r e n t . As a s t a r t I b u i l t what w a s , i n e f f e c t , a c r u d e mass s p e c t r o m e t e r i o n s o u r c e . Helium was used as a c a r r i e r g a s and t h e v o l t a g e a c r o s s t h e anode and c a t h o d e w a s s e t , h o p e f u l l y , t o i o n i z e t h e s o l u t e s b u t n o t t h e helium. The c u r r e n t w a s measured u s i n g a galvanometer and r e s u l t s were n o t e n c o u r a g i n g . The d e v i c e w a s u n d e r s t a n d a b l y n o i s y , and t h e f a c t t h a t i t had t o be o p e r a t e d a t reduced p r e s s u r e w a s a d e c i d e d i n c o n v e n i e n c e . (An e l e g a n t v e r s i o n w a s s u b s e q u e n t l y publ i s h e d by R y c e and Bryce ( 2 ) ) . During t h e c o u r s e o f t h e work my a s s i s t a n t , W . N e l , i n a d v e r t e n t l y d i s c o n n e c t e d t h e vacuum pump a n d ,

335

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Pt. electrodes ( h m apart ad 2mm aboveI jet) Jet ( O ~ I N ~ )

' "E-Air -NZ

F i g . 36.2 ( l e f t ) . The o r i g i n a l flame i o n i z a t i o n detector F i g . 36.3 ( r i g h t ) . O r i g i n a l s i g n a l observed f o r t h e flame i o n i z a t i o n d e t e c t o r , A , S i g n a l f o r 10 ng l a b o r a t o r y g a s ; B, s i g n a l f o r 10 pg l a b o r a t o ry gas. by c h a n c e , i n j e c t e d a sample of chloroform. A s t r o n g n e g a t i v e s i g n a l was o b s e r v e d , i . e . , a r e d u c t i o n i n t h e c u r r e n t , when t h e c a t h o d e was h e a t e d s t r o n g l y . The s i g n a l was n o t r e p r o d u c i b l e and, i n any c a s e , c o m p l e t e l y a t v a r i a n c e w i t h t h e p r i n c i p l e s o f gaseous e l e c t r i c a l conductance. I t h e r e f o r e r e f u s e d t o a c c e p t t h e r e s u l t s and s t o p p e d t h e work; t h e penny dropped when Lovelock p u b l i s h e d h i s c l a s s i c work on t h e e l e c t r o n c a p t u r e d e t e c t o r , about f o u r y e a r s l a t e r ( 3 ) . The h i s t o r y o f t h e flame i o n i z a t i o n d e t e c t o r merits t e l l i n g i n some d e t a i l . I had been aware of S c o t t ' s hydrogen flame d e t e c t o r i n which changes i n t h e flame t e m p e r a t u r e w e r e measured ( 4 ) and had, i n f a c t , b u i l t one a t t h e b e g i n n i n g of 1957. T h i s was done a t t h e r e q u e s t of D r . D. Horn o f t h e Council f o r S c i e n t i f i c and I n d u s t r i a l R e s e a r c h , i n P r e t o r i a , who was i n t e r e s t e d i n t h e a n a l y s i s o f e s s e n t i a l o i l s and needed a s e n s i t i v e d e t e c t o r which c o u l d o p e r a t e a t r e l a t i v e l y h i g h t e m p e r a t u r e s . I t was w h i l e I was t e s t i n g S c o t t ' s d e t e c t o r t h a t I remembered r e a d i n g t h a t t h e e l e c t r i c a l r e s i s t a n c e of a flame changes w i t h t h e composition of t h e g a s e s b e i n g combusted. I c o n s t r u c t e d a s i m p l e a l l - g l a s s d e t e c t o r f o r t h i s purpose and t h e f i r s t a t t e m p t t o measure a change i n t h e flame r e s i s t a n c e was a f a i l u r e because of i t s h i g h v a l u e and my l i m i t e d knowledge o f e l e c t r o n i c s . I t h e n c o n s u l t e d J . Harley who, a t t h a t t i m e , was a t e c h n i c a l a s s i s t a n t i n o u r department and had s e r v e d an a p p r e n t i c e s h i p i n e l e c t r o n i c s a t t h e P o s t O f f i c e . H e b u i l t a c i r c u i t f o r t h e purpose and t h e very f i r s t experiment was a s u c c e s s . A sample o f l a b o r a t o r y gas was i n j e c t e d almost s t r a i g h t i n t o t h e d e t e c t o r and from t h e s i g n a l observed I could c a l c u l a t e t h a t t h e d e t e c t o r had a s e n s i t i v i t y approaching g . I t was a p p a r e n t t h a t t h e r e s p o n s e was dependent on a v a r i e t y of p a r a m e t e r s which, a t t h a t t i m e , were p o o r l y unders t o o d ; n e v e r t h e l e s s I n e v e r c e a s e t o be amazed t h a t , by chance, t h e

336 detector geometry and operating parameters in that first experiment so closely approximated the optimum values which were ultimately determined after much work by many people. The project, up to that stage, was completed in four days towards the end of September 1957. After a discussion with Horn we decided to delay publication until he had used the detector for his essential oil project and we had also done some more work on the detector characteristics. For a variety or reasons (mostly unscientific) this took much longer than we had anticipated and I finally decided to submit a note ( 5 ) which appeared one month before the communication by McWilliam and Dewar

(6). It is well-known that I never filed a patent claim on the flame ionization detector, but that Harley did. I was, in fact, not aware that he had done so until my role in the invention was queried by I . C . I . who were involved in a claim on behalf of McWilliam. Harley had, by that time, left my department to join the South African Iron and Steel Corporation where there was an active patent division which encouraged employees to file patent applications at no cost to the employee. This, however, did not apply to me and for this reason, as well as the fact that I believed that the glow discharge detector I had developed (7) would rapidly supercede the FID, I waived my interest in any patent application. Just how disastrous this decision was, is now history. From discussions with many of my colleagues it would appear that several laboratories were exploring the possibility of using open tubular columns some time before Golay published his classic paper on the subject (8). My own attempts during 1956 failed for reasons which are now painfully apparent. The column consisted of a 20 m length of 1 mm I . D . copper tubing which had been coated with dinonyl phthalate by forcing a plug of pure stationary phase through the column and leaving it to drain overnight. The detector was a homemade katharometer which had an internal volume of about10 ml: The results were, understandably, not encouraging. For reasons which, with the wisdom of hindsight, are not now clear to me, I then attempted to increase the column capacity instead of, very obviously, constructing a detector of smaller volume. With this in mind I coated hard cotton threads with stationary phase and drew them into a glass tube - the idea being to create a large number of parallel capillaries. The system gave terrible results. It then occurred to me that plastic-coated multi-strand copper wire might provide the answer. The results were certainly more encouraging than anything I had previously obtained but the work was abandoned because I was devoting all my energy to the detectors. It was with great admiration, and not a little envy, that I later read of the beautiful work of Golay (8) and Desty (9). Gas chromatography in open tubular columns has always demanded exceptional experimental talents to which I cannot lay claim. Recently however I was searching for a suitable project for a Ph.D. student who was more practically inclined, and I put him onto the problem of modifying the inner surface of glass capillaries following the work of Novotny ( l o ) , Alexander ( 1 1 ) and others - with the difference

337

Fig. 3 6 . 4 . First successful whisker column. that we would use a scanning electron microscope to observe the effect of various etching techniques. Isolated whiskers were observed amongst thesodiumchloride crystals and this suggested using higher concentrations of the fluoro-ether to promote whisker growth which constituted the surface modification considered to be ideal by Desty. After much experimentation the conditions for obtaining uniform whisker growth were determined ( 1 2 ) and this structure should provided a springboard for exciting new advances in open tubular column gas chromatography. An area to which I have devoted considerable time is preparative chromatography. The fundamental basis of this subject has been developed in collaboration with a number of exceptationally brilliant students ( 2 3 - 2 6 ) but the practical implementation of these concepts has always been beyond our capabilities. To my great disappointment preparative chromatography, in the sense we outlined, has never been technologically developed t o the extent I believe it deserves. This has been particularly true in the Western countries but, to judge from the number of letters I receive from the Communist countries, there would appear to be more interest in the subject in that part of the world. The elegant simplicity of thin-layer chromatography has attracted many chromatographers including myself. Perhaps the greatest drawback to the technique is the fact that the solvent flow is caused by interparticle capillary action which leads to a flow which decreases exponentially with the distance it has traversed the plate. I have given much thought to devising alternative methods of controlling the solvent flow. This culminated in the use of electro-osmotic flow (27) which can be varied over wide limits and also possesses the

338 further advantage of exhibiting a flat velocity profile. At first sight the technique would seem to offer a real breakthrough but the problem of having to create a suitable zeta potential at the particlesolvent interface has limited its usefulness. Perhaps sometime, someone who is more skilled than I will further exploit this crude but, I believe, potentially worthwhile idea. Over the years it has become increasingly clear to me that chromatography is essentially a practical subject and that theoretical studies of the topic should avoid the temptation to use chromatography as a convenient peg on which to hang mathematical expertise. An example of this is the work my group did on high-performance liquid chromatography (18, 1 9 ) . I believe the essential elements of the subject were, in fact, disclosed in these papers, but that the practical issues were clouded by excessively complicated mathematical equations, with the result that we have received little credit for this work. REFERENCES 1 N.H. Ray, J . A p p Z . Chem. 4 (1954) 105. 2 S . A . Ryce and W.A. Bryce, Nature (London) 179 (1957) 541. 3 J.E. Lovelock and S.R. Lipsky, J . h e r . Chem. Soc. 82 (1960) 431. 4 R.P.W. Scott, iYature (London) 176 (1955) 793. 5 J. Harley, W. Nel and V. Pretorius, Nature (London) 181 (1958) 117. 6 I.G. McWilliam and R.A. Dewar, Nature (London) 181 (1958) 760. 7 J. Harley and V. Pretorius, Nature (London) 178 (1957) 1244. a M.J.E. Golay, in Gas Chromatography 1958 (Amsterdam S y m p o s h n ) , D.H. Desty, ed., Butterworths, London, 1958, pp. 36-55. 9 D.H. Desty, J.H. Haresnape and B.H.F. Whyman, Anal. Chem. 32 (1960) 302. 10 K. Tesarik and M. Novotny, in Gas Chromatographie 1968, H.G. Struppe, ed., Akademie-Verlag, Berlin, 1968, p. 575. 11 G. Alexander and G.A.F.M. Rutten, Chromatographia 6 (1973) 231. 12 J.D. Schieke, M.R. Comins and V. Pretorius, J . Chromatogr. 112 (1975) 97. 13 S.M. Gordon and V. Pretorius, J . Gas Chromatogr. 2 (1964) 196. 14 S . M . Gordon, G.J. Krige and V. Pretorius, J . Gas Chrornatogr. 2 (1964) 240. 15 S.M. Gordon, G.J. Krige and V. Pretorius, J . Gas Chromatogr. 3 (1965) 87. 16 G.J. Krige and V. Pretorius, J . Gas Chromatogr. 2 (1964) 115. 17 V. Pretorius, B.J. Hopkins and J.D. Schieke, J . Chromatogr. 99 (1974) 23. 18 V . Pretorius and T.W. Smuts, AnaZ. Chem. 38 (1966) 274. 19 T.W. Smuts, F.A. van Niekerk and V. Pretorius, J . Gas Chromatogr. 5 (1967) 190.

339

G.R. PRIMAVESI

G I U L I O RICHARD PRIMAVESI was born i n 1914

i n Northampton, England. H e s t u d i e d a t G o n v i l l e and Caius C o l l e g e , Cambridge, between 1932 and 1935, t a k i n g t h e N a t u r a l S c i e n c e T r i p o s . I n 1936 he p a s s e d t h e exa m i n a t i o n s and became a f e l l o w o f t h e B r i t i s h O p t i c a l A s s o c i a t i o n and t h e Spect a c l e Makers Company. I n l a t e 1937, he j o i n e d t h e A n a l y t i c a l L a b o r a t o r y of t h e Research Department o f D i s t i l l e r s Co., L t d . , a t Great Burgh, Epsom. H e remained t h e r e u n t i l h i s r e t i r e m e n t i n 1974 when he was t h e head o f t h e Gas Chromatography S e c t i o n of t h e A n a l y t i c a l Branch. I n 1967, Great Burgh was t a k e n o v e r , w i t h t h e rest o f D i s t i l l e r s Chemicals and P l a s t i c s , by B r i t i s h Petroleum Chemicals. M r . Primavesi h a s a number of p u b l i c a t i o n s i n a n a l y t i c a l c h e m i s t r y , mostly chromatography. He was a member of t h e Gas Chromatography p a n e l o f t h e Ins i t u t e of Petroleum and member of t h e Committee o f t h e (Gas) Chromat o g r a p h y D i s c u s s i o n Group between 1961 and 1973. M r . P r i m a v e s i was an a n a l y t i c a l chemist i n t h e same i n d u s t r i a l res e a r c h department f o r t h e whole of h i s p r o f e s s i o n a l c a r e e r . H i s i n volvement i n g a s chromatography s t a r t e d i n 1952 and he h a s been d i r e c t l y i n v o l v e d i n t h e e v o l u t i o n of t h e t e c h n i q u e u n t i l h i s r e t i r e m e n t i n 1974.

340 It must be very rare to find oneself suddenly in at the beginning of a real revolution in one's profession: "the time, the place, and the loved one all together." In 1951, I was in charge of a small analytical development section in a first-class research department, whose head, Dr. H.M. Stanley, was later elected a fellow of the Royal Society for his work in developing successful commercial chemical processes. Among the processes then being developed, was that for producing acetic acid from the lower hydrocarbons. To analyse the mixture of acids produced, we were trying liquid chromatography on silica gel. The apparatus was very simple - merely a vertical packed tube down which one poured the liquid. In those days, one produced one's own silica gel from water-glass, hydrochloric acid and what seemed like black magic: "Work only at full moon; stir four times widdershins and thrice deasil". Even then, only about one batch in three gave any sort of useful product. In autumn, 1952, I attended the I.U.P.A.C. Congress at Oxford. There I saw a crude but effective little machine, made by James and Martin, doing my very analysis in about fifteen minutes by a technique called "gas liquid partition chromatography". This was the first time the general scientific public (other than biochemists) had seen this marvel. One merely blew over Celite moistened with silicone fluid, and titrated what came out! On my return from Oxford, I got some Celite and silicone oil. In those days, one did not buy ready-prepared supports; one bought a sack of Celite and sieved it oneself. The yield was about 3%. Within a week, I had produced my first gas chromatogram. I was the detector and the recorder. I sat in front of a sheet of graph paper and a burette and plotted by hand the total titration every fifteen seconds. I got a better answer than I had ever had before in about quarter the time. I may say that having been a detector and a recorder myself gave me a certain empathy with the more temperamental sorts of both, which subsequently afflicted me; I have a sneaking fellow-feeling with their point of view! It was not very long before we had an automatic titrator fitted up and, in no time at all, we were indispensable to the acetic acid project. Analysts nowadays can have very little idea of what analysis was like 25 years ago. My particular class of samples arose from the oxidation, ammoxidation, or chlorination of the lower hydrocarbons. They consisted of the desired product containing oxygen, nitrogen and/or chlorine, and a surprising number of by-products, getting more numerous as their concentration fell. Chemical analysis presented some formidable problems. I shall briefly describe three, all simple, by gas chromatography: (a) Parts per million of alph-a methylstyrene in isopropylbenzene. This required careful bromination in the dark, and correction for side reactions, (b) 2,4,4-Trimethylpentene-1 in the presence of 2,4,4-trimethylpentene-2. We did this by exploiting the different rates of reaction with mercuric acetate. It needed about ten titrations at carefully measured times, and about two hours for one sample. (c) Approximately equal amount of 2-methylpropionaldehyde in butyraldehyde. This was a fiendish problem which produced one of

341 the few bits of research complete enough to publish (mostly we had no time to tie up loose ends). It may interest the reader to look up my 1953-paper ( 2 ) to see what has been replaced by a simple gas chromatographic separation. It was obvious that, provided one could detect the emergent material, gas chromatography could separate almost anything that was at all volatile from almost anything else. It was, by an amazing stroke of good fortune, exactly what we needed in our particular field. A.J.P. Martin had already developed a universal detector; from him I got details of his density balance and the revolution was really under way. From the very beginning, gas chromatographers enjoyed a sort of freemasonry among themselves, which remains to me one of the most delightful aspects of the art. For this free exchange of ideas and views, I think A.J.P. Martin himself was responsible more than anyone else. He started the co-operation and camaraderie among us. He was accessible to all and treated everybody as though they were as clever as he was. As a result, it was very early on that practitioners of the art tended to get together. The Gas Chromatography Discussion Group more or less gathered round Martin, James and Desty. In those early days, it really was a discussion group, meeting informally and fairly frequently at the Institute of Petroleum in London. These meetings were most stimulating; humble chemist, professor and Nobel prize-winner took part on equal terms. We all tended to know, or to have heard of, all the other practitioners. If we wanted details of new developments, we simply phoned and asked. In the matter of apparatus and methods, there was complete frankness; commercial reserve came in (unfortunately!) about the actual separations we were doing. As -the technique widened and spread, the numbers using it grew rapidly. The discussion group became more organised. It held informal symposia in Britain every six months, and, beginning in 1956, formal symposia every two years. We organised an internal Distillers Company discussion group, which also held six-monthly meetings. The potential and rapid development of gas chromatography was such that all these meetings were barely sufficient to keep up with advances, until at least the Rome formal symposium in 1966. It is difficult, after all these years, to recapture the splendid verve of those early days. Work was not only interesting and stimulating,it was actually fun: I must confess that even now, after 25 years, I still like watching a peak appear on a recorder trace (Icannotsay I get the same thrill out of a computer-typed percentage). Early 1953 was like an analyst's dream. Gas chromatography was almost confined to Great Britain. We users were in the unique position of having nobody except ourselves who could catch us out. This, of course, put us on our professional honour to be very careful about our results. We were constantly being visited by awed strangers who had not hitherto seen the technique in action. This halcyon period did not last very long! The spread of expertise and the development of new detectors was very rapid. Up to about 1960, we made most of our equipment, except recorders, ourselves. We started off being proud of being able to estimate 0.1% of a component. Our customers, of course, wanted us to go much lower. A steady SUC-

342 cession of new detectors - flame temperature, thermal conductivity, flame ionisation, and the various specialities such as argon, electron capture etc., got us down in a comparatively short space of time to parts per million and even lower. The flame ionisation detector, considered dispassionately, is an almost incredible analytical tool. It has a linear (or very nearly linear) range of lo6 and is practically a universal carbon detector with a fairly predictable response. Our revolution was now in full swing. Already in 1954, we were doing analyses in less than one hour which, two years earlier, would have taken days, or would have been impossible. Very soon, we could determine relative proportions of most of our reaction products and by-products with considerable accuracy and speed. "Relative proportions": this highlights one of the minor disadvantages of the technique. Without the word relative, gas chromatography could not have existed. Retention times or volumes, response factors, proportions - all were relative. Even today (let it be whispered), absolute answers are not all that easy to determine. However, in my particular field, it was only at certain stages in the investigation of a process that absolute values were necessary. We were still analytical chemists and had our absolute chemical methods for specific functional groups. Gas chromatography is a deceptively easy technique, which can be subtly imprecise if care is not taken. This became very apparent in collaborative experiments organised by the Gas Chromatography Panel of the Institute of Petroleum. This panel's job was to devise and test standard gas chromatographic methods for analysing particular classes of samples. Each class produced so much variation in interlaboratory results, and so much heated discussion on what parameters were or were not to be specified, that a sub-panel was set up in 1966, under my chairmanship, to consider the matter. It may be salutary for the modern reader to read its report (2), both to see how things have changed since 1966 and to learn how many subtle variables there are. As most of our samples derived from vapour phase oxidation etc. over catalysts, it was obvious that if we could sample the reactor effluent direct, a lot of messy handling would be avoided. We started this as soon as practical sampling valves were developed. For some reason, which I as an analyst cannot understand, there was (and is) considerable resistance to this idea from our customers, although a large minority were enthusiastic about it. We eventually did quite a lot of complete effluent analyses which involved multi-column set-ups and column switching. As gas chromatography is primarily a separation technique, we very early on used a 30 mm diameter column and trapped individual peaks for infrared identification. Nowadays, of course, this is usually done on-line with a mass spectrometer. The almost instant identification provided by this arrangement for impurities in the parts per million range, is a very good example of the gas chromatography revolution; such a thing was absolutely unthinkable in 1952. I sometimes wonder whether we are not nowadays providing the unfortunate research chemist with too much information and addling his brains for him; Boyle would never have discovered his law if he had had three places of decimals:

343 Of all the ancillary techniques which have been developed, the most useful to us was pyrolysis, especially with the Curie-point pyrolyser. The reproducibility and even the predictability of pyrograms has never ceased to amaze me. It extended the revolution to the analysis of polymers, co-polymers and various unclassifiable deposits and goos which are apt to appear on plates, and in various nooks and crannies in chemical plants. This has been a brief account of the revolution gas chromatography brought about in one particular field of analysis. It is, of course, not the only field, and analysis is not the only activity in which gas chromatography is useful and has been revolutionary. It has had such an enormous and so widespread an effect because it is in essence such an extremely simple technique. It is, after all, merely blowing through a tube packed or coated with "stationary phase", nothing could really be simpler. However, as in all scientific disciplines, the more precise one wants to be the more trouble one must take over details. Hence, there has been an immense amount of work done by hundreds or even thousands of workers; so many that it would be invidious, if not impossible, to select a short list to mention here. Long gone are the days when one knew all or nearly all the practitioners. Although gas chromatography is still advancing in details, it has now reached the status of an old and well-established discipline. Since I retired four years ago, the only major development, of which I saw only the beginning, is the widespread application of computers to the control of the apparatus and the calculation of results. As a retired old fogey, I regret the interpolation of yet another piece of complex electronics between the analyst and the sample. I get the feeling that the machinery is beginning to get the upper hand; but this is probably just what was said when pH meters began to take over from litmus. It was a great stroke of good fortune for me to be in the game (and this is in some ways l e mot j u s t e ) s o early. It made my working life exciting, even more useful than before, and ever-developing. There can be very few common undistinguished analytical chemists who have been so lucky. As a very busy analyst, I have had literally no time for work on the more theoretical and recondite aspects of gas chromatography, but we analysts were always very glad to benefit from the excellent work done by many academic gas chromatographers; even though sometimes, I fear, they were pained by our earthy practicality. I am very glad to have been asked to contribute to this volume as a representative of the ordinary industrial scientific worker. I think that gas chromatography is probably the aspect of modern chromatography that would most amaze Tswett. It is certainly the aspect for which the term chromatography is the most inappropriate. REFERENCES 1 G.R. Primavesi, Analyst (London) 78 (1953) 647. 2 G.R. Primavesi, N.G.McTaggart, C.G. Scott, F. Snelson and M.M. Wirth, J . .Tnnst. Petrol. 53 (1967) 367-381.

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345

N.H. RAY

NEIL HUNTER RAY was born i n 1921, i n Birkenhead, C h e s h i r e , England. H e was e d u c a t e d a t t h e Royal Grammer School, Newcastle-on-Tyne and t h e U n i v e r s i t y o f Manchester. H e j o i n e d t h e A l k a l i D i v i s i o n o f I . C . I . i n 1942 and began r e s e a r c h i n t h e f i e l d of i n o r g a n i c polymers, b u t from 1947 u n t i l 1960 he was mainly concerned w i t h s t u d i e s o f t h e e f f e c t s of h i g h p r e s s u r e s on t h e r e a c t i o n s of s i m p l e g a s e s (carbon monoxide, carbon d i o x i d e , n i t r o u s o x i d e and ammonia) w i t h o r g a n i c compounds. During t h i s p e r i o d he was a l s o i n volved i n an i n v e s t i g a t i o n of t h e e f f e c t s of i m p u r i t i e s i n e t h y l e n e on t h e p r o p e r t i e s o f p o l y e t h y l e n e . I n 1965 he was a p p o i n t e d an I . C . I . Research A s s o c i a t e and s t a r t e d t o work on t h e c h e m i s t r y and p h y s i c s of g l a s s and t h e i n t e r p r e t a t i o n o f i t s p r o p e r t i e s i n terms o f polymer s c i e n c e . I n 1968 he was seconded, and l a t e r t r a n s f e r r e d , t o t h e I.C. I . C o r p o r a t e L a b o r a t o r y ( t h e n t h e P e t r o c h e m i c a l and Polymer L a b o r a t o r y ) i n Runcorn t o work on i n o r g a n i c polymers, and i n 1976 he w a s a p p o i n t e d an I . C . I . S e n i o r Research Associate M r . Ray h a s p u b l i s h e d o v e r 40 s c i e n t i f i c p a p e r s on v a r i o u s s u b j e c t s and i s t h e a u t h o r of t h e s e c t i o n on i n o r g a n i c g l a s s e s i n t h e book " I o n i c Polymers" e d i t e d by H o l l i d a y . H e h a s been a v i s i t i n g l e c t u r e r a t Manchester U n i v e r s i t y s i n c e 1971, and i n 1974, he was e l e c t e d an I n d u s t r i a l Fellow Commoner a t C h u r c h i l l C o l l e g e , Cambridge. H e i s a f e l l o w o f t h e Royal I n s t i t u t e of Chemistry. M r . Ray's involvement i n g a s chromatography s t a r t e d i n 1950, i n c o n n e c t i o n w i t h h i s i n v e s t i g a t i o n s on t h e i m p u r i t i e s of e t h y l e n e . H e b u i l t t h e f i r s t g a s - l i q u i d p a r t i t i o n chromatograph w i t h a t h e r m a l cond u c t i v i t y d e t e c t o r and s y r i n g e i n j e c t i o n t h r o u g h a serum cap and cont i n u e d to c o n t r i b u t e t o t h e e a r l y development of gas chromatography u n t i l 1958.

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Polyethylene was discovered in the research laboratories of I.C.I. Alkali Division at Winnington, Cheshire, in 1933, but it was not until 1939 that continuous, full-scale production was begun. In those days, ethylene was produced by the dehydration of ethanol over a catalyst and purified by fractional distillation, and it was not until much later that reasonably pure ethylene became available from the first oil cracker at Billingham. During the early post-war years an important part of polyethylene research in the Alkali Division (now part of I.C.I. Mond Division) was concerned with the effects of different impurities present in trace amounts in ethylene on the properties of the polymer. It was for this reason that I spent a large part of 1949 and the first few months of 1950 tediously analysing five gallons (23 litres) of "still-bottoms" from the ethylene purification plant by fractional distillation. We finished up with nearly one hundred fractions of almost pure unsaturated hydrocarbons ranging from propene to 2,5,5,7-tetramethyloctene-1 (some of which represented no more than one or two parts per million of the original ethylene), and each of them was identified by ozonolysis and subsequent conversion of the resulting carbonyl compounds into crystalline derivatives. Although this procedure was reasonably successful in identifying and estimating the concentration of every significant higher-boiling impurity in the crude ethylene, it was obviously quite impracticable to analyse many samples this way and a much quicker and simpler procedure was needed for use on the polyethylene plant. After hearing C.S.G. Phillips' paper on the chromatography of gases and vapours which he presented to the Faraday Society in September 1949 ( I ) , I went to see him in Oxford and learned that, in principle at least, I should be able to perform the analysis of ethylene by displacement chromatography from a column of charcoal. We did, in fact, try this procedure and obtained some useful results with it; but in order to measure impurities at the level of a few tens of parts per million we had to pass two cubic feet ( 5 6 litres) of ethylene through the column, which was maintained at -8OoC during this part of the process, and then displace the adsorbed impurities very slowly at room temperature. A further disadvantage was that isomers were not distinguished on the chromatogram (even though they may have been separated) because they produced the same step height. I think it was Phillips who suggested that I went to see A.J.P. Martin at the National Institute for Medical Research, Mill Hill, which I did in October 1950, to learn about vapour-phase elution chromatography. At that time Martin and his colleague A.T. James were developing a gas-liquid partition chromatograph for the separation of fatty acids in the.vapour phase, and they were using an automatic titration Cell to record the elution curves of the individual acids as they emerged from the column. We discussed the possibility of applying a similar technique to the separation of hydrocarbons and it was agreed that I should attempt this. In the course of our conversation Martin mentioned the possible use of a catharometer as a detector, and he has always believed that it was he who gave me that idea. In fact I was already using a pair of thermal conductivity cells to

347 monitor the changes in composition o"f-the gas stream emerging from my charcoal adsorption column, having copied the idea from Phillips; but I did not reveal this to Martin at that time for the simple reason that I did not then know what the word "catharometer" meant and did not like to ask: I remember that our conversation that day was cut short by the news that Martin's son had been hurt in a road accident, and that I returned from Mill Hill with a lot of ideas. At this stage I knew that I had learned the essential features of a very powerful analytical tool which might not only solve my problem of ethylene analysis, but also many other similar problems throughout organic chemistry. It only remained to make it work. Some time later in 1951 having had some initial success, I returned to Mill Hill to keep Martin informed of my results. He kindly let me have an advance copy of the paper which he and A.T. James had just submitted for publication (2). I constructed the first gas chromatograph capable of separating and measuring hydrocarbons by partition chromatography in November 1950 in the I.C.I. Laboratories at Winnington. It consisted of a six-foot length of 4 mm bore glass tubing bent into a U-shape and immersed in a thermostatically-controlled oil bath contained in a three-foot long Dewar vessel. The column was packed with JohnsManville Celite which had been impregnated with bis(3,5,5-trimethylhexyl) phthalate. The choice of this high-boiling liquid for the stationary phase (which subsequent events proved to be a remarkably useful one) stemmed from the fact that I had received a sample of the material, together with a physical property data sheet, from I.C.I. Billingham Division (now Petrochemicals Division) who were then interested in developing it as a plasticiser for PVC. Having no special interest in PVC at that time, I could think of no use for this sample until the need arose for a high-boiling solvent for the gas chromatographic column packing. I reasoned that although a high-boiling hydrocarbon might be appropriate for the analysis of impurities in ethylene (which were nearly all hydrocarbons) nevertheless if it were successful the technique should also be applicable to a wide variety of organic compounds such as alcohols, ketones and esters, and so I wanted to use a more versatile stationary phase which would have a reasonable affinity for oxygen-containing compounds as well as hydrocarbons. Dinonyl phthalate seemed ideal since it contained a reasonable length of paraffin chain, two ester groups and an aromatic nucleus. For the detector I used the Same thermal conductivity cells that I had previously constructed for use with the charcoal column. These consisted of pairs of 48-S.W.G. nickel wires welded to short lengths of "Kovar" leads sealed through short glass tubes and contained in a vapour jacket so that they could be maintained at a different constant temperature from the column if desired. The thermal conductivity cells were connected together to form the arms of a Wheatstone bridge and this was supplied with 12 volts a.c. from a transformer. The out-of-balance signal was amplified by a rather ordinary audio amplifier originally designed for a radiogramophone (hi-fi enthusiasts may remember the Williamson anpli-

fier that was popular in those days) and fed to an ancient but extremely effective Evershed and Vignoles recording milliammeter in a beautifully-fashioned mahogany case. This instrument was actually capable of tracing a 50-cycle sine wave from the mains supply. So that I could introduce either liquid or gaseous samples into the column I decided to make use of hypodermic syringes of various sizes. When I went over to Winnington Works Medical Department and asked for some used hypodermic syringes (at that stage I had only a vague idea of what would be suitable) the sister-in-charge was extremely helpful and provided me with a selection ranging from 100 1.11 to 50 ml in capacity. The inlet end of the column was fitted with an ordinary serum cap (also from the surgery) and the carrier gas entered through a short tee-branch at right angles so that it swept the sample out from the tip of the hypodermic needle and on to the top of the packing, This was, I believe, the first time that a syringe was used for inserting samples on to a chromatographic column. I began to explore the capabilities of this equipment by separating mixtures of hydrocarbons. The first few trials immediately showed how sensitive the method was for detecting impurities and it proved quite difficult to obtain sufficiently pure samples of individual paraffins for calibration purposes. For this reason I had to spend quite a lot of time using the fractionating columns again, simply in order to purify specimens of the various isomeric pentanes, hexanes, heptanes and octanes. The olefins, of course, presented no such problem, because many of them had already been isolated from previous distillations of impure ethylene. I found that this column of dinonyl phthalate on Celite was very versatile, giving useful separations of homologuous series of hydrocarbons, alcohols, esters, ketones, and ethers ( 3 , 4 ) . By modern standards the chromatograms produced were rather poor, with quite a lot of overlap between adjacent peaks; but at that time the results were remarkable, for no other method of analysis that used such tiny samples existed, apart from mass spectrometry which could not always give unambiguous results with mixtures. Clearly the next step was to apply the technique to the analysis of ethylene. Since it was required to detect and measure some impurities that were present at levels of only a few parts per million, and with the thermal conductivity detector then in use the minimum detectable quantity of a hydrocarbon such as hexene-1 was judged to be about 0 . 0 5 pl of liquid (roughly 0.01 ml of gas), a direct analysis was going to need an injection of something like 10 litres of ethylene. In a fit of enthusiasm I did actually construct a one-litre hypodermic syringe; this was a fearsome device consisting of a Bakelite piston sealed with two "0"-rings, moving in a short lenght of 3-inch bore Q.V.F. pipeline and driven to and fro by vacuum and compressed air respectively, This was connected to a chromatographic column built up from a number of sections starting with 2-cm bore and decreasing in diameter gradually so that the last 10 cm were 2-mm bore. Needless to say, this system did not work because if the

349

sample was injected slowly enough to avoid a tremendous pressure surge, the ethylene peak was so broad that it obscured the peaks corresponding to propene and butene and all the later peaks were so broadened that the effective sensitivity was too low. Evidently it was going to be necessary to make some sort of preliminary concentration of the impurities. It would not be difficult to remove all the unsaturated hydrocarbons by absorption in bromine, and with this aim, I set up a short pre-treatment column packed with charcoal on to which 30% (w/w) bromine had been adsorbed. The quantity of bromine that can be taken up by activated charcoal to form an easily-handled solid with only a faint smell has always impressed me, and it seemed a good opportunity to make use of this reagent. The original intention was to pass a fairly large sample of ethylene through this pretreatment column and condense out the residual bromine-inert impurities from the gas stream in a cold trap; then by rapidly heating the contents of the trap, the mixture of impurities would be swept on to the chromatographic column and analysed. Although this procedure was reasonably successful, it had one serious disadvantage, which was a tendency to collect bromine in the trap and while this in itself did no harm (except to produce a spurious peak in the chromatogram), over a long period of time the nickel wires in the thermal conductivity cells became corroded and failed. The column packing was probably also altered to some extent by reaction with bromine, and for these reasons I did no further work on that idea until 1953 when I read of Janlk's work (5,6) in an English translation which C.S.G. Phillips kindly lent me. Janlk's idea of'using carbon dioxide as the carrier gas, and measuring the fractions eluted from the chromatogram in a gas burette filled with astrongpotash solution, solved my problem because potash would, of course, absorb bromine as well. Applying this idea to the analysis of bromine-inert impurities in ethylene I produced a simple, straightforward piece of equipment that was readily portable and could be used on the polyethylene plant by the process workers (7). This was, however, no more than a partial solution to the problem because it only recorded saturated hydrocarbons and "inerts" such as hydrogen, carbon monoxide and nitrogen. Furthermore, of course, the use of strongcaustic solutions compelled the operators to wear protective goggles which made it difficult to take accurate readings, and the apparatus, although robust, was never very popular. Meanwhile, the potential value of gas chromatography in the analysis of mixtures of volatile organic compound of all kinds had been recognised by I.C.I. Billingham Division (now Petrochemicals Division) and it was there that the prototype of the first commercially-available gas chromatograph was developed. This was subsequently manufactured for general sale by Messrs Griffin and George Ltd. of Wembley, Middlesex, and, although no longer available, this historic instrument became very popular in a number of our laboratories. With a somewhat modified version of the Griffin and George gas chromatograph containing two columns, one packed with Linde type 13 molecular sieve, and the other with Apiezon Oil sup-

350 ported on Johns-Manville type C-22 firebrick, we were able finally to provide an acceptable method for the analysis of ethylene which was capable of determining not only bromine-inert impurities, but also propylene and the butenes. REFERENCES 1 C.S.G. Phillips, Discuss. Furaduy Soc. 7 (1949) 241. 2 A.T.James and A.J.P. Martin, Biochem. J . 50 (1952) 679. 3 N.H. Ray, J. A p p l . Chem. 4 (1954) 21. 4 N.H. Ray, J. A p p l . Chem. 4 (1954) 82. 5 J. Janhk, C o l l e c t . Czech. Chem. Comun. 18 (1953) 798. 6 J. Janak, C o l l e c t . Czech. Chem. Comun. 19 (1954) 684. 7 N.H. Ray, Analyst (London) 80 (1955) 853.

351

LUTZ ROHRSCHNEIDER

LUTZ ROHRSCHNEIDER was born i n 1927, i n B e r l i n , Germany. H e s t a r t e d t o s t u d y i n Cologne and Konigsberg ( t o d a y : K a l i n i n g r a d , U.S.S.R.) b u t i n 1945, a f t e r three-months i n t h e German Army, he was c a p t u r e d by t h e Red Army and t h u s , had t o s t a y i n t h e Sov i e t Union f o r t h r e e y e a r s a s a p r i s o n e r of-war. I n 1948, he c o n t i n u e d h i s s t u d i e s , now a t t h e U n i v e r s i t i e s o f Miinster and Munich and r e c e i v e d h i s Ph.D. i n Chemistry from t h e U n i v e r s i t y of Munich i n 1957. I n t h e same y e a r he j o i n e d t h e A n a l y t i c a l Department o f Chemische Werke H u l s , i n Marl. A f t e r one y e a r , he took c h a r g e o f t h e gas a n a l y s i s l a b o r a t o r y ; a s p a r t o f t h i s app o i n t m e n t , he b u i l t - u p a g a s chromatography l a b o r a t o r y now comprising 36 g a s chromatog r a p h s , s i x o f which a r e working c o n t i n u o u s l y and a u t o m a t i c a l l y . I n 1968 he became t h e head of t h e I n s t r u m e n t a l A n a l y s i s Group and r e c e n t l y , o f t h e Anal y t i c a l Department. D r . Rohrschneider i s t h e a u t h o r of more t h a n 20 s c i e n t i f i c p a p e r s on g a s chromatography and a n a l y t i c a l methods. S i n c e i t s f o u n d a t i o n i n 1961, he i s a member o f t h e German A r b e i t s k r e i s Chromatographie; he was one o f t h e f o u n d e r s o f t h e i n t e r n a t i o n a l j o u r n a l Chromatographia and is now s e r v i n g a s one o f i t s e d i t o r s . H e i s a l s o a member o f s e v e r a l n a t i o n a l and i n t e r n a t i o n a l a n a l y t i c a l committees. D r . Rohrschneider's s c i e n t i f i c i n t e r e s t s a r e t h e c h a r a c t e r i z a t i o n of t h e s e l e c t i v i t y o f s o l v e n t s and s t a t i o n a r y p h a s e s , t h e t h e o r y of t h e thermodynamics of s o l u t i o n s , enhancement o f t h e speed o f a n a l y s i s , t h e s t a n d a r d i z a t i o n o f r o u t i n e a n a l y t i c a l methods and e n r i c h m e n t . t e c h n i q u e s f o r environmental a n a l y s i s . D r . R o h r s c h n e i d e r ' s a c t i v i t i e s i n gas chromatography s t a r t e d toward t h e end o f t h e 1 9 5 0 ' s . He h a s developed a system f o r t h e c h a r a c t e r i z a t i o n of s t a t i o n a r y p h a s e s which b e a r s h i s name, a widely used method f o r t h e GC d e t e r m i n a t i o n of monomers i n polymers by head-space a n a l y s i s of t h e polymer s o l u t i o n and an a u t o m a t i c GC method w i t h p r i n t o u t of t h e r e t e n t i o n i n d i c e s f o r each component.

"Build us a preparative gas chromatography apparatus" was the first task which, in 1957, brought me into contact with chromatography. I read the literature: Bayer, Kovhts, Kirkland and Tuey had built impressive apparatus. My apparatus looked completely different. The column lay, like a glass eel, in a long metal box through which flowed water. This apparatus was used to isolate the unstable compound diacetylene from the Hiils electric arc condensate, and to concentrate monochloroacetylene, an even more unpleasant compound which decomposes explosively on mere exposure to air, for calibration purposes. Analysis by means of gas chromatography in petrochemistry does not so much imply research into the basics of this fantastic analytical method, but, above all, the execution of rapid and correct analyses. We can look back on year-long battles about the "correct" calibration factors. The uncertainties were great: did every substance, every detector or even every concentration have its own calibration factor? It was only the paper by Messner, Rosie and Argabright ( 1 ) which shone light into this darkness.

PoZarity of the Stationary Phases The retention characteristics of the stationary phases were equally puzzling. We groped around helplessly, with the previously known stationary phases and with many other possible liquids. Every column proved to be different and no system could be found to describe the diverse and confusing retention characteristics. I was also searching for a suitable column, for what was at that time our most important problem, the complete separation of the C 1 to C5 hydrocarbons. Plasticisers, surfactants and high-boiling solvents were tried as stationary phases. Marlophene 87, a product of our Company (a nonylphenol ethoxylate) is, to this day, offered by several column manufacturers as a good separation phase. At the time about which I write, it was customary in our Company to provide, each month, a short report on the work carried out. The monthly report for February 1959 reads: "18 high-boiling liquids, including a number of Hills products, were examined for their suitability for the separation of gaseous hydrocarbons. It was found that the stationary phases can be classified in a series of increasing polarity. In this series, the retention data show a continuous progression, with the olefins and diolefins being retained more firmly with increasing polarity." . This fundamental relationship was first presented on April 17, 1959 at a symposium, and published in the same month ( 2 ) . Dal Nogare subsequently included it in his book (3). The basic diagram indicated how, with increasing polarity from right to left, the olefins are retained more strongly than the paraffins, the retention ratio of butadiene to butane being used as a measure of polarity.

Characterisation of Stationary Liquids From then on the problem of stationary phase characterization continuously occupied my thoughts. The graphic representation of the "polarity scale" already showed that silicone oils exhibit special solvent characteristics. With these oils, the paraffins are much

353 closer together than one would expect based on the polarity. However, the acetylenes are also evidently influenced by a further, unknown factor. Accordingly, several forces responsible for the retelition characteristics are at work in the solvent (the stationary phase). It was my belief from the beginning that it must be possible to provide an empirical quantitative description of these forces: During the winter of 1963-64, and beyond, we measured, calculated, thought and again calculated. My colleague Hubert Lorkowski helped me greatly in these intensive calculations. Finally, we were able to describe 600 retention data of 40 compounds on 9 or 20 columns by means of a system of three linear equations with three factors for the solute and three factors for the solvent ( 4 ) . My mathematical knowledge was inadequate for these matrix calculations. Accordingly, al: these statistical calculations were still carried out by conventional algebra with the aid of a mechanical table-top calculator. When, finally, this equation for calculating the retention index difference

AI = ax + b y + c z was tested on carefully considered, very comprehensive experimental data, it was found that two further factors had to be introduced in order to permit sufficiently accurate predictions:

AI

= ax

+ b y + c z + du + es

Thus, we now had a system of five linear equations with the parameters

a , b , c , d and e for the solute (the sample) and the parameters 5 , y , z , u and s for the solvent (the liquid stationary phase) and this system permitted the astonishingly accurate calculation of comprehensive retention data. This was exciting. It was also informative to learn that until then, such systems of equations had primarily been used in psychology for describing human characteristics. Accordingly, experience was available of the description of the diverse characteristics of human beings, like the diverse properties of liquid stationary phases, by a linear combination. Walter Supina immediately recognised the importance of this new method of characterising stationary liquids by means of five parameters and issued a catalogue with the "Rohrschneider constants", which undoubtedly did more to make my method known than did the original German publication ( 5 ) . Kovhts ( 6 ) also immediately appreciated the scientific possibilities behind this method.

An Improved Polarity Model Roy Keller then invited me to describe these discoveries in an article for the next volume in the Advances in Chromatography series. During this work (7) on the polarity of stationary phases in gas chromatography, the relationship between polarity and internal pressure became clear to me. Today, my belief is that the cause of this elusive phenomenon of "polarity" is in fact the internal pressure of the solvent.

354

355 For practical gas chromatography it suffices to describe the polarity by an index difference. The case is simplest with benzene. This gives us a polarity scale of about 600 index units between squalane (0) and tris-cyanoethoxy-propane (594), with Carbowax 20M (322) being in the middle. In 1968 I met E. Kovbts and W. McReynolds at the International Chromatography Symposium in Copenhagen. McReynolds had carried out further measurements with even more compounds and stationary phases. We had also tested my system on very complicated hydrocarbons and found certain differences between calculated and measured values. After intensive correspondence and mutual agreement, McReynolds published the results of his measurements, an outstanding piece of experimantal work ( 8 ) , which has proved exceptionally fruitful*. Today, this system represents the only possibility for predicting retention data from data available for the solute and the stationary phases. However, a complete theoretical breakdown of this system has hitherto not proved possible. Is it a meaningless mathematical description, or will the unknown parameters one day be replaced by measurable properties of the substance, such as dipole moment, polarisability, solubility parameters or molar volume? No doubt, the real case is somewhere between these extremes. Some properties of the substances will prove usable in calculating the retention, as has already been demonstrated ( 9 ) , while others will always have to be determined by chromatographic experiment. Above all, the influence of geometrical parameters on retention will always remain important for accurate retention data and if it ever becomes calculable from the actual properties of the substances, this will only be achievable with very extensive calculations.

Gas Chromatographic Analysis in the Industry The function of the gas chromatography laboratory of our Analytical Department is to be able to analyse every product suitable for gas chromatographic analysis, identifying every component including also those which are available only in trace quantities, and also to be able to determine each component quantitatively. This means that methods have to be developed for the analysis of over 400 different products. Furthermore, one cannot rely on a method developed a few years earlier because of two questions that must be considered continuously: are the separated and identified trace compounds still the same as earlier, and are the original calibration factors still valid? A particular analytical problem with which we have to deal is the purity of monomers used for polymerisation. Huls manufactures styrene, vinyl chloride, butadiene, ethylene and propylene, which are then polymerised, in some cases together with vinylpyridine, dibutyl maleate or acrylic acid. All these starting materials must be produced with extremely high purity. The purity is checked by *W.O. McReynolds, a scientist with the Celanese Corporation, who further developed the Rohrschneider system for expressing stationary phase polarity, died suddenly on September 17, 1976 (The Editors).

3 56

--

inlet

0.7 mllrnin

40 ml lmm

Phtholalll column

Fi -

-

-

Fig. 39.2. Injector-detector split for the production of a sample-size-independent internal marker in the chromatogram ( 1 3 ) . gas chromatographic analysis which has to meet exceptional requirements in respect of accuracy and the limit of detectability. For example, the butadiene content of vinyl chloride, which is in the order of 1 to 8 ppm, must, as a matter of routine, be determined on several samples per day to an accuracy of 0.1 ppm. This analysis additionally requires a special modification of the apparatus, such as a detector split ( 1 0 ) if the results are to be evaluated by means of a gas chromatograph-computer system. Many of these industrial analysis methods have not been published. No manufacturer of a particularly pure product likes to let his customers know the analytical methods used for the determination of the impurities. If, however, a manufacturer wishes to exploit a market advantage by being able to offer a particularly pure product, he has to disclose the methods of analysis so that the customer can convince himself of the exceptional quality of the product. Accordingly, a good method of analysis is often a valuable know-how advant age.

!.:npublished Results A substantial proportion of analytical research and development carried out in industrial laboratories remains unpublished. This is either because the intention is that the company shall retain the analytical know-how or that the publication of the results requires additional work for which there is no time available. I would like to give some examples to show what kind of important results obtained in industrial analysis remain unpublished. After the publication of Hal6sz and Heine ( 1 1 ) first describing packed capillary columns, I was able to see the method of column manufacture, at the neighbouring company of Scholven-Chemie. Dr. Halhsz then head of the analytical laboratories of this company - has always been outstanding in his willingness to share his experience with colleagues, including myself. Whilst Scholven-Chemie used aluminium

357

I

4

1

0

N

2

Fig. 39.3. Chromatogram of the lower hydrocarbons on a packed capillary column containing oxydipropionitrile on chromium/aluminum oxide. oxide containing a certain amount of water for the separation-of the C1-C4 hydrocarbons, we wanted to employ the packed capillary columns with oxydipropionitrile (ODP) on chromium/aluminium oxide, which we had ourselves developed. We had found, from extensive measurements, that a great variety of very useful columns could be produced with different loading of ODP on chromium/aluminium oxide. The original work we did on regular packed columns but we modified these to obtain packed capillary columns, resulting in a halving of the analysis time while maintaining the same separation efficiency. These hitherto unsurpassed columns have been used in our laboratories for over 10 years - with a working life of several years - for the analysis of C4-cuts; however, this is the first time that a chromatogram is shown. Another example: following an important investigation of Dr. D. Deans on the non-linearity of flame ionization detectors ( 1 2 ) we investigated this phenomenon thoroughly in our laboratory. In doing so, we found that at a certain concentration, the combustion process in the flame showed a "kink" in the linearity. These conclusions were not published either. Another piece of work which remained unpublished was a method of column optimisation, in which, in order to shorten the time of measurement, the columns to be tested were tested exclusively at the maximum possible column input pressure. This achieves the object more rapidly and it is not necessary to record a Van Deemter curve each time. An optimum separating column must be used under high

358 column input pressure, otherwise it is not an optimum column and it can be improved. The most important analysis in our gas chromatography laboratory is the so-called "standard analysis U" and this also has not been described in the literature: we have only published ( 1 3 ) the less extensively used automatic standard analysis "P" with a polar column and the calculation of the retention index for each peak. In our laboratory, three automatic gas chromatographic systems carry out about 50-80 similar analyses per day on a great variety of compound mixtures, using "standard analysis U". This analysis is extensively used by a staff of about 50 research chemists. Within 24 hours after submitting the sample, the chemist is receiving a print-out, with the attached chromatogram, on which is specified, for each component, the retention time, the retention index, the boiling point calculated therefrom, and the percentage concentration. The boiling point is an important piece of information to a chemist. The boiling differences permit him to assess what purification steps have to be applied to his product. Mistnlces Inevitably, I have also made mistakes. However, the case of an indignant letter to the editor of the Journal of Chromatographic Science by George H. Stewart, who considered it impossible that absolute retention volumes should be capable of calculation from the retention ratio of two adjacent homologous paraffins ( 7 4 ) , was not one of these mistakes. On the contrary, I am certain that such a relationship is correct,notonly empirically but also theoretically. I do not know whether my reply succeeded in convincing Mr. Stewart. One of my own letters, to Chrornatographia ( 1 5 ) , in which I expressed doubts about the assumption by Groenendijk and Van Kemenade that there was an absolutely linear relation between the carbon number and the logarithm of the retention time of homologous paraffins, was probably also not a mistake. The same Institute subsequently published an experimental investigation (16) which confirmed my view that a dead-time determination with methane is more accurate than a calculation from the retention of the lower paraffins. What was wrong, however, was my work ( 1 7 ) on the speed of separation of chromatographic columns, which fortunately was only published in German. It was Istvan HalLsz who delicately drew my attention to the mistakes. On the other hand, it was also HalLsz who had introduced me to this subject. In addition to the separating capacity of a column, the quality of a chromatographic separation must additionally be described by a time parameter if the speed of analysis is to be described. Even today, this problem has not yet been fully explored. For the characterisation of this speed, HalPsz is using the relationship N / t (where N is the number of effective plates and t is the retention time of that particular peak) originally described in 1959 by Desty ( 1 8 ) . In my view, this is not a universally valid measure since, at least in liquid chromatography, very high ?j/t values can be obtained if excessively high pressures are used

359 and the column is operated under conditions far removed from its optimum. However, whatever characterises the quality of a column, it should be the greatest when the separating capacity is optimum. My mistaken idea was to use the parameter hi/* (where n = number of theoretical plates) as a measure of speed, since fi is proportional to the resolution and ultimately, it is the resolution and not the number of plates (theoretical or effective) which counts. Barry Karger who visited me about this time, was - rightly - also unable to become enthusiastic about my idea. Today, I would be inclined to propose the parameter N 2 / t as a measure of the speed of separation. This parameter is virtually constant for all optimum analytical conditions (within a certain column type), at a given pressure, An even better parameter to maximize column optimization is

Qo = N 2 / t A

R P

where Qo refers to the column quality to be optimized, N is the number of effective plates, tR the retention time (from start) and A p the pressure drop along the column. This rather unknown relationship seems to be a good measure of column quality ( 1 9 ) .

Future Deve Zoprnents Gas chromatography has developed a long way. What we lack is simplification of the excessively diverse conditions of analysis. Most problems can be solved with a few standardised analytical conditions. It will also become possible to carry out analyses more rapidly by using high column input pressures. In that case, very precise electronic measurements of the retention time will be needed to ensure reliable peak allocation. Only integrated automatic injection will make it possible to provide the sufficiently accurate start time determination which this technique requires. The object of this development will be a gas chromatography apparatus which automatically and correctly analyses a variety of samples.

Friends Twenty years in gas chromatography from the beginning of the industrial use of the technique to the present time have brought me many happy and close contacts with colleagues in the same field. I esteem their skill of formulation, their technical originality and their scientific enterprise. These outstanding personalities have always inspired me in my own scientific research. Besides numerous professional relationships, many personal contacts, even friendships, have been established. To me each of the frequent meetings with my colleagues at chromatographic conferences and symposia is always a new pleasant and stimulating experience.

REFERENCES 1 A.E. Messner, D.M. Rosie and P.A. Argabright, Anal. Chem. 32 (1959) 230. 2 L. Rohrschneider, 2. Anal. Chem. 170 (1959) 256. 3 S. Dal Nogare and R.S. Juvet, Gas Liquid Chromatography, Interscience Publishers, New York, 1962. 4 L. Rohrschneider, J . Chromatogr. 17 (1965) 1. 5 L. Rohrschneider, J . Chromatogr. 22 (1966) 6. 6 E. KovHts, Chimia 22 (1968) 459. 7 L. Rohrschneider, in Advances i n Chromatography, J.C. Giddings and R.A. Keller, eds.,Vol. 4, M. Dekker, Inc., New York, 1967, pp. 333-36 8 W.O. IcReynolds, J . Chrornatogr. S c i . 8 (1970) 685. 9 P.H. Weiner and D.G. Howery, Anal. Chem. 4 4 (1972) 1189. 10 L. Rohrschneider, Chromatographia 7 (1974) 555. 11 I. Halhsz and H. Heine, in Advances i n Chromatography, J.C. Giddings : R.A. Keller, eds.,Vol. 4 , M. Dekker, Inc., New York, 1967, pp. 207-2' 12 D.R. Deans, Chromatographia 1 (1968) 187. 13 A. Jaeschke and L. Rohrschneider, Chromatographia 5 (1972) 333. 14 L. Rohrschneider, J . Chromatogr. S c i . 8 (1970) 105. 15 L. Rohrschneider , Chromatographia 2 (1969) 437. 16 C.A. Cramers, J.A. Luyten and J.A. Rijks, Chromatographia 3 (1970) 441. 17 L. Rohrschneider, Chromatographia 3 (1970) 431. 18 D.H. Desty, A. Goldup and W.T. Swanton, in Gas Chromatography (1959 Lansing Symposim), N. Brenner, J.E. Callen and M.D. Weiss, eds., Academic Press, New York, pp. 105-135. 19 L. Rohrschneider, 2. Anal. Chem. 277 (1975) 335.

361

KARL 1. SAKODYNSKII

KARL IVANOVICH SAKODYNSKII was born i n 1930, i n N o v o r o s s i j s k , t h e S o v i e t Union. H e graduated from t h e Physico-Chemical F a c u l t y of t h e Mendeleev I n s t i t u t e of Chemical Technology, Moscow, i n 1953, r e c e i v e d h i s c a n d i d a t e d e g r e e i n 1957 and h i s d o c t o r of chemical s c i e n c e s d e g r e e i n 1971. S i n c e 1957 he h a s been a s s o c i a t e d w i t h t h e Karpov Research I n s t i t u t e of P h y s i c a l Chemistry, i n Moscow, where he became a deputy d i r e c t o r i n 1974. Being a v i s i t i n g l e c t u r e r on chromatography s i n c e 1968, he a l s o became p r o f e s s o r of p h y s i c a l c h e m i s t r y a t t h e Mendeleev I n s t i t u t e . D r . Sakodynskii i s t h e a u t h o r of more t h a n 200 s c i e n t i f i c p a p e r s , a q u a r t e r of which i s r e l a t e d t o i s o t o p e s e p a r a t i o n w h i l e t h e rest deal. w i t h v a r i o u s a s p e c t s of gas chromatography. H e i s a l s o t h e aut h o r of six books: t h o s e on p r e p a r a t i v e g a s chromatography ( w i t h S. Volkov, 1972) and on i n s t r u m e n t s f o r chromatography (1973) have been s i g n i f i c a n t i n t h e development of t h e t h e o r y and p r a c t i c e of chromatography i n t h e U.S.S.R. D r . Sakodynskii i s t h e chairman of t h e Adsorbent Group of t h e S c i e n t i f i c Council f o r Chromatography i n t h e Academy of S c i e n c e s of t h e U.S.S.R., and s e r v e s a s a member of t h e e d i t o r i a l boards of t h e Z h m a Z Fizicheskoi Khimii and Chromatographia. H e h a s been t h e organ i z e r of a number of All-union and i n t e r n a t i o n a l chromatography confer e n c e s and symposia. D r . S a k o d y n s k i i ' s i n t e r e s t s i n c l u d e a number o f i m p o r t a n t problems i n chromatography. Among them h i s p i o n e e r i n g a c t i v i t i e s on t h e p r a c t i c a l r e a l i z a t i o n of p r e p a r a t i v e g a s chromatography a r e of s p e c i a l i n p o r t a n c e . H i s r e s e a r c h on t h e l i f e and a c t i v i t i e s of M.S. T s w e t t r e s u l t e d i n i m p o r t a n t i n f o r m a t i o n concerning t h e c r e a t i o n and development o f chromatography.

When, in September 1959, I visited the Organisch-Chemisches Institut fur Verfahrenstechnik in Leipzig, German Democratic Republic, I first saw a gas chromatograph with a flame ionization detector. At that time, the subject of my research work was the separation of the isotopes of light elements and although, naturally, I was aware of the publications on chromatography, I never dealt with it. It was in the Leipzig laboratory that the extremely great analytical possibilities of gas chromatography became evident to me. Early in 2960, it was decided to start activities on gas chromatography at the Karpov Institute of Physical Chemistry. We first belonged to the Laboratory for Adsorption which, at that time, was headed by Academician N. Javoronkov. The theoretical background of our activities was developed by Professor N. Tunitskii. We started with the development of the proper instrumentation. Manufacturing of chromatographs in the Soviet Union just started about that time. However, the type available then did not satisfy us and we created simple glass units, which worked quite satisfactorily. We utilized the wires in light bulbs as the filaments for catharometers and special bricks made €or industrial furnaces as the support materials. Believing in our own design abilities, we also developed a capillary gas chromatograph in 1962. The first scientists who were involved in gas chromatography at our Institute were young chemical engineers coming to our laboratory after graduating from the Mendeleev Chemical-Technology Institute in Moscow; V. Brazhnikov, S. Volkov and I. Udina should be mentioned particularly since they are still active in this field. The first successful analysis carried out was the determination of trace impurities in acrylonitrile, in the middle of 1960. L. Hohlova carried out hundreds of these analysis and always enjoyed doing it. The first commercial unit we acquired was a Pye Argon gas chromatograph. We have solved many analytical problems with it; however, we preferred to carry out our research and development work on the independent units developed by us. In 1963, our group was given an independent status within the Institute and the direction of our work was determined for the following years. Being born in a Laboratory of Separation, we considered preparative gas chromatography as the main direction of our studies. The first few years were spent on solving a number of design and automation problems. Part of these activities later served as the background for the development of the "Etalon" industrial instruments, In this period, we also solved the questions of preparing highefficiency columns and traps with 90-95% yield. The most important task with which we were faced was the separation of deuterated organosilicon compounds with boiling points over 35OoC. Great attention was also paid to the development of different types of preparative gas chromatography such as circular, dynamic heat chromatography, o r chromatography with modified sorbents, etc. In the period of 1966-1970, we carried out extensive research on the development of the linear theory under the conditions of injecting large volume samples, and established relationships for

363

Fig. 40.1. The gas chromatography group of the Karpov Institute of Physical Chemistry, MOSCOW, in Winter 1970. Left to right: V. Zelvenskii, V. Tchernoplekova, I . Yudina, Buy-Thi-Hong, K.I. Sakodynskii, Le-Chi-Le, S. Volkov, L. Panina, N. Klinskaya and 0. Trubotchkina. the calculation of the proper flow rate andscolumm length which could provide the maximum capacity. The general concepts developed for the quantitative calculation of preparative GC columns, including the estimation of the purity of the separated fractions permitted us to optimize the process of preparative separation by gas chromatography. We consider our results in using preparative gas chromatography for industrial purposes as a very important achievement. In collaboration with V. Avarin and his colleagues our experience was utilized in the creation of an industrial unit -.practicallythe first in the whole world which could separate about-100 reactive compounds with a purity over 99.9% ( 1 ) . The results of our activities have been summarized in a monograph of preparative chromatography (2). The second stage of our development work concerning the theory and practice of the calculation of industrial preparative columns started in 1970. These activities included research on the hydrodynamics of large diameter columns and the mathematical description of the chromatographic separation of large-volume samples, with high concentration of the sorbate. We succeeded in achieving an even distribution of the flow across the cross-section of 150-mm diameter columns.

-

364

Fig. 40.2. Industrial preparative scale gas chromatographic unit with a column of 0 . 5 m diameter.

On the basis of indirect and, for the last couple of years even direct, measurement of the local carrier gas flow rates it was shown that the decrease in efficiency when increasing the column diameter is caused not only by the changes in the flow profile (as was considered earlier) but also by local changes in the carrier gas flow rate. The methods developed by us made it possible to achieve an HETP value of 4 mm for columns with 150-200 cm in diameter. In 1972-1975 we used the plate model for the description of the chromatographic process when the isotherms are non-linear, and the program for computing the shape of the curve as well as for the calculation of a binary mixture with mutually influenced sorption was developed. On the basis of these experiments, regression equations linking the main parameters of asymmetric peaks (retention volumes, peak width, asymmetry value, etc.) to the separation conditions were obtained. The main parameter of the "plate model" is the value of the HETP. Our activities for increasing the industrial applications of preparative gas chromatography are still continuing. Recently, in cooperation with U. Kovanko, a unit with 0.5 m diameter columns was developed. Another direction of our research activities is the study of polymer supports and sorbents, aiming at the development of new materials for gas chromatography as well as establishing a relationship between the structure of polymeric sorbents and their gas chromatographic properties, We started these activities with the modification of a PTFE support. By various heat treatments of porous PTFE we were successful in developing the high efficiency and rigid support "Polychrom-1" which is now produced commercially (3). We also studied new types of porous polymer sorbents receiving the name "Polysorb". We have extended the investigations to a large number of polymer sorbents including those of the "Tenax" type and established their structure characteristics as well as obtaining scanning electron microscope photographs of their structure (4). As a result of this research, dealing with the synthesis of the polymers, their composition and structure, the methods of achieving high efficiency sorbents are no longer secret. By systematically controlling the

conditions, we could regulate the specific properties of the polymeric sorbents and create new selective substances (5). A further field of our activities concerns the selectivity of stationary liquid phases, We have investigated in detail the various polysiloxane type phases and also developed new substances of the carborane siloxane type, with phenyl groups, as well as siloxanes with phenanthrene groups. As a result of our activities, our group has published more than 150 articles on various aspects of chromatography. In addition to my activities at the Institute of Physical Chemistry, I should also mention the work of the Scientific Council on Chromatography of the Academy of Sciences of the U.S.S.R. There, our main activities were related to the question of sorbents and standard materials for chromatography. The close cooperation with many scientists and laboratories resulted in the wide utilization of our results. Finally, a favorite part of my own activities is related to collecting material about the founder of chromatography, M.S. Tswett. I started this activity in 1962 and it resulted in a number of publications, mainly in connection with the centenary of Tswett's birth (6). This activity not only helped to clear up some biographical information about this outstanding scientist but also showed the real aims of Tswett's activities. Many of us feel that although now 75 years old, chromatography is still young. Its task is to fulfill the requirements of science. Nowadays, chromatography meets the desires of a scientist and can help mankind. It is partly due to the application of the technique to biochemistry and medicine that we can eliminate a number of diseases. Chromatography will also help us in the future to develop new ways of providing energy sources to supply our needs and to improve our life on the Earth. I am sure that Michael Semenovich Tswett would have liked to have known how important his method will become when he discovered chromatography 75 years ago. REFERENCES 1 K.I. Sakodynskii e t a l . , Khim. Prom. 5 (1972) 331. 2 K.I. Sakodynskii and S . Volkov, Preparativnaya Gasovaya Khromatografiya (Preparative Gas Chromatography), MOSCOW, 1972. 3 V. Berezkin, V. Pakhamov and K.I. Sakodynskii, Tverdye NositeZi Dlya Gazovi Khromatografii (Supports for Gas Chromatography), MOSCOW, 1975. 4 K.I. Sakodynskii and L.I. Panina, Chromatographia 4 (1971) 113. 5 K. I. Sakodynskii and L. Panina, Polimernye Sorbenty Dlya MolecuZyarnoi Khromatografii (Polymer Sorbents for Molecular Chromatography), Nauka, MOSCOW, 1977. 6 K.I. Sakodynskii, in Uspekhi Khromatografii, Nauka, Moscow, 1972, pp. 9-25; see J . Chromatogr. 73 (1972) 303-360.

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367

GERHARD SCHOMBURG

GERHARD SCHOMBURG was born i n 1929, i n Bochum, Nordrhein/Westfalen, Germany. H i s s c i e n t i f i c e d u c a t i o n began i n 1951, w i t h s t u d i e s t o become a chemical e n g i n e e r and was c o n t i n u e d w i t h s t u d i e s i n c h e m i s t r y a t t h e U n i v e r s i t y o f Bonn between 1951 and 1954. H e r e c e i v e d h i s Ph.D. from t h e Techn i c a l U n i v e r s i t y o f Aachen i n 1956. H i s t h e s i s work was c a r r i e d o u t a t t h e MaxP l a n c k - I n s t i t u t f u r Kohlenforschung, i n Mulheim/Ruhr, under P r o f e s s o r K a r l Z i e g l e r , t h e Nobel l a u r e a t e ; i t s s u b j e c t was i n f r a red spectroscopy. After finishing h i s university s t u d i e s D r . Schomburg remained a t t h e I n s t i t u t f u r Kohlenforschung and s t a r t e d t o o r g a n i z e a g a s chromatography l a b o r a t o r y . H e developed i t i n t o a c e n t r a l i z e d and f u l l y computerized a n a l y t i c a l l a b o r a t o r y f o r chromatographywhich a l s o d e a l s w i t h r e s e a r c h and development on chromatographic metho d s . I n 1966 he took c h a r g e of a s i m i l a r l a b o r a t o r y a t t h e n e i g h b o u r i n g Max-Planck-Institut f u r S t r a h l e n c h e m i e d e a l i n g w i t h r a d i a t i o n and photoc h e m i s t r y of b i o l o g i c a l and n o n - b i o l o g i c a l m a t e r i a l . D r . Schomburg i s t h e a u t h o r and c o a u t h o r of o v e r 90 s c i e n t i f i c p u b l i c a t i o n s and is t h e a u t h o r o f a t e x t b o o k on gas chromatography ( V e r l a g C h e m i e , 1977). H e i s a member o f t h e E x e c u t i v e Board of t h e chromatography group o f t h e G e s e l l s c h a f t d e u t s c h e r Chemiker (GdCh) and one o f t h e e d i t o r s o f Chromatographia H e p e r i o d i c a l l y o r g a n i z e s c o u r s e s under t h e p a t r o n a g e o f t h e GdCh on c a p i l l a r y chromatography. H e was t h e c h a i r man o f t h e S c i e n t i f i c Committee o f t h e I n t e r n a t i o n a l Chromatography Symposium h e l d i n Baden-Baden, German F e d e r a l R e p u b l i c , i n September 1978. D r . Schomburg i s one o f t h e p i o n e e r s i n t h e a p p l i c a t i o n o f g a s chromatography t o t h e a n a l y s i s o f complex o r g a n i c samples. H e h a s c a r r i e d o u t b a s i c work on t h e p r e p a r a t i o n and u s e o f open t u b u l a r ( c a p i l l a r y ) columns p a r t i c u l a r l y t h o s e made o f g l a s s and on t h e v a r i o u s i n l e t s y s t e m s , on t h e development o f automated systems f o r b o t h a n a l y t i c a l and p r e p a r a t i v e g a s chromatography and on t h e improvement o f t h e s e p a r a t i o n e f f i c i e n c y of l i q u i d chromatographic systems.

.

368 After studies at the University of Bonn I began my work at the Max-Planck-Institut fur Kohlenforschung in 1954, with a thesis which was performed under the guidance of Professor 1)r. Karl Ziegler, the director of the Institute. The topic of my thesis was the infrared spectroscopy of organo-aluminium compounds and aluminium hydrides. In the course of this work I made my first steps toward instrumental analysis using one of the first self-registering infrared spectrophotometers (Perkin-Elmer Model 21) existing in Europe at that time. Professor Ziegler and his coworkers had just (1953) made the spectacular discovery of transition-metal aluminiumalkyl catalysis for olefin polymerization and oligomerization for which later (1963) he was awarded the Nobel Prize. Besides his experienced ingenuity and his successful enthusiasm ,for preparative chemistry in the field of aluminium and other metal alkyls and hydrides (at that time these compound classes were considered to be of purely academic interest and of no practical use until the polyethylene, polypropylene and polybutadiene patents!) Ziegler already had a positive attitude to the utilisation of physical methods supporting chemical research. He arranged the installation of the first commercial German mass spectrometer (Atlas Model CH l), one of the first IR spectrometers and last but not least the first Perkin-Elmer gas chromatograph, the Model 154A at the Institute in the years of 1952-1954, parallel to the introduction of other physical methods for the elucidation of chemical structures like NMR, Raman, UV, etc. After I received my Ph.I). in 1956 Professor Ziegler proposed that I dedicate my further activities at the Institute to the intoduction and application of gas chromatography. There was an urgent need for the analysis of complex olefinic hydrocarbon mixtures originating from the catalytical reactions performed at the Institute. At that time I was not very experienced and without a special education in analytical chemistry and, of course, chromatography. I had to learn nearly everything; consequently, I began to visit symposia and to contact instrument companies and met scientists with different experiences in chromatography. I missed the first International Symposium on Gas Chromatography in London 1956 but participated in every subsequent chromatography symposium, beginning with those held in Amsterdam (1958) and Edinburgh (1960). Both symposia were extremely important and stimulating to me regarding the further work to be done in our Institute. It was in Amsterdam that Golay and Dijkstra reported on the theory and the first experimental results with capillary columns. Faced with the problem of the separation and later also characterization of isomeric hydrocarbon mixtures, requiring maximum separation efficiency at optimum stationary phase selectivity, I was immediately impressed by the potentialities of capillary chromatography. Subsequent visits to the pioneering laboratories at British Petroleum in Sunbury-on-Thames (D.H. Desty), National Distillers (R.P.W. Scott) and also at the Technical University of Eindhoven (A.I.M. Keulemans) provided further stimulation. My first contact with the gas chromatographic scene in the U . S . A . was at the first "Zlatkis Symposium" at the Sheraton Hotel in Houston, in 1963.

369

Fig. 41.1. Separation of isomeric dimethylcyclododecanes (plus methyl- and ethylcyclododecane) by capillary column gas chromatography. Column: 100 m x 0.25 mm I.D. glass. Stationary phase: DC-200 methylsilicone oil. Temperature: 0-25OC. Carrier gas (He) inlet pressure 1.0 bar.

I attended this meeting accompanied by Professors Bayer and Halksz and stayed in the country for a few weeks to pick up information on the progress in gas chromatography with regard to instrumentation and application. In 1967 Professor Wilke became the director of our Institute and the designated successor of Professor Ziegler. Professor Wilke also patronized the development of the groups for instrumental analysis. He initiated the installation of the large DEC-PDP 10 timesharing computer system, the existence of which in the following years proved to be an absolute necessity for effective and high level applications of physical methods such as X-ray structure investigation, MS, NMR and, naturally, gas chromatography of complex mixtures. In 1966 I was also appointed as the head of the chromatography group in the Institut fur Strahlenchemie (radiation chemistry) which is affiliated to our Institute and located next to us. This new activity extended the range of application of chromatographic analysis in my group to different (mostly more polar) compound classes originating from the irradiation of organic material with special biochemical interest including fatty acids, sugars, amino acids and heterocyclic compounds. In many cases these compounds had to be separated, identified and determined while being present as trace components in complex matrices. In both chromatographic groups a large variety of complex mixtures has to be analysed. However, in the early years of capillary chromatography very polar compounds could not be easily chromatographed. Adsorption effects between solutes and the inside surfaces of the

370 stainless steel capillary columns preferably used at that time, deteriorated peak symmetry or even caused irreversible adsorption. These difficulties generally prevented the early and widespread application of capillary columns and were the reason for increased efforts in column manufacture with special emphasis on deactivation and homogeneous coating of glass surfaces with stationa,ry liquids of varying polarity. This kind of investigation is still part of our present activities ( 1 - 4 ) . Besides the use of high-resolution capillary columns for the separation of complex mixtures the identification and characterization of isomeric species of complex mixtures by means of retention parameters (e.g. Kovhts retention indices) turned out to be useful but also difficult because of reproducibility problems. During the entire 20-year period of chromatographic developments great efforts were made to improve reproducibility of retention data by better columns and improved instrumental techniques. For identification purposes it was moreover absolutely necessary to understand the retention behaviour of isomers ( 5 - 9 ) in dependence of their chemical structure. According to our experiences over many years, our methods

I0 min

Start--

12haurs

0"

D

2

Fig. 41.2. Separation of isomeric deuterated 4-vinylcyclohexenes obtained by the codimerisation of 1,3-butadiene and 1,s -Db-butadiene. Columns: 300 m x 0.25 mm I.D. glass, liquid phase: squalane. Column temperature: 25OC. Carrier gas (He) inlet pressure: 3.0 bar.

] 2

3

L

of retention data storage and interpretation have proved to be a valuable support especially if the characterization by spectroscopic methods like MS - being the best compatible spectroscopic method for combination with capillary gas chromatography - is insufficient because of non-characteristic patterns of spectra. We have tried different methods to use the compiled retention data for incremental interpretation of chemical structures; such investigations are still carried out with excellent practical results. The limitations of this empirical approach of understanding the relationships between retention and structure were also elucidated ( 1 0 ) . Systematical investigations on retention and structure have been performed with nearly all kinds of hydrocarbon isomers and with compounds containing polar functional groups ( 5 ) . Various deuterium-labelled compounds were separated from each other and from the corresponding unlabelled species by using capillary columns with very high separation efficiency ( 1 1 - 1 3 ) . For the general application of capillary columns in practical analytical work, improvements of the commercially available instruments were absolutely necessary because insufficient constructions

371 have caused poor results and consequently strong aversions to the application of capillary columns in quantitative analysis, and this prejudice still exists in some places, In order to change this situation we also directed our activities to this problem. A significant part of our investigations concerned the development of reliable sampling techniques including those which are suitable for trace analysis, with particular regard to reproducible qualitative and quantitative analysis ( 2 4 ) and to hightemperature work (15,16). This work was preferably executed with the aim of the separation and characterization of polynuclear aromatic hydrocarbons of "high temperature" coal tar. In recent publications (2) a new sampling technique was presented which permits the direct introduction of even very dilute samples into glass capillary columns without splitting and/or preliminary volatilisation of the sample in an overheated vaporizer. A significant part of our activities was also directed towards multidimensional gas chromatography using column switching, backflushing, trapping etc. ( 2 7 , 2 8 ) . These techniques which can be automated - have been specially adapted for use with glass capillary columns in order to achieve maximum chromatographic performance, i.e. separation efficiency and selectivity for only selected compound groups in very complex mixtures. Complete analyses of this type would be very time-consuming or even impossible, The same techniques have also been successfully incorporated into automated preparative-scale gas chromatographs ( 1 9 ) and meanwhile also to process chromatography. Six completely re-designed commercial preparative-scale instruments in which column switching and backflushing are applied are operating continuously and automatically in our Institute. A long and fruitful cooperation exists between my group and Dr. D. Henneberg who is in charge of the Mass Spectrometry Department operating for both Institutes for the past 20 years. This cooperation began with his first set-up for mass-fragmentation gas chromatography during his thesis work performed at the Institute ( 2 0 ) . A direct coupling of a capillary column with a CEC 620 monitor mass spectrometer operating at fixed masses was installed in 1958 and our first report on the results was presented at the 1960 Edinburgh Symposium (22). Further developments in this field concerned the so-called open-split connection for GC-MS interfacing ( 2 2 , 2 3 ) and the on-line hydrogenation of previously separated unsaturated species between the gas chromatograph and the mass spectrometer by means of a short piece of glass capillary with a Pd-dotted surface ( 2 4 ) . A few years ago modern HPLC was also introduced in the Chromatography Laboratory of our Institute. The bonded phases which are now available could be expected to be especially suitable for the separation of isomeric and - even more important - also homologous hydrocarbons and their functional derivatives because Van der Waals type interaction forces between the carbon skeleton of the solute and the alkyl groups of the stationary phase are effective. By adding silver salts to the mobile phase an increased intermolecular interaction of lipophilic unsaturated components in the mobile phase

372

Fig. 41.3. Separation of isomeric 1,5,9-cyclododecatrienes by HPLC. Column: 150 mm x 4 mm I.D. Stationary phase: 5 pm Nucleosil 5-C-18. Mobile phase: (A) methanol-water (3:l); (B) A + 0.003 N AgClOt,. Pressure: 80 bar. Flow-rate: 0.7 ml/min. Temperature: 24OC. Detector: RI at x4. Peaks: l=(Z,Z,Z)-; 2=(Z,Z,E)-; 3=(2, E,E)- and 4=(E,E,E)-1,5,9-cyclododecatriene; i=impurity. This chromatogram is a good example of Ag complexation in the mobile phase. In this way (chromatogram B), the four configurational isomers of cyclododecatriene have been separated. could be achieved by which special selectivities for configurational olefin isomers are also obtained (25,26). In 1968 the Chromatographic Group of the main Institute alone had grown up to a size of about 11 coworkers operating more than 20 commercial instruments. Today most of the chromatographic analyses for both institutes are handled in a centralized service organisation which is able to adapt within a short time the above-mentioned methods and techniques to the work of about 100 scientists doing research work in both institutes. More than 7 0 gas and liquid chromatographs are permanently used in the two groups. The necessary data handling is performed on the already mentioned “Mulheim“ computer system (27) which is based on a large version of the PDP 10 time-sharing computer: 50 analogue data lines are available to both chromatography groups ( 2 8 , 2 9 ) . The

373 software for chromatographic data handling has been specially designed for the type of analytical work which is done in a research institute and was developed by ourselves in cooperation with our computer group headed by Dr. E. Ziegler. Today, chromatographic methods are the most important tools for the qualitative and quantitative analysis of complex mixtures arising for example from environmental and biochemical research areas. In my opinion further efforts in gas and liquid chromatography will be directed towards further improvement of methods and techniques with regard to high resolution, reproducibility, reliability and ease of utilisation by chemists and analysts. Naturally automated instrumental set-ups will play an important role in practice. Although tremendous technical, methodological and applicational progress has been achieved in the past 20 years, even more chromatography has to and will be applied in the future to solve even more difficult analytical problems involving complex matrices.

REFERENCES 1 G. Schomburg and H. Husmann, J . Chromatogr. 99 (1974) 63. 2 G. Schomburg and H. Husmann, Chromatographia 9 (1975) 517. 3 G. Schomburg, R. Dielmann, H. Husmann and F. Weeke, J . Chromatogr. 122 (1976) 55. 4 G. Schomburg, H. Husmann and F. Weeke, Chromatographia 10 (1977) 580. 5 G. Schomburg, J . Chromatogr. 14 (1964) 157. 6 G. Schomburg, J . Chromatogr. 23 (1966) 1. 7 G. Schomburg, J . Chromatogr. 23 (1966) 18. 8 G. Schomburg, Anal. Chim. Acta 38 (1967) 45. 9 G. Schomburg, Chromatographia 4 (1971) 286. 10 G. Dielmann, Ph.D. Thesis, University of Bochum, 1977. 11 G. Schomburg and D. Henneberg, Z. AnaZ. Chem. 236 (1968) 279. 12 G. Schomburg and D. Henneberg, Chromatographia 1 (1969) 23. 13 G. Schomburg, D. Henneberg, P. Heimback, E. Janssen, H. Lehmkohl and G. Wilke, Ann. Chem. (1975) 1667. 14 G. Schomburg, H. Behlau, R. Dielmann, H. Husmann and F. Weeke, J . Chromatogr. 142 (1977) 87. 15 G. Schomburg, R. Dielmann, H. Borwitzky and H . Husmann, J . Chromatogr. 167 (1978) 337. 16 H. Borwitzky and G. Schomburg, J . Chromatogr.170 (1979) 99. 17 G. Schomburg and F. Weeke, in Gas Chromatography 1972 (Montreux Symposiwn), S . G . Perry and E.R. Adlard, eds., Applied Science Publishers, London, 1973, pp. 285-294. 18 G. Schomburg, H. Husmann and F. Weeke, Chromatographia 9 (1975) 205. 19 G. Schomburg, H. Kotter and F. Hack, Anal. Chem. 4 5 (1973) 1236. 20 D. Henneberg, Ph.D. Thesis, University of Aachen, 1960. 21 D. Henneberg, in Gas Chromatography 1960 (Edinburgh Symposiwn), R.P.W. Scott, ed., Butterworths, London, 1960, pp. 129-130. 22 D. Henneberg, U. Henrichs and G. Schomburg, Chromatographia 8 (1975) 449.

374 23 D. Henneberg, U. Henrichs and G. Schomburg, J . Chromatogr. 112 (1975) 343. 24 G . Schomburg, D. Henneberg, in Gas Chromatography 1968 (Copenhagen Symposium), C . L . A . Harbourn and R. Stock, eds., Inst. of Petroleum, London, 1969, pp. 4 5 - 5 4 . 25 B. Vonach, Ph. D. Thesis, Technical University Clausthal-Zellerfeld, 1978. 26 B. Vonach and G. Schomburg, J . Chromatogr. 149 (1978) 417. 27 E. Ziegler, D. Henneberg and G. Schomburg, Anal. Chem. 42 (9) (1970) 51A. 28 G. Schomburg, F. Weeke, B. Weimann and E. Ziegler, J . Chromatogr. S c i . 3 (1971) 735. 29 G . Schomburg, F. Weeke, B. Weimann and E. Ziegler, Angew. Chem. T n t . Z'd. E n g l . 11 (1972) 366.

375

G.-M. SCHWAB

GEORG-MARIA SCHWAB w a s born i n 1899, i n B e r l i n , Germany. A f t e r f i n i s h i n g h i g h s c h o o l , he s e r v e d i n t h e army d u r i n g t h e l a s t two y e a r s o f t h e F i r s t World War. Ret u r n i n g home, he s t u d i e d c h e m i s t r y a t t h e U n i v e r s i t y o f B e r l i n r e c e i v i n g h i s Ph.D. i n 1923. For two y e a r s he was t h e a s s i s t a n t o f P r o f e s s o r Bodenstein a t t h e I n s t i t u t e of P h y s i c a l Chemistry i n B e r l i n and t h e n , between 1925 and 1928 of P r o f e s s o r Dimroth a t t h e I n s t i t u t e o f Chemistry i n Wiirzburg. H e o b t a i n e d h i s h a b i l i t a t i o n i n Wiirzburg, i n 1927. I n 1928, he was i n v i t e d by Prof e s s o r Wieland t o t h e U n i v e r s i t y o f Munich where i n 1933 he became e x t r a o r d i n a r y prof e s s o r . E a r l y i n 1939, he l e f t Germany and was a p p o i n t e d t h e head o f t h e Department of I n o r g a n i c , A n a l y t i c a l and C a t a l y t i c Chemistry a t t h e newly o r g a n i z e d Research I n s t i t u t e "Niko'laos Kanellopoulos", i n P i r a e u s , Greece. T h i s was a l a b o r a t o r y o f t h e chemical i n d u s t r y , however, i t gave o p p o r t u n i t y t o f r e e b a s i c r e s e a r c h . I n 1949 D r . Schwab was a p p o i n t e d p r o f e s s o r o f p h y s i c a l c h e m i s t r y a t t h e T e c h n i c a l U n i v e r s i t y of Athens, Greece, and i n 1950, he r e t u r n e d t o Munich a s p r o f e s s o r and head o f t h e I n s t i t u t e o f Physic a l Chemistry a t t h e U n i v e r s i t y . H e s e r v e d i n t h i s p o s i t i o n u n t i l h i s r e t i r e m e n t i n 1967, b u t c o n t i n u e s h i s a c t i v i t i e s i n t h i s I n s t i t u t e a s an e m e r i t u s p r o f e s s o r . D r . Schwab i s t h e a u t h o r o f a number of p u b l i c a t i o n s and books on p h y s i c a l c h e m i s t r y , c a t a l y s i s and technology and t h e e d i t o r o f t h e Handbook o f C a t a l y s i s . He i s a member of t h e Academies o f S c i e n c e o f B a v a r i a , H e i d e l b e r g and Leopoldina and h a s honorary d o c t o r a t e s from t h e u n i v e r s i t i e s of P a r i s , B e r l i n and Lihge. D r . Schwab's main f i e l d s o f r e s e a r c h have been r e a c t i o n k i n e t i c s , h e t e r o g e n e o u s c a t a l y s i s , s o l i d s t a t e r e a c t i v i t y and chromatography. H i s i n v e s t i g a t i o n s i n t o t h e use o f chromatography f o r t h e s e p a r a t i o n o f i n o r g a n i c i o n s s t a r t e d i n Munich and were f i r s t r e p o r t e d a t t h e J u l y 8 , 1937 meeting o f t h e B u n s e n g e s e l l s c h a f t , i n F r a n k f u r t . T h i s work was c o n t i n u e d i n t h e subsequent y e a r s i n Munich and t h e n a t t h e I n s t i t u t e i n P i r a e u s , Greece.

376 The following quotation from the introduction of my first extensive paper of inorganic chromatography, written with my collaborator Kurt Jockers ( I ) who later became a promfnent chemist at BASF, characterized best the situation concerning the evolution of chromatography in general and its utilization in inorganic chemistry in particular up to 1937:

"Chromatography, i.e . the method of separation by adsorption during the passage of s o l u t i o n s through an adsorbent, has had a peculiar h i s t o r i c a l development. A f t e r t h e ingenious Russian b o t a n i s t Tswett had conceived t h e method and had applied it s u c c e s s f u l l y t o plant e x t r a c t s , i t slumbered f o r 25 years i n t h e bed of l i t e r a t u r e . I t was only when biochemistry under t h e pressure of modern p r o b l e m needed methods f o r a r e l i a b l e separation o f small q u a n t i t i e s of similar substances, t h a t chromatography saw a quick and splendid resurrection, f i r s t i n t h e Heidelberg laboratory and soon everywhere. However, again t h e events were peculiar. During t h e f i v e years of i n t e n s i v e chromatography a c t i v i t y t h e method remained e n t i r e l y r e s t r i c t e d t o organic substances, and even t h e t r a n s f e r t o adsorption from aqueous rather than organic s o l u t i o n s was admired as a progress. The question of separation o f the inorganic i o n s , known t o every chemist as t h e o r i g i n a l and educational problem of analysis, remained e n t i r e l y untouched by the development i n t h e neighboring organic domain . Only once has i t been proposed t h e o r e t i c a l l y t o separate by chromatography t h e rare earths. The undertaking of our broader-based i n v e s t i g a t i o n s was, however, uninfluenced by t h i s proposal; we even believe on ground of actual e q e r i e n c e t h a t s p e c i a l l y i n t h e domain of rare earth chromatography may only be u s e f u l as an a u x i l i a r y t o o l . Another description of an inorganic chromatogram, k i n d l y indicated t o us by Mr. Winterstein, can perhaps be seen i n Breddin's finding t h a t metal oxides added t o peat can be e l u t e d by hydrochloric acid i n f r a c t i o n s "comparable t o t h e scale of a spectrum". A c e r t a i n connection t o chromatography i s a l s o shown by the permutites. However, it is not due t o these previous observations but t o the close and productive r e l a t i o n s h i p between t h e d i f f e r e n t sectors of chemistry i n the Munich Laboratory t h a t we undertook t h e task t o examine the a p p l i c a b i l i t y of chromatographic methods t o inorganic anaZysis. " The relationship with other sectors of chemistry in the laboratory of the University of Munich mentioned above referred mainly to my close cooperation with Professor G. Hesse, now professor at the University of Erlangen-Nurnberg who has published many articles and books on organic chromatography and has shown me many stimulating demonstrations. On this occasion I may mention an interesting detail which perhaps offers part of the explanation for the delayed adoption of Tswetts invention: When the first chromatographic successes of Kuhn, Winterstein and Lederer were reported in a Colloquium at the Chemical

377 Institute of the University of Munich, its head, the famous Nobel laureate Heinrich Wieland said jokingly: "Up to now, we have learned with much effort to distill, crystallize and recrystallize, and now they come along and just pour the stuff through a little tube!" Indeed, we ourselves were somewhat afraid that the educational effect of chemical analysis on the material knowledge of the students might get lost by the introduction of such an unspecific physical method. However, during the development of the method, it soon became evident that, in order to overcome and to understand the many complications due to complex formation, solvation, hydrolysis, etc., a similar amount of chemical knowledge and reasoning is necessary in chromatography as in traditional analysis. The inorganic chromatography which we developed in the articles to be described here, was mainly or even almost exclusively performed with alumina as adsorbent. The ion exchangers, by which all the more modern progress in the field became possible, are zeolites on the one side and organic sulphonated or carboxylated resins on the other. They were both unknown at that time. Nevertheless we could conclude from the special experiments performed about the nature of the adsorption process in the case of inorganic cations: that it involves an exchange of sodium ions contained in the adsorbent alumina against the cations. Sodium ions are found in every technical alumina, and those samples prepared in the absence of any sodium compound have no adsorbing and separating capacity. On this point a certain controversy has subsequently arisen, but even today I see no reason to abandon our initial standpoint. Although chromatographic alumina has a pH around 9.4 the adsorption series is different from the series of precipitation pH of the hydroxides, so that the adsorbed species must be considered as superficial aluminates. Besides alumina a large number of other inorganic compounds and minerals were checked and none is comparable to alumina. The technique applied by us was very similar to the method already utilized in organ,ic chromatography at that time: the adsorbent was filled in a vertical tube, mostly as a viscous suspension in the solvent water, a small volume of the sample was added on the top, the column was washed with water until no further movement occurred, and finally, a suitable reagent was introduced to stain the zones, e.g., ammonium sulphide, or potassium cyanoferrate(I1). Since the adsorption is exchange-adsorption, no empty spaces could appear (as in organic chromatography) and the zones of the different cations touch each other in the final state. The chromatographic series achieved by this process and confirmed by all the necessary binary separations is the following:

378 A number of complications may be omitted here. However, it is impor-

tant to remark that the series is different, if instead of aquo-ions the respective ammino-ions are analysed and ammonia is used a s solvent instead of neutral water. The same applies to tartrato-complexes. These facts enable also the separation of ions appearing at the same place in the above series. It must be added that the hydrogen ion

Fig. 42.1. A chromatographic column showing the separation of various cations.

must be placed at the top (the left-hand end) of the above series because from acid solutions a white zone appears above the topmost metal zone, and this zone is acidic against indicators; H+ has here replaced Na+. It is clear that this white zone contains the anion of the applied acid and i t may be possible to replace this anion by others having a higher affinity for aluminium. Indeed, Gretl Dattler (2) was soon able to establish a similar series of anions on an acidified alumina column : 34

-

423- 2--SO4 --Fe(CN) 6 --C1---NO---MnO---C104--S 3 4 6 22CrO Cr207 4

OH---PO --F --Fe (CN)

In this case the staining of the zones (as far as they are not coloured themselves) is possible by Ag+, which migrates on the acidic column faster than any anion. After this passage the column may be illuminated for the detection of C1-. Thus, a chromatographic separation of anions is also possible.

379 In both cases, with anions and with cations, a white zone between coloured ones, natural13 indicates a colourless and not stainable ion like Zn2+, A13+ or F-, SO4-. It must be admitted that a full qualitative analysis of an alloy or a mineral, as often requested in industry or in student courses, is mostly not possible in the presence of more than 5-6 ions. This is due to co-adsorption (see the above series), to secondary adsorption of lower elements in upper bands, to chemical interaction (e.g. Ag+ and Mn2+) and to lack of suitable staining developers. Therefore, we have tried to combine column chromatography with the usual group separations of qualitative analysis (3). Thus, it is possible to separate on one column the elements of the hydrogen chloride group Pb-Ag-T1 with the developer (NH4)zS or K2Cr04. The hydrogen sulphide group Sb-As-Bi-Pb-Cu-Cd can be analysed in tartaric solution with H2S as developer, and the ammonium sulphide group Fe-Cr-U02-Zn-Co-Ni-Mn with the developer ammonia + air. The ammonium carbonate group of alkaline earths and alkali ions is not accessible to this type of chromatography but should be manageable with ion exchangers. The fact that the adsorption of ions on basic or acidified alumina takes place by displacement, leads to the conclusion, that the measurement of the zone lengths should permit a quantitative analysis. This has indeed be shown by Dattler ( 4 ) . A complication, discovered already in our early work ( I ) and extensively studied later ( 4 ) is the co-adsorption of anions in the cation zones on the basic column: nitrates give longer zones than sulphates, and in the presence of both anions cation zones may be divided into a more dilute and a more intense part. This difficulty can be avoided by a previous heating of the specimen with concentrated sulphuric acid until the disappearence of volatile acids. Then indeed equal concentrations of cations of equal valence give equal zone lengths and even equal concentrations of cations with different valence give equal zones, so that not equivalents but gram-atoms are measured, Control analyses of alloys (brass) and coins (Cu-Zn-Al-Ni) under these precautions gave satisfactory results within a few percents of error. Later, I was also be able to analyse the insecticide CuAs204 in this manner. Although the accuracy of quantitative chromatographic analysis is rather low, its specifity is remarkable. Together with Ghosh ( 5 ) I compared the identification limit (the smallest amount of substance still observable) with that of Feigl's spot tests. [The dilution limit (the greatest volume in which this amount is still observable) is, of course, practically unlimited, and therefore even the copper content of tap water can easily be measured). The identification limit exceeds the spot test sensitivity for Fe, Cu, U02, Ag and T1, and is about equal for Co and Ni. Naturally, it depends on the type of developer and the intensity of the respective compound colour. Separations are possible in the microgram-range. According to different observations the nature of the adsorbed state could probably be described as that of a sometimes basic double salt composed of an aluminate and a sulphate, chloride, etc. In order to approach this question I have measured with my collaborator Alice Issidoridis the reflection spectra of the cationic zones (6). Indeed

380 we could confirm that these spectra can be compared with such basic salts rather than with anhydrous aluminates (spinels). On this occasion we discovered an interesting photochemical interface reaction ( 7 ) : the yellow U02-zone turns brown by illumination with white light, and hydroperoxide can be traced. The probable mechanism is 2+ 4 U02 or

+

8 OH- + nhV

2+ 3 U 0 2 + 6 OH- + nhV

-

. 2 H20

U308

.

2 H20 + U04

U308

.

2 H20 + H 0 2 2 ’

i.e. a photochemical disproportionation. It also occurs on zinc oxide as adsorbent. At last we attacked the problem of separation of the platinum metals (although the separation of the lanthanides was considered improbable) (8). We knew already ( I ) that they are placed in the series between bismuth and iron. Mixtures of chlorides, prepared by heating ores or anodic precipitates in chlorine gave, however, chromatograms with ten or more zones. Therefore the single pure chlorides were tested. Here also great complications with a diversity of zones were observed, due to hydrolysis (hydroxo complexes), autocomplexation and change of valence, especially with ruthenium, iridium and rhodium. Ruthenium can be discarded, because it can be removed in practice like osmium as volatile tetroxide. The remaining four platinum metals can, starting from suitable (very fresh or stabilized) solutions, be well separated, according to the series Ir--Pt--Pd--Rh. No suitable staining developer is available here. Therefore, besides of the colour of the zones, the identification was performed by cutting the column, elution with hydrogen chloride solution, thermal decomposition of the chlorides and measurement of the DebyeScherrer interference (lattice constants) of the single metals which have differences in the order of 0.04 8 . Summarizing, we can nowadays say that the method of inorganic analysis by chromatographic adsorption on alumina has been extensively studied by our investigations, and it may be still useful for special problems. The discovery of ion exchangers, zeolites in the inorganic field and anionic and cationic resins in the organic field, have since made the task of inorganic chromatography much easier. The deionization of water for chemical purposes and for steam engines is a testimony to this. However, this does not belong to the early history of inorganic chromatography. REFERENCES 1 2 3 4 5 6 7

G.-M. Schwab G . -Y . Schwab G.-M. Schwab G.-M. Schwab G . - h i . Schwab G.-M. Schwab G.4. Schwab 1048. 8 G.-M. Schwab

and and and and and and and

K . Jockers, Angew. Chem. 5 0 G . Dattler, Angew. Chem. 5 0 A.N. Ghosh, Angew. Chem. 52 G. Dattler, Angew. Chem. 5 1 A.N. Ghosh, Angew. Chem. 5 3

(1937) 546. (1937) 691. (1939) 666. (1938) 709. (1940) 39. A. Issidoridis, Z. Phys. Chem. B53 (1942) 1. A . Issidoridis, Ber. Deut. Chem. Ges. 75 (1942)

and A.N. Ghosh, Z. A n o r g . Chem. 2 5 8 (1949) 323.

381

R.D. SCHWARTZ

ROBERT DONALD SCHWARZ was born i n 1924 i n t h e C i t y o f N e w York. H e r e c e i v e d a B.S. i n 1943 and a Ph.D. i n 1951 a t The S t a t e U n i v e r s i t y of New York a t B u f f a l o . During Worl War 11, he s e r v e d i n t h e U.S. Army A i r Force and worked on t h e Manhattan Proj e c t . H e was employed by S h e l l O i l Co., i n Houston, Texas from 1952 u n t i l 1966. During t h e s e y e a r s , he taught courses i n I n s t r u mental A n a l y s i s and Organic Chemistry a t t h e U n i v e r s i t y of Houston. I n 1966, he moved t o S h r e v e p o r t , L o u i s i a n a t o work a t t h e Research L a b o r a t o r y o f United G a s Corp. (now P e n n z o i l Co.) S i n c e 1968, he h a s been Manager o f t h e A n a l y t i c a l Research and S e r vice Division. D r . Schwarz i s t h e a u t h o r o f a number of p a p e r s on i n o r g a n i c a n a l y s i s , o r g a n i c a n a l y s i s and g a s and l i q u i d chromatography. H e h a s w r i t t e n c h a p t e r s on r e f r a c t o m e t r y and g a s chromatography f o r v a r i o u s books. H e h a s s e r v e d on t h e E d i t o r i a l Advisory Board of t h e Journal of Chromatographic Science ( f o r m e r l y t h e Jo wn a l of Gas Chromatography) since its inception. D r . Schwarz w a s one o f t h e p i o n e e r s on t h e p r a c t i c a l u s e of c a p i l l a r y columns i n petroleum a n a l y s i s . H i s achievements i n c l u d e t h e u s e o f mixed l i q u i d p h a s e s and t h e development of h i g h - t e m p e r a t u r e p o l a r s t a t i o n a r y phases.

In 1952, the year that James and Martin published their classic paper on gas-liquid chromatography, I started work at the Exploration and Production Research Laboratory of Shell Oil Company in Houston, Texas. My initial assignments were to develop new or modified analytical methods for the analysis of sedimentary rocks and crude petroleum. Liquid-solid chromatography was being used by Shell for the analysis of distillate fractions of petroleum and for the separation of the organic matter extracted from sediments. Elution techniques, with adsorbents such as Attapulgus clay, alumina, and silica gel, were employed for these separations. In general the procedures for crude oils, where plenty of samples were available, were satisfactory. However, for sediments, where the contents of organic matter and of hydrocarbons were low, more sensitive methods were required. These analyses were important for a major Shell research program, on the origin and migration of petroleum, directed by G.T. Philippi. In addition to scaling down oil analysis procedures so that they might be applicable to sediment extracts, we were interested in developing, o r modifying, procedures that would provide more detailed information and new parameters for the characterization of the hydrocarbons in the oils and in the extracts. The first assignment I had was to study, and scale down, a urea adduct procedure which had been developed at Shell's Houston Refinery Research Laboratory. This method provided a measure of the normal paraffins plus the lightly branched isoparaffins of the heavy fractions of oils and of sediment extracts. Also, this procedure provided a concentrate which could be analyzed by mass spectrometry, and eventually by gas chromatography, for the content of the individual normal paraffins. Further, the concentrate of isoparaffins and naphthenes, free of normal paraffins, was analyzed by mass spectrometry to provide useful parameters for Philippi's geochemical research. This urea adduct project was my introduction to the field of separation techniques and to the workers at Shell Laboratories in Amsterdam, Emeryville and Houston who were active in these areas. Later, when gas chromatography was applied to my work, these laboratories, and research workers, were still active in applying the latest developments to several problems of interest to Shell. After I completed the urea adduct work, I was asked to develop a modified fractional distillation procedure for crudeoilsamples. This work involved me, more deeply, in the area of separation techniques. The procedure, which was ultimately developed and used at Shell, provided fractions which were analyzed by a variety of liquid chromatographic and gas chromatographic procedures. At the time I started the distillation project, Shell hired a young chemist, D.J. Brasseaux. Don Brasseaux did most of the experimental distillation work and also studied the liquid chromatographic separation of distillate fractions. He continued later to work with me in gas chromatography and is currently District Sales Manager for Varian in Houston, Texas, Don's skill and enthusiasm were such that he made many major contributions to the research program.

383

Liquid-Solid Chromatography The urea adduct procedure, mentioned earlier, was not applicable to the gasoline-range fractions of crude oil produced by the modified fractional distillation procedure. Therefore, we were interested in developing procedures for the determination of normal paraffins in these gasoline-range materials. At about this time, we learned that Linde Air Products Co. (a Division of Union Carbide) had developed a synthetic zeolite which acted as a molecular sieve for hydrocarbon molecules. We requested, and received, samples of the Linde molecular sieve 5A and started experiments with the distillate fractions of petroleum. Our initial results, of experiments performed by Don Brasseaux, indicated that the Linde zeolite 5A quantitatively removed normal paraffins (cg to Clo) from these gasoline-range distillates. We found that a slight adsorption of aromatic hydrocarbons also occurred upon the 5A zeolite. In order to avoid the recovery of the adsorbed normal paraffins fron the sieve or the weighing of the adsorbent with the normal paraffins, and to eliminate the effect of aromatic adsorption, we developed a rapid, convenient method based on the measurement of refractive index. The details of this method were presented at the National American Chemical Society Meeting, Spring 1957 and the paper was published in A n a l y t i c a l Chemistry later that year ( 2 ) . This paper was the first Shell publication on the use of molecular sieves for analytical purposes. It appeared in the same journal issue as the Phillips Petroleum Co. publication on a gravimetric molecular sieve method for the analysis of heavier normal paraffins (2). In rapidly moving fields, such as molecular sieve separations, or gas chromatography, it was difficult for researchers at Shell to compete for early publication with people in academic institutions. First of all, the work had to be written up, approved and/or revised technically and editorially by Management, and then circulated throughout the Shell organization. Then, after a variable time period, depending upon the nature of the work, it had to be cleared by the Patent Department of Shell before it could be submitted to a journal for publication. Despite this handicap, the Shell researchers in chromatography in Houston, Emeryville, Thornton, and Amsterdam were able to publish many timely and important publications. The technical information had to appear first in a Progress Report and then in a Shell Technical Report, before-it could be proposed as an outside report. Our work with molecular sieves indicated that the pore-size distribution of adsorbents was a very important factor in the adsorption separation of hydrocarbon mixtures. Philippi's work with sediment extracts and crude oils required the preparation, separation, and analysis of many heavy mixtures which contained hydrocarbons of all types plus such %on-hydrocarbons" as "resins, "asphaltenes , etc. Brasseaux and I studied the literature on the separation and analysis of such mixtures. We concluded that the detailed analysis of the non-hydrocarbons and of the aromatics would be much more complicated, and difficult than the analysis of the heavy saturated 'I

384 hydrocarbons. Therefore, we worked to develop a simple, rapid, routine procedure for the preparation of a saturated hydrocarbon concentrate that would be applicable both to crude oil residues and to sediment extracts. We found that a combination of a large-pore adsorbent plus a small-pore adsorbent, permitted the removal of non-hydrocarbons and of aromatic hydrocarbons. Elution of the saturated hydrocarbons was achieved with cyclohexane which acts as a weak,eluent but a good solvent. This procedure was published as a Shell report and later appeared in AnalyticaZ Chemistry ( 3 ) . By this time, gas chromatographic work was being persued by Shell at several locations, in Amsterdam (Netherlands), Thornton (United Kingdom), Emeryville (California), and at Houston.

Gas Chrornatogmphy a t She1.7, i n Houston Gas chromatography research at the Exploration and Production (E & P) Research Lab was initiated by Dr. T.C. (Theo) McCoy. The0 constructed a homemade instrument. His work was concerned with injection loops, columns, and detectors for hydrocarbon analyses. This work was concurrent with, and paralleled, the corresponding activities at Emeryville and the Houston Refinery Research Laboratory. After this early work demonstrated the feasibility of hydrocarbon analysis by gas chromatography, other workers, using early commercially-available instruments, entered this area. D r . G.O. Guerrant ordered and evaluated an early Perkin-Elmer Model 154 instrument. Later, a Burrell Kromo-Tog was purchased. The evaluation of this instrument was conducted by A1 Zlatkis, one of the editors of this volume, A1 had worked at Shell's Houston Refinery Research Laboratory. When he left Shell, he started teaching at the University of Houston. The following summer, he did this evaluation at the Shell Exploration and Production Research Laboratory in Houston. In 1956, a major expansion was completed at the Shell E 8z P Laboratory. Don Brasseaux and I were transferred from the Chemistry Department to the Geology Department. There, we were to set up a new laboratory for the routine analysis of oils and sediments and were also to continue work on the development of analytical methods for Philippi's research. By this time aversatilegas chromatograph, developed by the Instrument Department at Emeryville, was available as the ShellHallikainen Gas Chromatograph. We ordered this instrument and Don went to a chromatography school in Chicago conducted by the Podbielniak Co. and Seaton Preston. Don returned from this school full of enthusiasm. Then we started to study methods for the analysis, by gas chromatography, of the gasoline fractions of petroleum. It appeared that one of the major requirements for our work would be the preparation of efficient columns which would separate the complex mixtures of light hydrocarbons in these distillates. As we progressed, we went from heavily-loaded (20-308) columns of large diameter (1/4 inch) to longer, 1/8 inch columns with 5-10s of liquid phase. However, even 48 ft. of 1/8 inch column containing a good hydrocarbon solvent, such as hexadecane, did not provide complete resolution of the c6 and C7 isomers present in

385

Fig. 43.1. Don Brasseux in 1960, with the Barber-Colman Model 20 gas chromatograph. crude oils. Fortunately, at about this time, Golay developed the capillary column. We started to prepare capillaries and to study their performance in the instruments we had available. Golay and other pioneers in this field stressed the need for highly-sensitive, low dead-volume, detectors for capillary column separations. Our first capillary separations were performed with a new instrument, the Fisher-Gulf Partitioner, which had a low dead-volume thermistor detector. The results were, indeed, encouraging. Later, with splendid cooperation from E.C. Reinhardt of Shell's Machine Shop, we modified a standard Gow-Mac T.C. cell so that the capillary effluent could be brought in, through the top of the cell, very close to the detector filaments. This provided very good capillary column peaks. At about this time, we read the report of R.P.W. Scott on the use of long plastic tubing. We studied the separations obtained with nylon and other plastic tubing on conventional chromatographs with the modified Gow-Mac detectors. These results were presented at an Instrument Society of America Meeting in Houston and were scheduled for publication in their Proceedings. However, after some delays, because of changes in the editorial staff of I . S . A . , we submitted the results to Seaton Preston who was planning the new JournaZ of Gus Chromatography. The work finally appeared in the first issue of the new journal ( 4 ) . One of the other items we studied was the preparation and use of capillary adsorption columns. These are columns coated with a solid adsorbent rather than partition liquid. By this time, G.R. (Gerald) Shoemake joined our research project. He and Don worked together on the preparation and evaluation, of various capillary adsorption columns. These columns were tested with the small volume T.C. cells and also with the new ionization detectors (argon and

386

Fig. 43.2. G.R. Shoemake in 1961, with the Barber-Colman Model 20 gas chromatograph. hydrogen flame). This work was presented at the first Houston International Symposium on Advances in Gas Chromatography in January, 1963, and published in Anal. Chsm. in April, 1963 ( 5 ) . Shell was very active in the planning, sponsorship, and support of the first Houston International Meeting. I recall meeting with A1 Zlatkis and I. Dvoretzky of Shell's Houston Refinery Research Lab, in the spring of 1962, to plan a first-class technical meeting and exhibit. We wanted to review the papers prior to the meeting in order to eliminate promotional presentations by instrument companies. Shell was a co-sponsor of the first meeting with the University of Houston and there were four Shell papers on the program (out of 23). Also, Gerry Shoemake, who was by then a graduate student at the University of Houston, was co-author of a paper. After the financial and technical success of the first meeting, A1 Zlatkis and the University of Houston have continued to plan, sponsor, and present these gatherings of the chromatography fraternity. After Shoemake left Shell, R.G. (Rod) Mathews was transferred to our research group. Rod, Don and I studied other capillary adsorption columns. One type that was particularly efficient were the columns coated with a colloidal, hydrophobic silica (6). Years later, when glass capillaries became popular in the drug and medical area, several workers added small amounts of colloidal adsorbents to improve the coating process, for polar liquids that are difficult to coat, and to provide films that were more stable at elevated temperatures. Because many of these workers were not familiar with the hydrocarbon literature, they made no mention of the earlier uses of colloidal silica for these purposes.

387 The successful resolution of the 39 components of crude petroleum, which boil in the 28OC to 114OC range, was achieved by Don Brasseaux in 1962. By coating a special grade of stainless steel, with a mixture of hexadecane and one of several fluorocarbons, we were able to separate all of these compounds on a.200 ft. x 0.010 in. I . D . column (7). This procedure was adopted by Shell for the analysis of a wide variety of crudes and to help compare the light hydrocarbons in sediments with those in the oils. Don, Rod, and I completed two more capillary column projects before we left Shell. We developed a rapid procedure for the separation of all of the aromatic hydrocarbons in the 80 to 18OoC boiling range of petroleum. For this purpose, we constructed a column coated with di-propyl tetrachlorophthalate (a strong pi-complexer) plus squalane (8). Finally, we developed a capillary column for the rapid separation of the C1 to Cs alcohols ( 9 ) . This last project was carried out because so many of our friends who worked with "polar" compounds challenged us to prepare steel columns for "polar" compounds. In December, 1966, I left Shell to join the Research Laboratory of United Gas Corporation (now Pennzoil Co.) in Shreveport, Lousiana. Rod Mathews went to the University of Houston at the same time to attend some classes and to work on a research grant with A1 Zlatkis. Later, in 1968, he joined me in Shreveport where we have collaborated on further research projects in chromatography. I cannot finish the summary of our work at Shell in this period without quoting the poem written by Rod in May 1966. This was finally published in the July 1967 issue of the Journal of the American O i l Chemists' Society with the following remark: "The following verse found its way to AOCS Headquarters recently, and describes so aptly the daily frustrations in the quest for reasonable accuracy - to say nothing of perfection - that we are reprinting it here. We would welcome hearing from the author, in order to give credit where it is due." Now, over 10 years later, I am happy to give again credit to its author, Rod Mathews:

The Chromatographer's Lament (Tune: My Bonnie Lies Over the Ocean) I've heard of chromatograph systems That give separations so fine, But the beautiful peaks in those pictures Bear little resemblance to mine. We bought a new phase for our column With a high-temperature guarantee They said we could cook it forever, But it conked out at 200C.

...

We coated it ever so slowly; Conditioned with greatest of care... But the baseline kept rising and rising And our sample got lost way up there.

388 Now the oleate's in with the stearate, And the peak shapes are slightly askew. . . The flowrate just fell off to zero. So I really don't think it will do. We've avidly read all the journals, Advise we have never refused ... But after you've plugged all that stainless, It's hard to stay really enthused. We've tried this thing over and over And we feel that success must be near... All we need are a few more good substrates And a couple more bottles of beer.

Chorus : To-morrow, to-morrow, We'll coat the new phase that they say is best ... But I'll still bet you It'll bleed even worse than the rest.

Gas Chromatgraphy a t Pennzoi Z My initial work at United Gas (now Pennzoil) involved the purchase, installation, and calibration of gas chromatographs for the analysis of natural gas and of various hydrocarbon samples. The standard instruments, columns, and procedures were adequate for most of this work. However, there were two items where much improvement was possible: the inert supports and liquids for high-temperature work. After Rod Mathews joined Pennzoil, he proposed and initiated the synthesis of polymers specifically designed as liquid phases for high-temperature gas chromatography. He prepared and evaluated a series of polyamides ( l o ) , polyimides ( I ] ) , and polyphenyl ether sulfones (12). These phases were suitable for the separation of heavy hydrocarbons, but their major uses have been for the separation and analysis of heavy "polar" organics. We were assisted in the evaluation of these polymers by a number of workers who specialize in the analysis of drugs, lipids and other biological compounds. It is a pleasure to acknowledge the splendid cooperation of.Drs. J. Stouffer, E.C. Horning, M.G. Horning, C. Pfaffenberger, B.C. Pettitt and S. Lin of Baylor College of Medicine and A1 Zlatkis and Milos Novotny of the University of Houston (Milos is now at Indiana University). Later, arrangements were made with Applied Science Laboratories, State College, Pennsylvania to market these phases as Poly-A, Poly-I, and Poly-S materials. In addition to promoting the use of these phases, R. Henly, Ramachandran, and A.J. Webber have extended the evaluation and thus the applicability of these materials. In the area of solid supports, we studied the properties of a "dendritic" form of sodium chloride ( 1 3 ) aud of a special spherical alumina of low surface area ( 1 4 ) . J.E. Rountree, D.M. Irvine and N . A . Pedro were active enthusiastic collaborators in this work. Also, Dr. D . E . Durbin, formerly of Shell E & P Research, now with Honeywell, and Mrs. Durbin (J. Fruge) assisted in the evaluation of the spherical alumina.

389

Fig. 43.3. R.G. Mathews in 1964, with the BarberColman Model 20 gas chromatograph

.

Our latest research project has involved the drawing, etching, and coating of glass capillary columns for the separation of the 28OC to 114OC hydrocarbons occurring in petroleum. The results have been excellent and will be presented at the next "Zlatkis symposium" (15). Rod Mathews and J. Torres have performed experiments which indicate that the hexadecane-fluorocarbon mixture, coated upon a specially etched glass column can provide much more rapid separation of the 28OC to 114OC hydrocarbons than stainless steel columns.

People, Places and Pleasures As I conclude this historical summary of my involvement with chromatography, I find that there are many people, companies, groups and activities that were quite important to my work although they were not directly connected with my projects at Shell or Pennzoil. Chromatography was a rapidly moving field in the 1950's and early 1960's. New advances in instrumentation appeared very often and the very latest information could be obtained from the ambitious, talented sales-people. I recall the excitement brought to our laboratories by Jim Corbin and Jack Baudean of Perkin-Elmer, Bob Faley of F & M (now Hewlett-Packard) and the late Haskell Lilly of BarberColman. The late Ken Clough of the Curtin Co. in Houston was a very early researcher in gas chromatography and, with the help of his son, provided good quality crushed supports for our early work at Shell. Ken presented some of his data at local meetings of the Gulf Coast Spectroscopic Group (now Gulf Coast Analytical Group) a very informal, but active group, which continues to meet twice a year in the Gulf Coast region. While in Houston, I participated in the organization of an analytical sub-group for the local ACS section. This group had many speakers on chromatography and contributed to the rapid diffusion of new developments. My relationship with the Barber-Colman Co. was particularly close. DonBrasseaux and I participated in the evaluation of the Barber-Colman Model 10 in A1 Zlatkis' laboratory at the University of Houston. On this occasion Haskell was able to arrange to bring

Dr. J.E. Lovelock from England, with an argon detector and a capillary column to help install and operate this large piece of equipment. We were joined by the late Royce Johnson, from Rockford, Illinois who was the Barber-Colman engineer for this project. Although we were impressed with the Model 10, we desired a smaller compact instrument. During the next two weeks, Royce Johnson, the late M.C. (Mac) Simmons of Shell's Houston Refinery Research Laboratory, and I sketched a number of design features that we wanted in a capillary columnionization detector instrument. Eventually, this instrument was built and marketed as the Barber-Colman Model 20. Needless to say, the active, agressive Haskell sold many Model 20's to Shell and to other companies. The Model 20 was a rugged, well built instrument. Its cabinet was made of such heavy metal that Don Brasseaux used to say during the days of the Cuban Crisis, "if we ever have an air-raid, I'm going to climb inside the Model 2 0 . " Finally, my involvement with chromatography has taken me to many of the most interesting places in the world. The Zlatkis symposia have moved from Houston to New York, Las Vegas, Miami, Toronto, Munich and Amsterdam. I have really enjoyed each and every one of them and hope to be able to attend and contribute to those of the future .

Acicnow Zedgements I would like to express my thanks to Mr. W.E. Ellington of Shell Development Co., Houston, Texas, for supplying the photographs used in connection with this contribution. REFERENCES 1 R.D. Schwartz and D.J. Brasseaux, Anal. Chem. 29 (1957) 1022. 2 K.H. Nelson, M.D. Grimes and B.J. Heinrich, AnaZ. Chem. 29 (1957)

1026, 3 R.D. Schwartz and D.J. Brasseaux, Anal. Chem. 30 (1958) 1999. 4 R.D. Schwartz, D.J. Brasseaux and G.R. Shoemake, J . Gas Chromatogr. 2 (1963) 32. 5 R.D. Schwartz, D.J. Brasseaux and G.R. Shoemake, Anal. Chem. 35 (1963) 496. 6 R.D. Schwartz, D.J. Brasseaux and R.G. Mathews, Anal. Chem. 38 (1966) 303. 7 R.D. Schwartz and D.J. Brasseaux, Anal. Chem. 35 (1963) 1374. 8 R.D. Schwartz, R.G. Mathews and D.J. Brasseaux, J . Gas Chromatogr. 5 (1967) 251. 9 R.D. Schwartz and R.G. Mathews, J . Chromatogr. S c i . 7 (1969) 593. 10 R.G. Mathews, R.D. Schwartz, J.E. Stouffer and B.C. Pettitt, J . Chrornatogr. S c i . 8 (1970) 508. 11 R.G. Mathews, R.D. Schwartz, M. Novotny and A. Zlatkis, Anal. Chem. 43 (1971) 1161. 12 R.G. Mathews, R.D. Schwartz, C.D. Pfaffenberger, S.-N. Lin and E.C. Horning, J . Chromatogr. 99 (1974) 51. 13 R.D. Schwartz, R.G. Mathews, J.E. Rountree and D.M. Irvine, Anal. Chem. 4 3 (1971) 2000. 14 R.D. Schwartz, R.G. Mathews, D.M. Irvine, D.E. Durbin and J.M. Fruge, Anal. Chem. 45 (1973) 1280. 15 R.G. Mathews, J. Torres and R.D. Schwartz, to be published.

391

C.D. SCOTT

CHARLES DAVID SCOTT was born i n 1929 i n C h a f f e e , M i s s o u r i , U.S.A. H e r e c e i v e d t h e B.S. d e g r e e i n chemical e n g i n e e r i n g from t h e U n i v e r s i t y of M i s s o u r i i n 1951 and subs e q u e n t l y t h e M.S. and Ph.D. d e g r e e s from t h e U n i v e r s i t y of Tennessee. H e a c c e p t e d employment w i t h t h e Union Carbide Corpor a t i o n i n 1953 a s a development e n g i n e e r working i n a n u c l e a r p r o d u c t i o n f a c i l i t y ; a few y e a r s l a t e r , h e j o i n e d t h e s t a f f of t h e Oak Ridge N a t i o n a l L a b o r a t o r y , which i s a l s o o p e r a t e d by Union Carbide Corpor a t i o n f o r t h e government. A t t h e O a k Ridge N a t i o n a l L a b o r a t o r y , he h e l d s e v e r a l d i f f e r e n t p o s i t i o n s i n r e s e a r c h and development i n t h e Chemical Technology D i v i s i o n and was a p p o i n t e d A s s o c i a t e D i r e c t o r o f t h a t d i v i s i o n i n 1976. D r . S c o t t i s t h e a u t h o r and c o a u t h o r o f o v e r 75 s c i e n t i f i c and t e c h n i c a l p a p e r s and numerous t e c h n i c a l rep o r t s i n t h e g e n e r a l a r e a s o f chemical p r o c e s s development, b i o t e c h n o l o g y , and advanced i n s t r u m e n t a t i o n . H e i s a member o f s e v e r a l s c i e n t i f i c , t e c h n i c a l and honorary s o c i e t i e s and h a s t w i c e been t h e r e c i p i e n t of an IR-100 Award f o r t h e development o f one q f t h e 100 m o s t - s i g n i f i c a n t new t e c h n i c a l p r o d u c t s d u r i n g a g i v e n y e a r . Within h i s g e n e r a l a r e a s of i n t e r e s t , D r . S c o t t h a s s t u d i e d s p e c i f i c r e s e a r c h problems i n h e t e r o g e n e o u s k i n e t i c s , chromatography, o t h e r separ a t i o n s c i e n c e s and b i o e n g i n e e r i n g . As a l e a d e r i n t h e development o f h i g h - p r e s s u r e l i q u i d chromatography i n t h e rnid-l960s, he was one o f t h e f i r s t t o i n c o r p o r a t e t h i s t e c h n i q u e i n t o a n a l y t i c a l systems f o r b i o medical a p p l i c a t i o n s . H i s achievements i n c l u d e d t h e d e s i g n o f v a r i o u s h i g h - r e s o l u t i o n columns a s w e l l a s many of t h e system components t h a t a r e n e c e s s a r y f o r modern h i g h - p r e s s u r e l i q u i d chromatography.

392

I was educated as a chemical engineer. After a two-year stint in the U.S. Army, my entire professional career has been in association with government facilities operated by Union Carbide Corporation. Although I had early experience with gas chromatography in the late 1950's, my involvement in real development work in this field started in the 1960's when I became involved in high-pressure liquid chromatography, with specific emphasis on biomedical applications. The higher operating pressure made in possible to use very small sorption media in the chromatographic column thus resulting in highly resolving separation systems. Pressures up to 5000 psi were tested in a series of experiments designed to establish the feasibility and possible advantages of this approach. This early work was certainly influenced by pFrsona1 interactions with Dr. George E. Boyd and Dr. Waldo E. Cohn, both of Oak Ridge National Laboratory, who had had much experience with low-pressure ion-exchange chromatography * The high-resolution separations of various body fluids for their ultraviolet-absorbing constituents was of particular interest in this early development ( 2 ) . Techniques were developed to sizeseparate ion-exchange resins into an ultrafine fraction, not then available commercially, which would permit high-resolution separations to be obtained (2). Various chromatographic system components also had to be designed since high-pressure systems could not then be purchased from the instrument companies. These included highpressure stainless steel columns, high-pressure sample injection valves, and highly sensitive, double-beam, flow-through photometers ( 3 ) . The development of this necessary hardware made it possible to separate up to 100 ultraviolet-absorbing constituents from body fluids such as urine, and later modifications and improvements resulted in even higher resolution. We received an IR-100 Award for this system in 1971 as one of the most-significant technical products of that year. During the late 1960's and early 1970's, we succeeded in developing a whole family of high-resolution analytical systems for use in the biomedical area. These systems, all of which were based on high-pressure ion-exchange chromatography, provided accurate detection and analysis of carbohydrates, organic acids, biogenic amines, and other constituents important clinically ( 4 ) . In these as well as other separations, it was necessary to develop concepts and equipment for column monitoring in which a reagent stream was continuously mixed with the column effluent to yield reaction products with the separated constituents that could, in turn, be photometrically monitored ( 5 ) . During the time of these early developments, there was almost continuous collaboration with various researchers in the biomedical and separation sciences. Notable among these were Dr. Norman G. Anderson of the Oak Ridge National Laboratory, who was primarily concerned with biochemical separations; Dr. Paul B. Hamilton of the Alfred I. duPont Institute, who was a pioneer in chromatography, particularly with biomedical applications; and Dr. Donald S. Young of the National Institutes of Health, who is an outstanding

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394 clinical laboratory scientist. These interactions were largely responsible for the determination of numerous applications for these new high-resolution chromatography systems. The first-generation high-resolution chromatographic systems required relatively long periods of time to achieve a separation. With a view toward increasing sample throughput, we investigated alternative separation concepts such as the use of multiple separation columns in sequence with column switching and the use of multiple columns in parallel. The latter technique led to the development of a unique differential chromatography concept that allowed the direct comparison of two samples being separated simultaneously on dual columns (6).

Fig. 4 4 . 3 . Separation of a nickel and two cobalt complex ions in a simulated ammonia process liquor by the continuous annular chromatograph(Z0).

395 Several other innovations in analytical liquid chromatography were also introduced by our group. Two potentially promising concepts included the coupling of electrophoresis with elution chromatography for the separation of biological macromolecules ( 7 ) , and the application of a centrifugal field to force eluent through many channels of liquid chromatography simultaneously while utilizing a single, stationary effluent monitor for all channels (8). Our group also contributed to the development of continuous, preparatory liquid chromatography by developing an annular chromatograph based on the original concept of Martin. In this system, a continuous feed stream was separated into a series of component helical bands, each of which could be collected as a product stream ( 9 ) . Both pressurized and gradient types of elution operation were possible with this development. We are continuing to investigate new concepts for liquid chromatography, especially for biochemical separations and for preparative procedures. REFERENCES 1 C.D. Scott, J.E. Attrill and N.G. Anderson, Proc. Soc. E q . BioZ. Med. 1 2 5 (1967) 181. 2 C.D. Scott, AnaZ. Biochem. 24 (1968) 292. 3 C.D. Scott, R.L. Jolley and W.F. Johnson, Amer. J. CZin. PathoZ. 53, No. 5 (11) (1970) 701. 4 C.D. Scott, Advan. C Z i n . Chem. 1 5 (1972) 1. 5 C.D. Scott, in Separation and P u r i f i c a t i o n Methods, Vol. 3 , E.S. Perry, C.J. Van Oss and E. Grushka, eds., M. Dekker, Inc., New York, 1974, pp. 263-297. 6 C.D. Scott and W.W. Pitt, Jr., J . Chromatogr. S c i . 10 (1972) 740. 7 C.D. Scott and N.E. Lee, CZin. Chem. 21 (1975) 1217. 8 C.D. Scott, W.W. Pitt, Jr. and W.F. Johnson, J . Chromatogr. 99 (1974) 35. 9 C.D. Scott, R.D. Spence and W.G. Sisson, J . Chromatogr. 1 2 6 (1976) 381.

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397

R.P.W. SCOTT

RAYMOND PETER WILLIAM SCOTT was born i n 1924, i n E r i t h , Kent, United Kingdom. H e s t u d i e d a t t h e U n i v e r s i t y o f London, obt a i n i n g h i s B.Sc. d e g r e e i n 1946 and h i s Doctor o f S c i e n c e d e g r e e i n 1958. A f t e r s p e n d i n g more t h a n a decade a t Benzole P r o d u c e r s , Ltd. where he became t h e head o f t h e P h y s i c a l Chemistry L a b o r a t o r y , he j o i n e d W.G. Pye i n 1960. I n 1961 he moved t o U n i l e v e r Research L a b o r a t o r i e s a s mana g e r o f t h e P h y s i c a l Chemistry Department. I n 1969 he immigrated t o t h e United S t a t e s and s i n c e t h a t t i m e , he h a s been t h e d i r e c t o r of t h e P h y s i c a l Chemistry Department a t Hoffmann-La Roche I n c . , i n N u t l e y , N . J. D r . S c o t t i s t h e a u t h o r and co-author o f o v e r 100 s c i e n t i f i c p a p e r s l a r g e l y i n v o l v i n g t h e t h e o r y and p r a c t i c e o f b o t h g a s and l i q u i d chromatography. H e e d i t e d t h e p r o c e e d i n g s o f t h e 1960 Edinburgh symposium and i s t h e a u t h o r o f two books, Contemporary Liquid Chromatography and Liquid Chromatography Detectors. D r . S c o t t was a founding member of t h e Gas Chromatography D i s c u s s i o n Group and e x h i b i t e d high-speed columns a t t h e Royal S o c i e t y T e r c e n t a r y E x h i b i t i o n i n 1961. H e r e c e i v e d t h e American Chemical S o c i e t y Award on Chromatography (1977) and t h e M.S. T s w e t t Chromatography Medal (1978). H e i s f e l l o w o f t h e Royal I n s t i t u t e of Chemistry. D r . S c o t t ' s a c t i v i t i e s i n gas chromatography s t a r t e d p r a c t i c a l l y a t t h e i n c e p t i o n o f t h e t e c h n i q u e . H e p i o n e e r e d i n t h e development o f highr e s o l u t i o n columns, h i g h s e n s i t i v i t y d e t e c t o r s and p r e s e n t e d fundament a l d i s c u s s i o n s on t h e r e l a t i o n s h i p between t h e t h e o r y and p r a c t i c e of t h e t e c h n i q u e . H e j o i n e d t h o s e e a r l y chromatographers who became i n volved i n t h e development of modern high-performance l i q u i d chromatog r a p h y ] and p i o n e e r e d i n many o f t h e achievements which a r e t o d a y r o u t i n e l y u t i l i z e d by many a n a l y t i c a l l a b o r a t o r i e s .

398 My first introduction to gas chromatography occurred early in 1954 at a lecture given by Professor A.J.P. Martin, in which he showed a chromatogram of a benzole mixture, the product of the company for which I worked. In that slide, he demonstrated that he knew more about benzole mixture than my own company, who had carried out extensive research on the material for over twenty years. The day after the lecture, I telephoned Archer Martin and asked whether I could visit him to discuss more details of his technique and equipment. That same afternoon, I arrived in his laboratory and there met Dr. A.T. James. Archer Martin and Tony James did more than invent gas chromatography. They also introduced an attitude toward research which was maintained in the field of gas chromatography for nearly a decade. Their attitude was that all research information in methodology should be free for the asking, whether published or not,but its origin should be respected. Further, as they practiced this principle, all the other workers in the field at that time followed their lead. During that afternoon, Archer Martin and Tony James showed me how to make a gas density balance, how to pack a column and how to assemble a chromatograph. I went back to my laboratory musing on the problems involved in having to make my own gas density balance. Although I did not realize it at the time, Dennis Desty was also wrestling with the same problem. The gas density balance consists of a Wheatstone network of capillary tubes drilled out of a solid block of high conductivity copper. The block was 6 in. x 2 in, and anyone familiar with the problem of drilling in high conductivity copper will know that drills snap in it very easily. We filled three copper blocks with snapped drills before we eventually made a working gas density bridge. It worked very well, but it also stimulated me to look for an alternative and simpler method of detection. My colleagues and I did not want to face the difficulties of making another density balance. In 1955, I devised the flame thermocouple detector which, in its original form, consisted of a thermocouple (supported in a cocoa tin) situated over a jet through which the carrier gas from the column was passing. The carrier gas used was hydrogen or a mixture of hydrogen and nitrogen and this was burned at the jet so the thermocouple was heated to a steady temperature. When solutes were eluted the temperature rose, the thermocouple output increased and could be recorded. This detector in fact was the forerunner of the flame ionization detector, but was far less sensitive. However, sensitivities of 106-107 g/ml were readily attainable and this was high sensitivity in those days. During this period Dennis Desty assembled together a group of workers interested in gas chromatography and formed the Gas Chromatography Discussion Group, which had its first informal meeting in Ardeer, Scotland. At this meeting, I introduced the flame thermocouple dtector and met for the first time the other enthusiastic workers in gas chromatography, Dennis Desty, Howard Purnell, Courtenay Phillips, Tony Littlewood, Cliff Scott and many others, and of course, met again the originators of the technique, James and Martin.

399 It is difficult to impart the excitement and enthusiasm that was involved in gas chromatography at that time. There was so much to be learned, so much to be found out and so many exciting things to do. Every sample one placed on the column gave information that nobody was aware of before. Essential oils were found to be far more complex than they had been considered. The so-called pure solvents were found to be very impure indeed and things like chloroform were found to be present in toothpaste. Subsequent to the meeting at Ardeer, a form of chromatographers' club was formed; these workers in the first instance were largely British, but were rapidly followed by workers in Holland, such as Keulemans, Kwantes, Van der Craats, Boer and others. Among this group, it was almost unnecessary to read the literature because one was in contact with almost all of them, either by letter or telephone on a weekly or montQly basis and new developments were heard of prior to being published. I can remember exciting interchanges between Dennis Desty and myself by telephone almost daily, as we sought to improve detector sensitivity, column efficiency, and analysis time, keeping each other completely informed as we went. It was the most exhilarating atmosphere to carry out research that I have ever known. At that time, the team I worked with consisted of Bob Maggs, Ernie Omerod, Graham Hazeldean, Neville Coup and Colin Cummings. In 1956, the international symposium on gas chromatography was held at the Institute of Electrical Engineers in London. The meeting had an audience of about 250 people, only about 50 or 70 of which were probably actively engaged in gas chromatography. The excitement of the meeting was tremendous. On the eve of the first international meeting at which I was to present a paper, I was quite nervous and I can well remember my concern increasing by each step as I ascended to the platform to present my paper on the flame thermocouple detector. After the meeting, I mentioned to Archer Martin my nervousness and I can still remember his answer, "You will always be nervous, however long you live and however many lectures you give. You just learn to conceal it better." At the meeting Professor A.I.M. Keulemans and his colleague, Kwantes, presented the first paper on the HETP equation. Although this had been worked out by others such as Van Deemter Klinkenberg and Zuiderweg, the first presentation to gas chromatographers generally was made by Keulemans at the meeting. In 1957, Dr. J. Lovelock was working on the argon ionization detector. Jim Lovelock worked in a laboratory next to Archer Martin. When I visited him, he, 'like Archer Martin, provided all the information I asked for to make an argon detector. The construction of an argon detector at that time also had interesting ramifications. The argon detector required a 10 mCi strontium 90 source which was indeed a "hot source". It arrived in a silver sheet fairly rigid and contained between lead blocks. It had to be bent into a tube in order to insert it into the cavity of the detector. This was carried out by two of my colleagues, holding (with long crucible tongs) a sheet of glass in front of my face to protect me from beta rays and by wrapping my hands in lead sheet obtained from the wrappings from

400

Fig. 45.1 ( l e f t ) . At the laboratory of Benzole Producers in the 1950's. Left-to right Ernie Omerod, Bob Maggs and R.P.W. Scott. Fig. 45.2 (right). At the entrance of he Institute of Engineering Auditorium during the 1956 Symposium on "Vapour Phase Chromatography". Left-to-right: Dr. Van der Craats and Dr. Kwantes. Far right: R.P.W. Scott.

several two-ounce packets of pipe tobacco. I managed to bent the silver sheet into a cylinder and inserted it into the argon detector. Considering the safety requirements necessary today, our safety inspectors would be horrified by my technique. However, I must say at this stage that I took far less risks than Madame Curie and with any luck I hope I will live as long. Incidentally, the micro-argon detectors that were developed subsequently, by Jim Lovelock, used a 5 0 VCi radium source and these we were quite happy to cut up with pairs of scissors as at that time they only represented the activity in a half a dozen luminous dials from a wristwatch. The argon detector showed extreme sensitivity and, in fact, to this day is still the most sensitive general detector. It has about an order of magnitude greater sensitivity than the flame ionization detector, at g/ml. That detector allowed my colleagues and I to produce high-efficiency columns whichwedescribed at the Amsterdam Symposium in 1958. These columns had efficiencies of 40,000 theoretical plates, were 50 ft. long and operated at 250 psig. They were packed in the form of ten foot loops and at that time produced a separation of the isomeric heptanes and octanes that even to this day have not been surpassed by a packed column. In Amsterdam between the 19th and 23rd of May, 1958 the Gas Chromatography Discussion Group held its second international symposium at the Royal Tropical Institute and this meeting was the most

401

impressive that I have ever attended. Nearly 500 participants from eighteen different countries were present, all enthusiastic workers in the field of chromatography. In that meeting, Golay introduced capillary columns, much work on the theory of chromatography was clarified and McWilliam introduced the flame ionization detector for gas chromatography which has been the bullwark of detectors since that time. The originators of the detector were in fact Harley, Nel and Pretorius, but McWilliam had continued in the development of the detector. Grant described his emissivity detector and the possibilities of process monitoring by gas chromatography were first considered. All the pioneers of gas chromatography were there, Purnell, Littlewood, Golay, Phillips, Primavesi, Martin, C.G. Scott, Adlard, Ray, Keulemans, Van der Craats, Dijkstra, Boer and many others. My most vivid recollection of the meeting in Amsterdam was the reception at the Rijksmuseum where all the delegates of the symposium had the works of Rembrandt, Vermeer, Hals and other famous classical Dutch artists all to themselves for three hours with the accompaniment of unlimited excellent wine. As the evening progressed the pictures became more beautiful and the wine more exotic. After the meeting in Amsterdam, effort tended towards improving column efficiency, detector sensitivity and analysis time. During this period, an amusing incident occurred to me involving the Dutch customs. About that time, brickdust, made from Celite and ground to a suitable size, was found to produce very good efficiency in gas chromatographic columns. It suffered however, from the disadvantage of having fairly high adsorptive activity. One of my co-workers, Ernie Omerod and I set about trying to reduce this activity by plating it with gold. In practice, we soaked it in auric chloride and reduced it in a current of hydrogen at an appropriate temperature. The product did indeed exhibit a very small amount of activity and although not comparable with present-day low activity supports, at that time, it was very effective. I happened to mention this to one of the Dutch workers who requested a sample, and so on my next visit to Amsterdam, I took a small bottle of gold-plated brickdust with me. This was in my briefcase, and was found by the customs. I was asked what it was. I replied quite innocently it was gold-plated brickdust. I was immediately escorted very firmly, but very politely to a little office where ensued the most unbelievable conversation imaginable. I won't go into details, but after approximately two hours, the customs officials concluded they had not apprehended an international gold smuggler, but some scientific nut who had actually gone to the trouble to gold plate brickdust. In 1958, my colleagues and I investigated the use of Nylon capillary tube for chromatographic purposes and were the first to obtain a million theoretical plates from a 1000 ft. of Nylon tubing 0 . 0 2 in. in diameter. By pure chance, we had purchased our Nylon tubing from a small vendor on the south coast of England. As a result of our work many people asked us where we obtained our Nylon tubing and we directed them to the small Sussex Company, which was then inundated with orders for Nylon tubing. This particular tubing was normally used for drainage in abdominal operations and at one time after, I had ordered 1000 ft. of the material I received a phone call from the company agent expressing some concern at the magnitude of the number of operations that appeared being carried out in this area

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In 1960, the Third International Symposium on Gas Chromatography was held in Edinburgh, where analysis time, high efficiencies and detector sensitivities were the subjects most discussed. My colleagues and I demonstrated for the first time separation of complex mixtures in such a short time (seconds) that a cathode ray tube was necessary to display the chromatogram. At the same meeting, Dennis Desty showed rapid separations on fine bore glass capillaries that realized 10,000 effective plates in ten seconds. The meeting at Edinburgh had its spectacular moments, but personally I found it less exciting than Amsterdam, although Iwas involved in its organization. The meeting at Edinburgh to me appeared to be the peak of activity in gas chromatography. Subsequent to 1960, the excitement and development in gas chromatography began to wane. In 1961, I left Benzole Producers and joined W.G. Pye and was made responsible for the research and development in gas chromatography. My co-workers at that time were Bob Maags who had come with me from Benzole Producers, Ian Fowliss and Tom Young. At that time, I was beginning to turn my attention toward liquid chromatography and it was there that we initiated the development of the wire transport detector. After a year at W.G. Pye, I moved on to Unilever Research Laboratories at Colworth House largely as a result of contact with Tony James, who had already moved there from his Mill Hill laboratory. It was a great pleasure to work with Tony James and John Ravenhill to complete the development of the wire transport detector which we described at the international gas chromatography symposium in Brighton in 1964. I was fortunate to again have a small research team working almost exclusively in the development of chromatography which comprised of Trevor Wilkins, Ian Fowliss, who joined me from W.G. Pye, Colin Cummings, who joined me from Benzole Producers and Graham Lawrence. The trend at that time was to associate gas chromatography with spectroscopic techniques. During my period at Unilever, we developed a GC/IR/MS that operated on a step-start procedure. The solute was eluted into a chamber, the carrier gas stopped, the infrared spectra of the solutes obtained simultaneously with mass spectrum and the chamber purged and the next peak examined when it was eluted. At that time, Ted Adlard was also developing GC/MS as were many other workers in the United States. At the Sixth International Symposium in Rome (1966), we carried our equipment over the Alps, down through Italy to Rome and demonstrated the apparatus in operation throughout the whole meeting. As a result of our demonstration in Rome, we were able to show that very little band-spreading occurs in a column when there is no flow of mobile phase providing the temperature is relatively low. In our last demonstration, we turned off the chromatograph half way through the development of the chromatogram of the solute mixture. We took out the column, CaDDed the ends, carried it in a truck over the Alps, and back to Colworth House. There we reassembled the equipment, heated the column to its normal temperature, started the flow of gas and completed the development of the chromatogram that we had started in Rome. We found that the efficiency drop was less than 50% of its original theoretical plates. It became very apparent at that meeting that the excitement in gas chromatography was declining, the research and development was far less and most papers were involved in the application of the technique as opposed to its development. By the

403 end of the sixties, I was almost exclusively working in the field of liquid chromatography. During the 1960 Symposium in Edinburgh, I met Professor S.R. Lipsky, who invited me to visit his laboratories at Yale University. This visit to America was to be first of many that took place over the next 8 years. As a result of my friendship with Sandy Lipsky, I eventually decided to emmigrate to the United States. In 1969, I left the Unilever Research Department and joined the Chemical Research Department of Hoffmann-La Roche, Nutley, N.J. in the U . S . A . as Director of the Physical Chemistry Department. There I joined another of the old guards in gas chromatography who was actively employed in its development in the early fifties, namely Dr. C.G. Scott. At Roche, my research became more of a side interest as my main responsibility was the organization and management of the Physical Chemistry Service Group. And with only one co-worker, Mr. Paul Kucera, my activities in research and development in chromatography are to some extent restricted. However, the development of liquid chromatography since 1969 to the present day has been so exciting and so rapid that the old atmosphere of the early fifties in gas chromatography was almost recaptured. With the work of Lipsky and Horvhth, the modern high-performance liquid chromatography began to evolve and was consumated by the work of Ron Majors and Jack Kirkland in their methods for packing microparticulate columns. From columns of a few hundred plates we were into columns of a few thousand plates, then to ten thousands of plates and now columns of a few hundred thousand plates are readily obtainable. In the early days, Paul Kucera and I worked on solvent systems to improve gradient work and later examined in detail the characteristics of the silica gel proprietary packings and also the nature of solvent/solute interactions. We developed a series of solvents that could be used for gradient elution to develop solutes having a polarity range from that of squalane to that of glucose. By using twelve solvents, the gradient elution could be carried out without any displacement steps obvious in the chromatogram. Unfortunately, the solvents for a large part were opaque to the UV and, therefore, the wire-transport detector had to be employed which had limited sensitivity. The system, however, as a whole was shown to be useful in monitoring cosmetic products and any mixtures having wide polarity ranges. In the early seventies, I had the good fortune of having Professor Grushka of the New York State University at Buffalo joining me for a short stay at Roche. We found we had many ideas in common and since then have maintained constant contact in the field of liquid chromatography development. In fact, as I used to regularly telephone Dennis Desty to discuss developments in gas chromatography, I find myself regularly contacting Eli Grushka, and freely discussing ideas and results that we have obtained in the field of liquid chromatography. As the development of liquid chromatography progressed and columns of high efficiency were produced the need to identify the substances eluted became more and more apparent. And so as the concepts of GC/MS evolved from the development of high-efficiency gas chromatographic columns, so the concept of LC/MS arose from the

404 development of high-efficiency liquid chromatography columns. At the International Meeting on Advances in Chromatography held at Toronto (1973), I suggested to a number of people, the possibility of running a wire-transport system through the source of a quadrupole mass spectrometer to form a suitable interface. Except for two instances the response was one of amused disbelief. However, Professor A.J.P. Martin and Mr. T.Z. Chu of Finnigan Corporation believed my concept possible and Mr. Chu went further, offering to lend me a quadrupole mass spectrometer for a few months to try the idea out. In this venture, I was joined by Cliff Scott and two instrument engineers, Marion Munroe and John Hess. Between us, we developed interfaces that would permit the wire to pass through the ion source and out again while maintaining a pressure inside the mass spectrometer at torr. The venture was highly successful and the device was taken over by Dr. McFadden of Finnigan who developed it into the commercial instrument which is now available. More recently, Paul Kucera and I have turned our attention to microbore columns in an attempt to see just how high were the efficiencies, that could be obtained from a liquid chromatography column. Using microbore columns 1 mm I.D. packed in lengths of 1 meter and joined together, we found that extremely high efficiencies could be obtained. A 10 meter column, packed with 20 pm Partisil produced a quarter of a million theoretical plates and recently using 14 meters of 1 mm bore column packed with 5 pm Spherosorb, an effiency of three quarters of a million plates was obtained. From the results so far, it would appear that the goal one million theoretical plates is certainly obtainable and ten million plates well within the realm of possibility. The feasibility of a hundred million plates remains to be seen. Chromatography has given me the most exciting and rewarding professional life as a scientist, that I could possibly hope for. One of the most interesting aspects of chromatography is its multidisciplinary nature. The design and operation of a chromatograph requires a keen knowledge of electronics, physics, chemistry and engineering. For instance, d.c. amplifiers, analogue-to-digital converters, flow programmers, flow controllers, high-pressure pumps, optical systems, ionization techniques and even the manufacture of bonded phases are just a few of the devices and procedures with which one needs to be familiar. Indeed, the whole gamut of scientific disciplines seems to be involved. I feel very fortunate indeed that my active professional life span has encompassed the invention and development of gas chromatography as well as the development of liquid chromatography. I am very thankful that I went to that lecture by Professor A.J.P. Martin in 1954 and was able to appreciate its significance. I also realize, like many others who are writing in this book, that serendipity has played an important part in our contribution to chromatography. I had the good fortune to be in the right place, at the right time, and with enough knowled.ge to be able to play a part in the development of this facinating technique. I hope the reader also senses from what I have written some of the fun that I have derived from working in the field of chromatography over the last quarter of a century.

405

G.T. SEABORG and G.H. HlGGlNS

GLENN THEODORE SEABORG w a s born i n 1912, i n Ishpeming, Michigan. H e s t u d i e d a t t h e Univ e r s i t y o f C a l i f o r n i a , a t Los Angeles, where h e was named P h i Beta Kappa i n h i s j u n i o r y e a r and r e c e i v e d t h e A.B. d e g r e e i n chemist r y i n 1934. In 1937 he was awarded t h e Ph. D. d e g r e e from t h e U n i v e r s i t y o f C a l i f o r n i a a t Berkeley. Between 1937 and 1939 h e w a s t h e p e r s o n a l r e s e a r c h a s s i s t a n t o f Berkel e y ' s famous p h y s i c a l c h e m i s t , G i l b e r t Newt o n L e w i s . S i n c e 1939, h e h a s been on t h e F a c u l t y of t h e U n i v e r s i t y o f C a l i f o r n i a a t Berkeley. Between 1958 and 1961, he was Chancellor of t h e Un i v e r s i t y of C a l i f o r n i a a t Berkeley and p r e s e n t l y he is U n i v e r s i t y P r o f e s s o r o f Chemistry and A s s o c i a t e Direct o r of t h e Lawrence Berkeley L a b o r a t o r y . During World War 11, D r . Seaborg headed t h e group a t t h e U n i v e r s i t y o f C h i c a g o ' s M e t a l l u r g i c a l L a b o r a t o r y which d e v i s e d t h e chemical e x t r a c t i o n p r o c e s ses used i n t h e p r o d u c t i o n of plutonium f o r t h e Manhattan P r o j e c t . H e s e r v e d between 1946 and 1950 as a member of t h e Atomic Energy Commiss i o n ' s f i r s t General Advisory Committee and from 1959 t o 1961 on t h e P r e s i d e n t ' s S c i e n c e Advisory Committee. Between 1961 and 1971 h e s e r v e d a s t h e Chairman o f t h e United S t a t e s Atomic Energy Commission under t h r e e p r e s i d e n t s . I n 1963 he headed t h e U.S. d e l e g a t i o n t o t h e U.S.S.R. f o r t h e s i g n i n g o f t h e "Memorandum on Cooperation i n t h e F i e l d o f U t i l i z a t i o n of Atomic Energy f o r P e a c e f u l Purposes" and was i n t h e U.S.A. d e l e g a t i o n t o Moscow f o r t h e s i g n i n g o f t h e Limited Nuclear T e s t Ban T r e a t y . I n September 1971, he was p r e s i d e n t o f t h e F o u r t h United N a t i o n s Conference a t Geneva on t h e P e a c e f u l Uses o f Atomic Energy. I n 1973, D r . Seaborg v i s i t e d t h e P e o p l e ' s Republic o f China as a member o f t h e f i r s t s c h o l a r l y d e l e g a t i o n sponsored by t h e U.S. Committee on S c h o l a r l y Communication w i t h t h e P e o p l e ' s Republic o f China w h i l e i n S p r i n g 1978 he s e r v e d a s t h e chairman o f t h e D e l e g a t i o n on Pure and Applied Chemist r y v i s i t i n g t h e same c o u n t r y . D r . Seaborg i s t h e a u t h o r o f o v e r 300 s c i e n t i f i c p a p e r s and a dozen books as w e l l as works on t h e p e a c e f u l u s e s of n u c l e a r e n e r g y . H i s w r i t i n g s have been t r a n s l a t e d i n t o many f o r e i g n l a n g u a g e s . I n 1951, a t t h e age o f 39, D r . Seaborg was awarded ( w i t h E.M. M c M i l l a n ) t h e Nobel P r i z e f o r Chemistry f o r h i s work on t h e c h e m i s t r y o f t r a n s u r a n i u m e l e m e n t s . H i s o t h e r r e c o g n i t i o n s i n c l u d e t h e Atomic Energy Commission's E n r i c o Fermi Award, t h e Arches of S c i e n c e Award of t h e P a c i f i c S c i e n c e C e n t e r , t h e U.S. Department of S t a t e D i s t i n g u i s h e d Honor Award, t h e John E r i c s s o n Gold Medal o f t h e American S o c i e t y of

406 Swedish E n g i n e e r s , t h e F r a n k l i n Medal, t h e L e i f E r i k s o n Award, t h e John S c o t t Award and Medal o f t h e C i t y o f P h i l a d e l p h i a , t h e P e r k i n Medal o f t h e S o c i e t y o f Chemical I n d u s t r y , t h e Chemical P i o n e e r Award and t h e Gold Medal Award o f t h e American I n s t i t u t e o f C h e m i s t s . The American Chemical S o c i e t y h a s honored him w i t h i t s Award i n P u r e C h e m i s t r y , t h e William H , N i c h o l s Medal, t h e C h a r l e s L a t h r o p P a r s o n s Award, t h e W i l l a r d Gibbs Medal, t h e Madison M a r s h a l l Award, and w i t h i t s h i g h e s t h o n o r , t h e P r i e s t l e y Medal. D r . Seaborg s e r v e d a s P r e s i d e n t o f t h e American Chemical S o c i e t y ( 1 9 7 6 ) , t h e American A s s o c i a t i o n f o r t h e Advancement o f S c i e n c e ( 1 9 7 2 ) , and Chairman o f t h e AAAS Board o f D i r e c t o r s ( 1 9 7 3 ) . H e h a s r e c e i v e d o v e r 40 honorary d o c t o r a l d e g r e e s . H e i s a member o f t h e U.S. N a t i o n a l Academy of S c i e n c e s and n i n e f o r e i g n n a t i o n a l academies i n c l u d i n g t h a t of t h e U . S . S . R . I n 1973, h e w a s d e c o r a t e d as an O f f i c e r i n t h e L6gion d'Honneur o f t h e R e p u b l i c o f F r a n c e . In 1940-1941 D r . Seaborg c o - d i s c o v e r e d e l e m e n t 94 ( p l u t o n i u m ) , t h e f i r s t of t h e t r a n s u r a n i u m e l e m e n t s which h e and h i s coworkers d i s c o v e r e d , Am (element 9 5 ) , Cm (96), Bk ( 9 7 ) , Cf ( 9 8 ) , E s ( 9 9 ) , Fm (loo), Md ( 1 0 1 ) , No (102) and e l e m e n t 106. H e and h i s c o l l e a g u e s have d i s c o v e r e d more t h a n 100 i s o t o p e s t h r o u g h o u t t h e p e r i o d i c t a b l e s . A s t h e aut h o r ( i n 1944) o f t h e " a c t i n i d e c o n c e p t " of t h e heavy e l e m e n t e l e c t r o n i c s t r u c t u r e , D r . Seaborg i s t h e o n l y p e r s o n s i n c e Mendeleev t o have made a major change i n t h e p e r i o d t a b l e o f e l e m e n t s . D r . S e a b o r g ' s involvement i n chromatography i s r e l a t e d t o h i s a c t i v i t i e s i n t h e p i o n e e r i n g atomic e n e r g y r e s e a r c h : h e u t i l i z e d i o n - exchange chromatography f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of uranium and p l u t o n i u m from e a c h o t h e r and from f i s s i o n p r o d u c t s , s e p a r a t i o n of t r a n s p l u t o n i u m e l e m e n t s from r a r e e a r t h s and from e a c h o t h e r . -0-o-o-

GARY HOYT HIGGINS w a s born i n 1927, i n S t . James, Minnesota. H e s t u d i e d a t M a c a l e s t e r C o l l e g e , i n S t . P a u l , Minnesota, where he r e c e i v e d h i s A.B. d e g r e e i n 1949. A f t e r b e i n g employed a t Minnesota Mining & Man u f a c t u r i n g Co. and s e r v i n g a s a t e a c h i n g a s s i s t a n t a t M a c a l e s t e r C o l l e g e , he j o i n e d t h e U n i v e r s i t y of C a l i f o r n i a a t Berkeley a s a t e a c h i n g a s s i s t a n t i n 1949. H e r e c e i v e d h i s Ph.D. t h e r e i n 1952 and t h e n j o i n e d t h e Lawrence Livermore L a b o r a t o r y . H e h a s been associated with t h i s laboratory ever since; h i s p r e s e n t p o s i t i o n i s t h a t of a technical. a d v i s o r t o t h e a s s o c i a t e d i r e c t o r f o r Ener-. gy & Resource Program. D r . H i g g i n s i s t h e and c o a u t h o r of o v e r 50 p u b l i c a t i o n s . H e h a s r e c e i v e d t h e Guggenheim F e l l o w s h i p Award and a h o n o r a r y D.Sc. d e g r e e from Macalester C o l l e g e . H i s r e s e a r c h i n t e r e s t h a s been c e n t e r e d on t h e c h e m i c a l and n u c l e a r p r o p e r t i e s o f t h e t r a n s u r a n i u m e l e m e n t s . H e i s t h e c o d i s c o v e r e r o f e l e m e n t s 99 and 1 0 0 , e i n s t e i n i u m and fermium. D r . H i g g i n s h a s been i n v o l v e d i n t h e u s e o f ion-exchange chromatography f o r t h e separation of transuranium elements.

407 In the decade spanning the mid-1940's to the mid-1950's uses of chromatography in our laboratory were divided into three major areas. These were, of course, designed to solve three chemical separation problems, namely, separation and purification of uranium and plutonium from each other and from fission products, separation of transplutonium elements from rare earths and separation of transplutonium elements from each other. There are alternative chemical separation processes to solve each of these problems; however, because we were dealing with isotopes whose half lives were quite short, speed of separation was crucial. For example, we could not have used the fractional crystallization techniques to study isotopes of curium separated from americium. Chromatography, as we used the method, was restricted to use of various ion-exchange resins with complexing agents as eluants. In the uranium/plutonium purification and separation process chloride complexes which are anionic were alternately adsorbed and desorbed from anion-exchange resins of the quaternary amine type. Since plutonium in its higher oxidation state forms these complexes the Pu-U could be isolated as a group. Plutonium in its plus three state forms a very weak chloride complex while uranium always remained in its higher oxidation states in aqueous solution and is strongly complexed to anionic form at high chloride concentration. These facts make for easy separation of uranium and plutonium. Neptunium generally follows uranium but can be separated using ion exchange by strong reduction. During this period, particularly during the forties, the actinide concept was not universally accepted; however, the work in our laboratory by Burris Cunningham on ionic radii in compounds of the heavy elements confirmed the view that the 5f electron shell begins with actinium similar to the 4f shell with lanthanum. His work further convinced us that the stable aqueous ions of the transplutonium actinides would all have plus three oxidation states. Hence we started very early to adopt the ion-exchange techniques, used by F. Spedding for purification of the rare earths, to the new and then unknown actinides with atomic numbers 97 through 103. This technique involved use of sulphonic acid type cation-exchange resins with chelating agents for eluants. Citric acid with carefully adjusted pH was most common but EDTA, lactic acid, sodium thiocyanate and later, a-hydroxyisobutyric acid were also used. The early elution columns required 24 or more hours for separation of elements 95 and 96, americium and curium, with room temperature citric acid. This precluded study of isotopes with half lives shorter than several hours so development of faster separation was important. The problem was solved by two separate developments. The first, made in our laboratory, was achieved by increasing the column and eluant temperature to 87 OC. The temperature was maintained constant by condensing trichloroethylene vapor in a jacket surrounding the ion-exchange column. The second was due, probably, to the commercialization of ion-exchange water softeners. The quality of the resins became both better, in that more regular crosslinkage and

active sulfonic acid sites were produced, and more uniform in that different lots were alike. The result of both developments was separations which were complete in about 2 hours, a factor of ten improvement. By the mid-1950's further improvements due to use of a-hydroxyisobutyric acid reduced separation times of the same elements to about 10 minutes. The third area of application was separation of the plus three valence lanthanides from the plus three actinides. Early in the study of various eluants HC1 was used to separate rare earths from each other although the separations were not very clean. At first it was thought that the elution was due to competition between H30+ and the rare earths for the sorption sites but studies with various acids disclosed a special effect of HC1. When the HC1 concentration was raised to 13 N studies with Am, Cm, and Pm showed that the actinides were apparently complexed by the C1- ion, while the lanthanides either were not complexed or were weakly complexed.

Fig. 46.1. Simple ion-exchange equipment in protection box used for actinide-lanthanide or anioncation separations. The sleeved curved roads used by the manipulators are shown in the foreground. The exchange column shown in this figure just left of center and immediately over the 1-liter polyethylene bottle is of the unjacketed type. The tubes in the circular tables were used to sequentially collect eluate.

409

Thus separation of the two groups could be achieved by eluting the mixture on cation-exchange resin with concentrated HC1. By the decade of the 1950’s separations were improved by using the ethyl alcohol azeotrope saturated with HC1 gas. It was also found that anionic complexes of the actinides were formed in 36 N LiCl and that the actinides could be selectively adsorbed from an actinidelanthanide mixture thus providing an alternative separation procedure useful for handling massive amounts of the actinides. The first gram-quantity purification of curium was accomplished by this method. Construction of ion-exchange columns was, except for the jacketed column, very simple for the handling of the tracer quantities we usually processed. A 1 or 2 mm diameter thick-walled capillary tube 5 or 10 cm in length was drawn to a tip at one end and joined to a centrifuge cone test tube at the other. The tip was broken off and a small plug of glass wool inserted as a stopper for holding the resin. The test tube was closed with a one hole

Fig. 4 6 . 2 . Ion-exchange equipment in protection box used for higher temperature separation of members of the actinide and lanthanide series from each other. Note again the sleeved curved rods used for manipulations. The column seen here on the mount attached to the backwall directly in the center, swung above the right lazy Susan tube holder over tube No. 9 is typical of the jacketed type used for higher temperature separation of this type.

410

Fig. 4 6 . 3 . D r . Seaborg in early 1950's with jacketed ion-exchange column used for the separation of actinides or lanthanides from each other. stopper and a tube leading to a separatory funnel reservoir of eluant was inserted through the hole. These were used for cationic resins mostly. Simple batch separations, for example in cases where cations were separated from anions, were carried out in fritted glass filter funnels with or without provisions for vacuum filtration. Because of concern for cross-contamination, resins were rarely regenerated for repeated uses and column, resin and all were discarded after use. Our ion-exchange equipment in use during the mid-fifties was placed in dry boxes to protect against the alpha particle radioactivity. Such equipment is typical of those used for remotely operated separation of actinide elements from reactor irradiated samples. The assemblieswere inserted in lead shields, for protection against gamma radiation, and the equipment operated with manipulators via sleeved curved rods. The ion exchange columns belonged to two general types. The first type was unjacketed and used for actinide-lanthanide or anion-cation separations. The jacketed columns were used for

411 higher temperature separation o f members o f t h e a c t i n i d e o r lanthan i d e s e r i e s from each o t h e r . This b r i e f report was r e s t r i c t e d t o d i s c u s s i o n only on the major ongoing chromatographic techniques used i n our laboratory, Naturally, there were a l s o numerous experimental procedures which were s h o r t - l i v e d or u n s u c c e s s f u l .

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413

M.S. SHRAIBER

MARIA SEMENOVNA SHRAIBER w a s born i n 1904, i n L i t i n , near Vinnitsa, i n t h e southwestern c o r n e r of t h e Ukraine. A t t h e c o n c l u s i o n of h e r s t u d i e s i n pharmacy, s h e worked a s an a s p i r a n t ( g r a d u a t e s t u d e n t ) a t t h e Ukraini a n I n s t i t u t e f o r Experimental Pharmacy under p r o f e s s o r Izmailov, f i n i s h i n g h e r t h e s i s i n 1939. Five y e a r s l a t e r D r . S h r a i b e r w a s appointed a s e n i o r researcher i n t h e l a b o r a t o r y of a n a l y t i c a l c h e m i s t r y of t h e Khar'kov Chemistry and Pharmacy Research I n s t i t u t e , an i n s t i t u t i o n which succeeded t h e Ukrainian I n s t i t u t e f o r Experimental Pharmacy. D r . S h r a i b e r i s t h e a u t h o r and c o a u t h o r of a number of p a p e r s . H e r main f i e l d i s p h a r m a c e u t i c a l a n a l y s i s such a s complexometry,non-aqueous t i t r a t i o n and p a p e r and t h i n - l a y e r chromatography. On t h e o c c a s i o n of h e r 7 0 t h b i r t h d a y , t h e C o l l e c t i v e of t h e Khar'kov Chemistry and Pharmacy I n s t i t u t e honored D r . S h r a i b e r f o r h e r d i s t i n g u i s h e d career of 40 years i n a n a l y t i c a l research. D r . S h r a i b e r , t o g e t h e r w i t h P r o f e s s o r N . A . Izmailov, developed t h i n l a y e r chromatography. T h e i r f i r s t p u b l i c a t i o n on t h e t e c h n i q u e appeared i n 1938.

414 In 1938, as a post-graduate student at the Khar'kov Chemistry and Pharmacy Institute, I worked in the laboratory of the Institute for Experimental Pharmacy (now the Khar'kov Chemistry and Pharmacy Research Institute). The head of the physical chemistry laboratory of the Institute was N . A . Izmailov, aged 30; already known for his work on adsorption and physical-chemical methods of analysis*. The main task of the Institute at that time was to improve the methods of control of pharmaceuticals. Working in the field of pharmaceutical analysis we encountered difficulties in the control of the so-called galenic preparations (tinctures, extracts, etc.) because of the lack of suitable methods of analysis. This impelled us to search for specific analytical methods that could be used for the detection of physiologically active substances. Chromatography appeared especially promising in this respect. It was in this early period of chromatography development that we became attracted to this refined method. For the separation of complex mixtures that form part of pharmaceuticals, we used column chromatography according to Tswett which made it possible to check the content of substances in vegetable materials and the quality of galenical preparation. The separated substances were further analyzed by fluorimetry. For example, in this way, by applying fluorimetry to the separated fractions, we could determine the harmine content in harmine tincture accurately to within 2% at the 5 ng/liter level. My first two publications with Professor Izmailov originated in this field: they were entitled "Harmine as a fluorescent indicator" ( I ) and "Quantitative determination of some alkaloids by luminiscence" (2). However, in spite of the obvious advantages of the method, the combination of column chromatography with fluorimetry required too much time; thus, it could not be adapted to routine pharmaceutical analysis. Therefore, our efforts were directed toward accelerating the separation of complex mixtures. Our main idea was to take advantage of the high selectivity of the sorbents in combination with the separation of the substances in a flat bed. We selected a flat layer of the adsorbent as the analogy of Tswett's chromatographic column. In this, we were directed by Tswett's ideas -

*NIKOLAI ARKAD'EVICH IZMAILOV was born in 1907, in Suchumi, Russia. He earned his doctor's degree in chemistry and was professor and the head of the Physical Chemistry Department at Khar'kov State University as well as the head of the physical chemistry laboratory at Khar'kov Chemistry and Pharmacy Research Institute. Dr. Izmailov was elected a corresponding member of the Academy of Sciences of the Ukrainian Soviet Socialist Republic and, in recognition of his scientific work, was awarded the Mendeleev Prize of the Academy of Sciences of the U.S.S.R. He died suddenly in 1961, at the height of his professional life. Professor Izmailov's main field was the physical chemistry and electrochemistry of solutions, adsorption and chromatography,

415

Fig. 47.1. N.A. Izmailov (1907-1961).

comparing the properties of a column of sorbent and a strip of paper (3). It occurred to us that a thin layer of the sorbent could be used in lieu of ,a strip of paper; also, we felt that this flat bed could be considered as a cut-out of the adsorbent column. We believed that in carrying out the separation process in such a layer the process would be accelerated significantly. In our work, we deposited a drop of the solution being investigated on the flat adsorbent layer and observed the separation into concentric circular zones which could became visible because of their fluorescence in the light of an UV lamp. In this way, the so-called ultrachromatogram was obtained which was identical to the results obtained in column chromatography. For better separation we washed the frontal chromatograms by spotting a drop of pure solvent after depositing the drop of sample solution. In this way, an even more complete separation could be obtained. This method follows from Tswett's ideas that complete separation of the components of a mixture can only be ensured through the use of a dynamic method i.e., due to uneven migration of adsorption zones under the stream of pure solvent. With the help of our newly developed method we studied the following tinctures; absinthium, belladonna, red pepper (capsicum), cinchona, foxglove, ipecacuanha, mint, rhubarb, strophantus, poison nut, valeriana, hellebore, lily-of-the-valley, Spanish fly, cinnamon, etc. The summary of our investigations was published in 1938 in a paper entitled "A spot chromatographic method of analysis and its application in pharmacy"(4). This paper represents the beginning of thin-layer chromatography. Circular thin-layer chromatograms of different tinctures when viewed in ultraviolet light exhibit such a bright and specific type of fluorescence that we considered this property to be suitable for the rapid analysis of pharmaceuticals. For this purpose, we prepared coloured drawings on paper of the zones obtained. These drawings

416

I

I1

Yellow

W l e blue

Fig. 4 7 . 2 . Chromatograms of extract of belladonna. I , Column chromatograms: (a) before washing, (b) after washing. 11, Ultrachromatograms on thin adsorbent layer. could be used as a reference in the identification of tinctures. The tinctures could be characterized by the number of zones and their positions and colours. The first two characteristics could be adequately described but the indication of the colors was vague. This was especially true for fluorescing colours. It was our intention to compile a manual for the quantitative characterization of galenical preparations by means of colour drawings of the zones, but the war interfered with our plans. Nevertheless our paper did not remain unnoticed. In 1941, Crowe ( 5 ) , making reference to the abstract of our article, reported on the use of TLC in his laboratory for similar separations. Meinhard and Hall (6) also referred to our method calling it "surface chromatography". Stahl - who can be credited with improving TLC and helping in its wide-spread use by standardizing the materials and techniques - in his book (7) stressed the priority of Soviet scientists in the development of TLC, giving quotations and reproducing a number of figures. In a copy of it, he wrote, "I dedicate my book to Prof. N. Izmailov and M. Shraiber, pioneers of thin-layer chromatography. '' In the past 25 years, thin-layer chromatography underwent an exponential development. While by 1950, only about 20 papers existed on this technique and by 1960, their number still did not exceed about 60, today, they number many thousands. The development of TLC in this period had been carried out in many directions: its theory was studied, the application fields expanded and various devices were constructed to facilitate its use. Nowadays, the technique of thin-layer chromatography is utilized in almost every field of chemistry and analysis: in inorganic and organic chemistry, pharmacy, biology and biochemistry, in purity control, diagnostic investigations, as well as in industrial control.

417 REFERENCES 1 N.A. Izmailov and M.S. Shraiber, Farm. Farmakol. 4 (1938) 8. 2 N.A. Izmailov and M.S. Shraiber, Farmatsiya 6 (1939) 1. 3 M.S. Tswett, KhromofilZy v Rastitel'nom i Zhivotnom Mire, Izd. Karbasnikov, Warsaw, 1910. 4 N.A. Izmailov and M.S. Shraiber, Farmatsiya 3 (1938) 1; English translation of this paper has been published as part of the following chapter: N. Pelick, H.R. Bollinger and H.K. Mangold, in Advances in Chromatography Vol. 3, J.C. Giddings and R.A. Keller, ed., M. Dekker, Inc., New York, 1966, pp. 85-118. 5 M.O'L. Crowe, Ind. Eng. Chem. Anal. Ed. 13 (1941) 845. 6 J.E. Meinhard and N.F. Hall, Anal. Chem. 21 (1949) 185. 7 E. Stahl (ed.), i?iiiir,nschichtchromatographie, Springer Verlag, Berlin, 1962.

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419

LLOYD R. SNYDER

LLOYD ROBERT SNYDER w a s born i n 1931, i n Sacramento, C a l i f o r n i a . H e r e c e i v e d h i s Ph.D. d e g r e e s from t h e U n i v e r s i t y of C a l i f o r n i a i n 1952 and 1954. Following two y e a r s i n t h e r e s e a r c h department of S h e l l O i l Company i n Houston, Texas, h e w a s a t t h e Union O i l Company Research C e n t e r i n Brea, C a l i f o r n i a , f o r 14 y e a r s . S i n c e 1971, he i s w i t h Technicon I n s t r u m e n t s Corporat i o n and i s c u r r e n t l y v i c e p r e s i d e n t and d i r e c t o r of t h e C l i n i c a l Chemistry Department. D r . Snyder i s t h e a u t h o r o r c o a u t h o r of o v e r 100 s c i e n t i f i c p a p e r s i n t h e a r e a s of chromatography, t h e a n a l y s i s of petroleum and c l i n i c a l a n a l y s i s . H e h a s a u t h o r e d o r coauthored t h r e e books (on a d s o r p t i o n , l i q u i d chromatography and s e p a r a t i o n s c i e n c e ) each of which h a s become t h e s t a n d a r d t e x t i n t h a t a r e a . H e h a s been on t h e e d i t o r i a l board of f i v e j o u r n a l s o r series d e a l i n g w i t h a n a l y t i c a l c h e m i s t r y and chromatography, and i s c u r r e n t l y an a d j u n c t p r o f e s s o r a t N o r t h e a s t e r n U n i v e r s i t y . H e r e c e i v e d t h e American Chemical S o c i e t y Petroleum Chemistry Award i n 1970 and t h e Stephen D a l Nogare Award i n Chromatography f o r 1976. D r . S n y d e r ' s involvement w i t h gas chromatography d a t e s from 1955 and i n 1957 he began h i s major work i n t h e f i e l d of l i q u i d chromatography. S i n c e t h e n he h a s made c o n t r i b u t i o n s t o t h e compositional a n a l y s i s of petroleum, t h e t h e o r y of r e t e n t i o n i n a d s o r p t i o n chromatography, and t h e development of high-performance l i q u i d chromatography. H i s i n t e r e s t s a r e c u r r e n t l y focused on t h e a r e a s of l i q u i d chromatography and c l i n i c a l chemistry.

My introduction to chromatography in mid-1955 came about as a stroke of luck: being in the right place at the right moment. At that time, Dr. Zlatkis, Max Simmons and I shared a small laboratory in the middle of the Shell Houston refinery, and Dr. Zuiderweg (a coworker of Van Deemter) was on leave from Amsterdam as a visitor to the Shell group in Houston. The Shell Amsterdam group had pioneered the rapid development of GC at that time, and their results were quickly shared with other Shell laboratories in the U.S.A. I remember also the pleasure of reading the galley proofs of Dr. Keulemans' book as part of that contact with our European associates. Shortly after learning about this new analytical technique, we were busily engaged in building our own GC units. Then we turned to solving a number of practical problems, and trying to rationalize the large amount of data we were soon generating. An early problem triggered my life-long interest in the technique later to be called "column switching." This problem was that of analyzing very complex mixtures containing a large number of components; specifically gasoline samples from catalytic reformers. We quickly discovered that no single column would provide good resolution of these samples, due to a problem of "limited peak capacity." However, it was apparent that different stationary phases led to considerable changes in selectivity. In a flash of inspiration, it occurred to me that the problem could be solved by using two columns packed with different stationary phases, and connected together with a switching valve. This allowed fractions from the first column to be diverted to the second column, with complete (or near-complete) resolution occurring in the second column. So far as I am aware, this was the first apDlication ( I ) of what later became a general and powerful technique for both gas and liquid chromatography. At the same time I was intrigued by the possibility of predicting retention data for a large number of compounds from actual measurements on a much smaller number, mainly by use of the Martin relationship. However, others were making similar observations, and my followup of this interest had to wait another two years. In early-1957 I found myself in the Union Oil Research Center, with responsibility for developing compositional-analysis methods for high-boiling petroleum fractions and products. Our main tool was a medium resolution mass spectrometer, and my experience in Houston convinced me that narrow compound-class separations of the sample would be required prior to mass spectral analysis. After a brief attempt with displacement (FIA) separations on silica, I turned to liquid-solid chromatography (LSC) in the elution mode. I t then became clear that useful separations required some understanding of retention as a function of molecular structure and other experimental conditions. The books on chromatography at that time were of little help, and in fact the general belief was that quantitative predictions of retention were near-impossible, due to the isotherm nonlinearity associated with LSC systems. Fortunately, some widely overlooked articles from the 1940's suggested that isotherm linearity was in fact possible, and was facilitated by addition of water to the ad-

sorbent. Subsequent work in my laboratory confirmed this fact and led to two important developments. First, with sample size now removed as a key variable in affecting sample retention, it became possible to collect linear-isotherm retention data, and to begin to develop a general theory of retention in LSC separation. The appearance of the first papers (2) which described how linear-isotherm separations by LSC could be routinely attained, and retention in LSC predicted, aroused immediate and widespread interest. I was encouraged to persevere in this area. The second practical effect of my early work on linear-isotherm LSC was the development of practical assays for several compound types present in petroleum: the so-called linear elution adsorption chromatography or LEAC (3). At that time (and even now) this technique provided a unique answer to the problem of assaying a number of important petroleum constituents, and eventually several such assay procedures were reported. The technique required precise absorbent standardization, and in turn completely predictable separation was achieved. As a result, several samples could be simultaneously separated into fractions containing specific compound types. The resulting fractions were then analyzed by ultraviolet absorption measurements or colorimetry. Typically, up to eight samples could be assayed for as many as five different compound types in a halfday, In the early-1960's my group at Brea turned its attention to one of the last frontiers in petroleum compositional-analysis: the nitrogen and oxygen-containing compounds which occur as traces in various petroleum products. On the one hand, we saw that the availability of high-resolution (one part in 20,000)mass spectrometers would be invaluable in characterizing these enormously complex materials. On the other hand, however, we anticipated that a much greater reliance on prior LSC separation would be required. Not only would it be necessary to devise more sophisticated separation procedures, it would prove necessary to use retention data as an additional qualitative analysis tool. This provided the incentive for a major effort aimed at a complete theory of retention in LSC, at least for sample compounds of the type suspected to be present in petroleum. The culmination of this latter effort was the publication of my first book in 1968 ( 4 ) . By applying this theory to the original problem of petroleum oxygen and nitrogen compounds, the first detailed description of these compounds in a typical crude oil was eventually achieved ( 5 ) . The potential of automated, high-performance liquid chromatography (HPLC) was apparent to a number of people by 1965. In the following year I acquired a refractive index detector and assembled a crude HPLC system from scratch. I was completing my first book at the time, and I wanted to be able to say something about column efficiency and performance' in LSC. So the first application of my crude LC unit was to a systematic study of column plate number and permeability as a function of the usual variables; relatively fast separations with column efficiencies of 2000-4000 plates were readily

422

7 65 3

0.3onhec

003 c m l s a

I

I

Y.

cmlsec

i I V /

LJ-U-2

4 2 0 TIME (MIN.)

Fig. 48.1 ( l e f t ) . Column efficiency data for two adsorbent columns. 1958: 20-cm column of 80-400 mesh alumina (3); 1968: 132-cm column of 20-pm silica (7). Fig. 48.2 ( r i g h t ) . Separation of a mixture of aromatic compounds on silica column (same column as in Fig. 48.1. "1968"). achieved (6). This work may have been the first reported example of HPLC with retentive (i.e. non-exclusion) packings, and it much preceded later applications of high-performance LSC. Equally important, it was possible from this study to extrapolate column performance to pressures higher than the 1500 psi possible with our first unit, and to particles smaller than the 40 pm particles available to us at that time. From this it was apparent that the combination of high pressure plus small particles could provide very exciting performance indeed. I should note in passing that my first study of column performance in liquid-solid chromatography was carried out in 1958, as reported in ref. 3. It is interesting to compare column efficiencies for that original column (packed with 80-400 mesh alumina) with one of the better 1968 columns (20-pm silica), as shown in Fig. 48.1. While the 1958 column is obviously "slower", efficiencies as measured by H were worse by a factor of only three. By early-1968 I was working with smaller-particle columns, and I had discovered with other workers that these could not be packed into efficient columns by the usual dry-fill procedures. Borrowing a page from earlier work in ion exchange and gel permeation, I prepared a narrow-mesh silica fraction of average diameter equal to 20-Um by elutriation. After some initial failures, I was able to

423 prepare an efficient column by the balanced-density slurry-packing procedure (7). Fig. 48.2 shows a typical chromatogram obtained with this column in early-1968. Several years were to pass before slurrypacked small-particle columns would come into widespread use in HPLC. By late-1968 there was a flurry of activity and interest in this new field of HPLC. The next few years were marked by frequent symposia, short courses and international meetings devoted to this technique. It was during this time that I met most of the "names" in chromatography, many of them in transit from the matured field of gas chromatography to frontier areas of liquid chromatography. As a result of these contacts and the work then in progress in my own laboratory, I soon became almost totally immersed in HPLC. We continued to rely on home-made equipment for the most part. A major innovation was the conversion of a spectrophotometer for use as an LC detector. While the sensitivity was only of the order of 0.002 A, it was clear that the versatility of such a detector more than made up for its other shortcomings. We were also using the Valco sample valve at pressures up to 1500 psi, with complete satisfaction. With this liquid chromatograph, we were soon looking at a wide variety of practical applications. Because of my earlier interest in LSC, most of our work at that time was concentrated on this separation mode. We were able to transfer much of the technology developed in the pre-HPLC area in my laboratory for use with HPLC as well. Among the examples of this that come to mind are: addition of water to the mobile phase to control adsorbent activity; use of TLC to pilot HPLC separations; use of column-switching as an alternative for gradient elution. About this time Dennis Saunders joined my group, and we were soon looking at gradient elution from both a practical and theoretical viewpoint. The result was the first practical and complete theory of this technique for use with HPLC, complete with several experimental examples (8). The years since 1970 were marked on my part by an increasing interest in the teaching of HPLC to the next generation of practitioners. With Jack Kirkland, I went on to develop and present the popular American Chemical Society short course on "Modern LC." This was followed in a few years, first by an audio,course under the auspices of the ACS, and a year later by our book with the same title. I would venture to say that about 10,000 LC students were eventually to be influenced by one or more of these three educational vehicles. I also moved in 1971 from petroleum to clinical analysis, as it seemed to me that the latter field offered greater scope for the further application of HPLC. This opinion has since been confirmed, and clinical LC is expected to become a major future growth area for HPLC. My own involvement in this area, however, i s too recent to comment on at this time. REFERENCES 1 M.C. Simmons and L.R. Snyder, Anal. Chem. 30 (1958) 32. J. Chromatogr. 5 (1961) 430; 6 (1961) 22.

2 L.R. Snyder,

424 3 L.R. Snyder, Anal. Chem. 33 (1961) 1527, 1535, 1538. 4 L.R. Snyder,

Principles o f Adsorption Chromatography, Marcel Dekker,

New York, 1968. 5 L.R. Snyder, Ace. Chem. Res. 3 (1970) 290. 6 L.R. Snyder, Anal. Chem. 39 (1967) 698, 705. 7 L.R. Snyder, J . Chromatogr. S c i . 7 (1969) 352. 8 L.R. Snyder and D.L. Saunders, J . Chromatogr. Sci. 7 (1969) 195.

425

EGON STAHL

EGON STAHL w a s born i n 1924, i n Eberach on t h e Neckar ( n e a r H e i d e l b e r g ) , Germany. H e s t u d i e d pharmacy a t t h e T e c h n i c a l Univers i t y of K a r l s r u h e , o b t a i n i n g h i s Ph.D. i n 1952. I n 1954, he went t o Z u r i c h , S w i t z e r l a n d , f o r h i s p o s t d o c t o r a l y e a r a t t h e ETH, t h e F e d e r a l T e c h n i c a l U n i v e r s i t y . H e rec e i v e d h i s h a b i l i t a t i o n i n 1957 a t t h e Univ e r s i t y of Mainz, w i t h a p u b l i c l e c t u r e on t h i n - l a y e r chromatography and i t s a p p l i c a t i o n s . H e was t h e n a p p o i n t e d as a P r i v a t dozent a t t h e I n s t i t u t e f o r P h a r m a c e u t i c a l Chemistry i n Mainz. In 1958 h e moved t o t h e University of t h e Saarland, i n Saarbrucken, a s an a s s o c i a t e p r o f e s s o r and s e n i o r lect u r e r . S i n c e 1965 he h a s been F u l l P r o f e s s o r and D i r e c t o r o f t h e I n s t i t u t e o f Pharmacognosy and A n a l y t i c a l Phytochemistry a t t h i s University. D r . S t a h l is t h e a u t h o r and c o a u t h o r of more t h a n 150 s c i e n t i f i c p a p e r s a p a r t o f which d e a l s w i t h t h e components o f m e d i c i n a l p l a n t s and t h e rest w i t h chromatography and r e l a t e d methods. He i s a l s o t h e a u t h o r of t h r e e b o o k s : t h e Textbook of Pharmacognosy, t h e well-known l a b o r a t o r y handbook on Thin-layer Chromatography (1st e d i t i o n i n 1962 and 2nd e d i t i o n i n 1967, w i t h more t h a n 1000 pages) and a t h i r d book, on Drug AnaZysis by Chromatography and Microscopy. P r e s e n t l y , h e i s working on a book on t h e a n a l y s i s of m e d i c i n a l p l a n t s . D r . S t a h l is a member and h o n o r a r y member o f numerous s c i e n t i f i c s o c i e t i e s . For h i s p i o n e e r work i n chromatography h e r e c e i v e d a number of awards, p r i z e s and honours, such a s a honorary d o c t o r a t e o f medicine from t h e U n i v e r s i t y o f Louvain, Belgium ( 1 9 7 3 ) ) t h e F r e s e n i u s P r i z e and Gold Medal of t h e German Chemical S o c i e t y ( 1 9 6 6 ) ) t h e Ludwig Schunk P r i z e o f t h e Medical F a c u l t y o f t h e U n i v e r s i t y o f Giessen (1967), t h e T a l a n t a Gold Medal (1967), t h e F l u c k i n g e r Gold Medal ( 1 9 7 1 ) , t h e Kolth o f f Gold Medal (1973), t h e American Chemical S o c i e t y Award i n Chromatography (1975) and t h e C a r l Mannich Medal o f t h e German P h a r m a c e u t i c a l S o c i e t y (1977). D r . S t a h l i s t h e German Expert f o r N a t u r a l Drugs i n t h e European Pharmacopoeia Commission, S t r a s s b o u r g . H e i s a l s o chairman of t h e P h a r m a c e u t i c a l - B i o l o g i c a l Committee of t h e German Pharmacopoeia Comm i s s i o n and member of v a r i o u s groups and committees: t h e Pharmacopoeia

426 Commission, t h e S c i e n t i f i c Advisory Board of t h e I n s t i t u t e f o r Drugs of t h e F e d e r a l German O f f i c e of H e a l t h , and t h e Committee of t h e German Chromatography Group a f f i l i a t e d w i t h t h e German Chemical S o c i e t y . Between 1965 and 1972 he h a s s e r v e d a s t h e p r e s i d e n t of t h e Academic O f f i c e f o r Foreign S t u d e n t s a t t h e U n i v e r s i t y of t h e S a a r l a n d , i n Saarbrucken. D r . S t a h l ' s involvement i n t h i n - l a y e r chromatography s t a r t e d i n t h e f i r s t p a r t of t h e 1 9 5 0 ' s b u t i t was n o t u n t i l t h e end of t h a t decade t h a t t h e t e c h n i q u e won wider i n t e r e s t . D r . S t a h l p i o n e e r e d i n t h e development of s i m p l e and e f f i c i e n t systems and equipments and i n t h e s t a n d a r d i z a t i o n of a d s o r b e n t s ; t h e r e d u c t i o n of p a r t i c l e s i z e s by a f a c t o r of t e n was a l s o i n i t i a t e d by him. H i s a c t i v i t i e s i n t h e disseminat i o n of i n f o r m a t i o n on t h e new t e c h n i q u e a r e a l s o very i m p o r t a n t : t h e two e d i t i o n s of h i s l a b o r a t o r y handbook s e r v e d a s t h e most i m p o r t a n t r e f e r e n c e material t o thousands of n o v i c e s and e x p e r t s a l i k e . D r . S t a h l i s c o n t i n u i n g t o develop new and i m p o r t a n t a n a l y t i c a l t e c h n i q u e s based on t h i n - l a y e r chromatography.

427 To earn money for studying after World War I1 I did testing work in the night hours for a pharmaceutical wholesale company in Karlsruhe. Daily routine comprised of analyses of drugs, tinctures and extracts of medical plants. One day in the homoeopathic pharmacopoeia I came upon a method that was to make life easier for me: capillary analysis, I also happened to get the fundamental book by Goppelsroeder, CapiZZary anahjsis, written in 1906 which proved to be very helpful, leading me directly to paper chromatography in the modern way. A few years later, during the experimental work of my thesis (1950-1952), the problem arose of separating the contents of single glandular hairs of plants. These glands have a content of only about 5 0 ng and are smaller than a grain of potato starch. Such a grain disappears in the large interspaces of the cellular fibres under the microscope on the usual chromatographic paper of that time. I realised quickly that layers with very small pores and fine grains are needed here. It was only after some wrong moves, e.g., chromatography on cigarette paper, on magnesium grooves and -rods and on anodic aluminum oxides, that I succeeded with the selfprepared layers of very fine-grained (1 to 5 pm) aluminum oxides and silica gel which I spread on glass plates. The reproducibility of the separating power and of the results caused difficulties. It took years of repeated tests to find out the secrets of chromatography on thin layers. It was already clear at that time that the method

Fig. 49.1. Stahl, as a student, in his homemade laboratory, in 1948. In the background at right, on the wall: box for capillary analysis.

428

Fig. 4 9 . 2 . Progress in chromatography becomes obvious in comparing the particle sizes of the various procedures. Left top: A1203 for column chromatography; right top: cellulose fibers for paper chromatography; left bottom: silica gel for TLC; right bottom: cellulose powder for TLC. involved essentially the old Tswett's column chromatography, though not with a "closed" but with an "open" column of the thin layer. The foundation of thin-layer chromatography is the silica gel of narrow grain-size range which was then self-prepared with the help of wet sedimentation and which resulted in layers of high separating power. Slowly, I learned of further influences, such as thickness of the layer, length of run chamber saturation, etc., and in 1956, I dared to publish a first work with the title Thin-Layer Chrornatography in a professional magazine, called Die Pharmazie ( I ) . However, neither this pub1 cation nor those of other authors (e.g. Izmailov and Shraiber, Meinhard and Hall, Kirchner and Miller, Reitsema etc.) found resonance: the "experts" were not interested. During my years at the Pharmaceutical-Chemical Institute of the University of Mainz I had the opportunity to test numerous substances with my "hobby", thin-layer chromatography. Separation of ergot alkaloids was one of the problems, and it became obvious that thin-layer chromatography offered itself as the most versatile, universal and least demanding chromatographic fast method of the future. I asked myself why it did not find acceptance and thought the following reasons responsible: - absence of commercially available standard adsorbents of narrow range of grain size for thin-layer chromatography;

429

-

absence of suitable equipment for preparing thin layers; and absence of suitable examples stimulating the use of the method.

I kept these points in my mind and continued my work, publishing the second informative article in 1958, in Chemiker-Zeitung (2). At the same time, the basic kit for thin-layer chromatography (DESAGA, Heidelberg) and "silica gel according to Stahl €or TLC" (E. Merck, Darmstadt) were shown at the ACHEMA exhibition of chemical equipments, in Frankfurt. Now, the situation became different: the method quickly won a wide interest and the number of publications per year on thin-layer chromatography increased exponentially. As a result of the assumption that thin-layer chromatography is only adsorption chromatography on "open" columns, the range of application was limited to the separation of lipophilic compounds; the domain of hydrophilic separations was reserved for paper chromatography.

Fig. 49.3. The first steroid separations on thin silica gel layers (1959). 1 = Lactone of dihydroxycholic acid; 2 = lactone of acetoxy-20-oxycholic acid; 3 = pregnene-3B-ol-20-on; 4 = cholesterol; 5 = cholesterol acetate; 6 = mixture of 1-5; 7 = serum lipids. T = color test mixture,

430

D i g i t oxose

Rho m nose

Ribose Xylose

Fructose Glucose Sacchar ose Lactose

Fig. 4 9 . 4 . Sugar separation, an example for the separation power of TLC for strongly polar substances (1961). We kept experimenting and in 1961, we succeeded with four publications (3-6) in entering the hydrophilic domain, 1t.could be shown that mixtures of sugars, heart glycosides and further hydrophilic plant components were better and more rapidly separable by TLC than by PC. These publications recommended the use of TLC in trace analysis and demonstrated that certain indole derivatives, such as heteroauxin, could be detected in amounts down to 5 ng. By 1962, so many publications had appeared and so much experience had been won that a team of experienced specialists under my editorship was able to bring out the first laboratory handbook on TLC (7). In this, Professor Brenner, Basel, discussed theory and the analysis of amino acids; Dr. Bolliger, Basel, vitamins; Dr. Ganshirt, quantitative evaluation and synthetic drugs; Professor Mangold (then U . S . A . ) , the technique of isotopes, aliphatic Lipids, nucleic acids and nucleotides; Dr. Seiler, Basel, inorganic ions; and Dr. Waldi, Darmstadt, steroids, clinical diagnostic and spray reagents. The book became a bestseller and was translated in a short timetomanylanguages, including Russian. It helped spread the method all over the western and eastern world. Although originally printed in a numerically large edition, a second edition had to follow in 1967; this time, the team counted 25 coauthors. In 1969, the English translation of Professor Ashworth, Saarbrucken, followed with more than 1000 pages (8).

Tntroduction of S p e c i a l Working Techniques In the first decade of TLC, we extended the application to practically all types of mixture and also devoted special attention to various working techniques. Thus, the circular and wedged tip

431

Fig. 49.5. Gradient layers yield three different possibilities for chromatography. techniques were described in 1958, and stepwise development and chamber saturation followed in 1959. About this time, I also introduced the sandwich chamber (S-chamber) as a new chamber system. To study the inactivation of pyrethrins through light, the two-dimensional SRS (Separating-Reaction-Separation)-technique was developed in 1960. Later on, it was called reaction chromatography by different authors. The so-called gradient-TLC ( 9 ) brought a climax and genuine step forward in 1964. Gradient layers can be easily and rapidly prepared using a special gradient-spreader, which I developed. Three different separating surfaces are available on a gradient layer: development can be carried out at right angles to the gradient (T-gradient-technique) or in two different directions along the line of the gradient. This cleared the way for new possibilities. A few years later, we succeeded in preparing defined pH-gradient layers. On such layers, chromatography of, e.g. basic or acidic substances, perpendicular to the gradient, furnishes typical curves which resemble titration curves. This is a new possibility for substance identification. For the application of substance mixtures in the form of a band we developed a special appliance, the Autoliner which is also suitable for the preparative application of dissolved substances. Special attention was given to the techniques of transfer since from the very beginning I had considered the R -values as f guide values only and never as physico-chemical figures ( 1 0 ) . Transfer methods are techniques that allow microgram amounts of substance in a spot to be transferred to another identification procedure, e.g. IR-spectroscopy, gas chromatography etc. In collaboration with the firm of Zeiss, Oberkochen, we developed a chromatogram-spectrophotometer which allows scanning of a thin-layer chromatogram in remission as well as in transmission in the UV and visible range and recording of the extinction values. My former student, Professor H. Jork, has continued the detailed study of quantitative evaluation.

Standardisation and TerminoZogy I recognised early the necessity for standardisation of the TLC method. It began with the plate size ( 2 0 x 20 cm), the length of run (10 cm), the position of the start points, chamber saturation

432

3 F i g . 49.6. P o s s i b i l i t i e s f o r t h e i d e n t i f i c a t i o n of chromatographically s e p a r a t e d subs t a n c e s by d i r e c t coupling with v a r i o u s ident i f i c a t i o n procedures. GC = gas chromatography; PC = paper chromatography.

e t c . This enabled work t o be performed everywhere under t h e same c o n d i t i o n s , i n c o n t r a s t t o paper chromatography. This y i e l d e d g r e a t advantages f o r t h e manufacturer of TLC equipment a s w e l l a s f o r t h e u s e r , considering t h e i n t e r c h a n g e a b i l i t y of t h e a p p l i a n c e s . I n t e r n a t i o n a l understanding demanded a l s o t h a t we concerned o u r s e l v e s with terminology and d e f i n i t i o n s . With t h e acceptance of TLC a s an o f f i c i a l method i n t h e pharmacopoeias i t became necessary t o f i n d a s p e c i a l s t y l e and wording. Proposals f o r t h i s were made. Suggestions were worked o u t f o r a series of t e s t procedures f o r t h e TLC of drug components f o r t h e European Pharmacopoeia. These and f u r t h e r procedures f o r drug c h a r a c t e r i s a t i o n and a n a l y s i s w e r e summarised i n a s p e c i a l book (23). The emphasis of my xork i n t h e second decade of t h i n - l a y e r chromatography, t h a t i s , a f t e r 1967, was on t h e micro-extraction of substances from complex samples, e . g . from medicinal p l a n t s , and a l s o t h e d i r e c t t r a n s f e r onto t h e t h i n l a y e r . Furthermore, I was i n t e r e s t e d i n t h e problem of c h a r a c t e r i s a t i o n , i . e . a n a l y s i s of n a t u r a l and s y n t h e t i c polymeric m a t e r i a l with t h e h e l p of TLC. So f a r , t h i s had not been p o s s i b l e .

YfiS procedure I n t h e process of t e s t i n g p l a n t m a t e r i a l with t h e h e l p of TLC, t h e p r e p a r a t i o n of t h e sample, i . e . e x t r a c t i o n , shaking procedure, and evaporation down, is o f t e n t h e most time-consuming p a r t . Cons e q u e n t l y , w e made many attempts t o develop a d i r e c t e x t r a c t i o n and t r a n s f e r method. Stimulated by t h e work on microsublimation done by L . Kofler and R . F i s c h e r i n t h e t h i r t i e s , we f i r s t t e s t e d o u t thermal e x t r a c t i o n procedures. Step by s t e p , t h i s l e d t o t h e TAS procedure* ( 1 4 - 1 5 ) patented i n 1967. In 1968 and t h e following y e a r s , t h e

* TAS

r e p r e s e n t s an acronym of a number of words: T = thermomicro, t r a n s f e r ; A = a p p l i c a t i o n ; S = substance and/or S t a h l .

433 possibilities of application in various fields were demonstrated in twelve publications. Special study was devoted to the procedures taking place during extraction, to the optimisation and to the quantitative side. The TAS method was quickly adopted in the European laboratories and is the preferred procedure both in industry and in pharmacognosy teaching for fast extraction coupled with TLC.

Thermofractography A logical further development of the TAS procedure is thermofractography ( 1 6 ) leading to an apparatus, called the TASOMAT. In the TASOMAT, the temperature of a few mg of sample is raised linearly from room temperature to 45Q°C. The volatile products are collected as a starting band on a thin layer, and are then chromatographed as usual. The chromatogram, the so-called thermofractogram, then yields the substances separated according to their volatility (boiling or sublimation temperatures) along the abscissa, and according to their chromatographic behaviour along the ordinate. This fractional thermal extraction is in principle a distillation or sublimation in a carrier gas in the microgram range. This is the first time that microgram amounts of many substances of high boiling or sublimation points were transferred directly onto a thin layer. The separation effect corresponds roughly to that of a distillation under 0.1 torr. This is shown in particular by our work on rapid analysis of lipid mixtures, e.g. of ointments, suppositories and cosmetic preparations, such as creams, lipsticks, etc. Basically, all substances which can be subjected to gas chromatography are amenable to thermofractography. Naturally, the procedure does not apply to a number of polar and involatile substances. However, during thermofractography (TFG) these undergo thermal decomposition within a particular temperature range; in other words, pyrolysis or, better, thermolysis, occurs. Definite fragmentation products are thus yielded which can serve for identification on the thermofractogram. For example, we have had good success in distinguishing different lignins (27). The characterisation of tannins by means of TFG was also very valuable, and we also succeeded in establishing how the tanning of leather samples was carried out. After these encouraging results we turned to the investigation of plastics and were able to carry out the rapid identification of condensation polymers (nylon and perlon types), phenol resins, vinyl polymers and also of plasticisers and other additives ( 1 8 ) . In further studies, we then performed classical thermal reactions in the temperature gradient of the TFG. This had the advantage that such reactions can be conducted with microgram amounts as found, for example, in chromatographic zones. All the substances formed are found on the corresponding thermofractograms and this enables the course of the reaction to be recognised. Work in the ultramicro region was done on dehydrogenation with sulphur and with selenium, catalytic dehydrogenation and zinc dust distillation. These techniques can be regarded also as a carbon-skeleton TLC, analogous to that in gas

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F i g . 4 9 . 7 . Techniques f o r t h e t r a n s f e r o f v o l a t i l e s u b s t a n c e s from t h e sample d i r e c t l y t o TLC. GC = g a s chromatography; EC = e l e c t r o chromatography ( e l e c t r o p h o r e s i s ) . chromatography. A review o f t h e r m a l work c o u p l e d w i t h TLC was p r e p a r e d on t h e o c c a s i o n o f r e c e i v i n g t h e ACS Award i n Chromatography ( 1 9 ) . L a b i l e n a t u r a l p r o d u c t s a r e , however, n o t e x a c t l y i d e a l s u b j e c t s f o r s e p a r a t i o n u s i n g t h e r m a l methods. W e t h u s l o o k e d f o r less d r a s t i c e x t r a c t i o n methods which p e r m i t t e d d i r e c t c o u p l i n g w i t h TLC; t h i s l e d us t o t h e s u p e r c r i t i c a l g a s e s .

Fluid Extraction coupled with TLC The s o l u b i l i t i e s o f many s u b s t a n c e s i n s u p e r c r i t i c a l g a s e s i n c r e a s e a s t h e p r e s s u r e i s r a i s e d . I t was hence o f i n t e r e s t t o employ such g a s e s under p r e s s u r e i n t h e compressed s t a t e , i . e . , i n t h e s u p e r c r i t i c a l r e g i o n , f o r e x t r a c t i o n . The problem o f d i r e c t c o u p l i n g w i t h TLC c o u l d be circumvented by p r e s s u r e r e l e a s e t h r o u g h very f i n e c a p i l l a r i e s ( i n n e r d i a m e t e r o f 50 pm). We f i r s t developed an a p p a r a t u s f o r t h i s new t y p e of c o u p l i n g p r o c e d u r e (20) and t h e n c a r r i e d o u t many e x p e r i m e n t s on model m i x t u r e s t o test t h e i r ext r a c t i b i l i t y . We were a b l e t o e s t a b l i s h rules-of-thumb which p e r mitted the estimation of the e x t r a c t a b i l i t y (21). A t f i r s t , we worked i n t h e p r e s s u r e r e g i o n up t o 500 b a r and s u b s e q u e n t l y ext e n d e d t h i s t o 2500 b a r . S u p e r c r i t i c a l c a r b o n d i o x i d e was used and also, especially for alkaloid extraction, supercritical nitrous o x i d e . T h i s work on f l u i d e x t r a c t i o n concluded f o r t h e t i m e b e i n g o u r e f f o r t s t o f i n d s u i t a b l e c o u p l i n g p r o c e d u r e s w i t h TLC. The scheme shown i n F i g . 4 9 . 7 summarises o u r endeavours i n t h i s domain, t o e x t r a c t sample components and t r a n s f e r them d i r e c t l y t o TLC. I n c o n c l u s i o n I s h o u l d l i k e t o s a y t h a t t h i n - l a y e r chromatography, i n t h e two decades s i n c e i t s development, h a s become an i n d i s p e n s a b l e t o o l i n l a b o r a t o r i e s a l l o v e r t h e w o r l d . Of a l l c h r o m a t o g r a p h i c proc e d u r e s , i t is t h e most o f t e n u s e d , b e i n g i n e x p e n s i v e , s i m p l e t o h a n d l e and g i v i n g f a s t r e s u l t s . By u s i n g TLC, many new s u b s t a n c e s have been d i s c o v e r e d and t h e p u r i t i e s o f commercially a v a i l a b l e c h e m i c a l s and p h a r m a c e u t i c a l s i n c r e a s e d . TLC w i t h i t s h i g h s e p a r a t i n g

435 power s t i m u l a t e d a f u r t h e r development o f t h e c l a s s i c a l T s w e t t l i q u i d column chromatography t o HPLC. I t w i l l keep i t s f i r m p l a c e i n t h e l a b o r a t o r y , being t h e only procedure which allows t h e s e p a r a t i o n of numerous s u b s t a n c e s under e x a c t l y t h e same c o n d i t i o n s i n s e r i e s w h i l e employing hundreds of d e t e c t o r s . REFERENCES 1 E . S t a h l , Pharmazie 1 1 (1956) 633. 2 E . S t a h l , Chem.-Ztg. 82 (1958) 323. 3 E . S t a h l and U. Kaltenbach, J . Chromatogr. 5 (1961) 351. 4 E . S t a h l and H. Kaldewey, Hoppe-SeyZer's Z. PhysioZ. Chem. 323 (1961) 182. 5 E . S t a h l and P.J. Schorn, Hoppe-SeyZer's Z. PhysioZ. Chem. 325 (1961) 263. 6 E . S t a h l and U. Kaltenbach, J . Chromatogr. 5 (1961) 458. 7 E. S t a h l ( e d . ) , Diinnschicht-Chromatographie, e i n Laboratoriwnshandbuch, 1. Aufl. Springer-Verlag, B e r l i n , Gottingen, Heidelberg, 1962. 8 E . S t a h l ( e d . ) , Thin-Layer Chromatography, 2nd e d . , T r a n s l . P r o f . Ashworth, Springer-Verlag, B e r l i n , Heidelberg, New York, 1969. 9 E . S t a h l , Angew. Chem. I n t . Ed. EngZ. 3 (1964) 784. 10 E . S t a h l , Z. Anal. Chem. 221 (1966) 3. 11 E . S t a h l and P . J . Schorn, Arzneim.-Forsch. 17 (1967) 1288. 12 E . S t a h l , J. Chromatogr. 33 (1968) 273. 13 E . S t a h l , Drug Analysis by Chromatography and Microscopy, Ann Arbor Science P u b l . , Ann Arbor, Michigan, 1973. 14 E . S t a h l , J. Chromatogr. 37 (1968) 99. 15 E . S t a h l , Analyst (London) 94 (1969) 723. 16 E . S t a h l , Z. Anal. Chem. 261 (1972) 11. 17 E . S t a h l and F. Karig, Holzforschung 27 (1973) 89. 18 E . S t a h l and V. B r u d e r l e , i n Advances i n Polymer Science, H . J . Cantow, e d . , Springer-Verlag, B e r l i n , Heidelberg, N e w York, 1978, i n p r e s s . 19 E . S t a h l , Accounts Chem. Res. 9 (1976) 75. 20 E . S t a h l and W. S c h i l z , Z. Anal. Chem. 280 (1976) 99. 2 1 E . S t a h l and W. S c h i l z , Chem.-Ing.-Techn. 48 (1976) 772.

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H.H. STRAIN

HAROLD HENRY STRAIN was born in 1904 in San Francisco, California. He studied at Stanford University receiving his Ph.D. in Chemistry in 1927. From 1927 to 1949 he was associated with the Carnegie Institution, for two years with its Coastal Laboratories, in Carmel, California, and then with its Department of Plant Biology, at Stanford. In 1937-1938 he was a Rockefeller Foundation Fellow at the Carlsberg Laboratories, in Copenhagen, Denmark. Dr. Strain joined the Chemistry Division of Argonne National Laboratories, Argonne, Illinois, in 1949. He officially retired from his position on Januari 1, 1969, but continues to serve as a guest investigator at Argonne. Dr. Strain is the author of almost 200 papers and several books among them the first American book on chromatography published in 1942, by Interscience Publishers. In 1958 he was the Priestley Lecturer at Pennsylvania State University and in 1963, a senior fellow at the Australian Academy of Science. He was the first recipient of the American Chemical Society Award in Chromatography and Electrophoresis; he was also honored by the Midwest Award of the St. Louis Section of the American Chemical Society. In November 1968, before his retirement, Argonne National Laboratories held a special symposium in Dr. Strain's honor, the Conference on Photosynthetic Pigments, with international participation. Dr. Strain's career centered almost completely on the isolation and study of the chemical constituents of plants, especially plant pigments. His contributions have been extensively recognized both in plant biology and in chemistry, and have had great impact on many workers in broad areas of both disciplines. Dr. Strain's activities in chromatography started in the early 1930's and he is one of the early pioneers who have brought this technique to its present usefulness. He was also one of the first who developed electrochromatography and then went on to pioneer in the development of one-way, multiway and continuous techniques.

As a young man I had c a r r i e d o u t many s t u d i e s w i t h D r . Edward C . F r a n k l i n . I n t h i s w e made great use o f a n a l o g y . S h o r t l y b e f o r e I o b t a i n e d my Ph.D., D r . F r a n k l i n c a l l e d m e i n t o h i s o f f i c e a t h i s S t a n f o r d L a b o r a t o r y . H e t o l d m e c h a t D r . Spoehr of t h e C a r n e g i e I n s t i t u t i o n o f Washington P l a n t P h y s i o l o g y ' s L a b o r a t o r y a t C a r m e l , C a l i f o r n i a , was t o have a new l a b o r a t o r y b u i l t a t S t a n f o r d . H e s a i d t h a t he t h o u g h t t h a t t h i s l a b o r a t o r y would be a good p l a c e f o r m e . As you may see f r o m t h i s s h o r t r e p o r t , i t remained a good p l a c e f o r m e f o r a long t i m e . A t t h e C a r n e g i e I n s t i t u t i o n l a b o r a t o r y I w a s soon i n v o l v e d i n t h e s t u d y o f t h e c a r o t e n e s . The y e l l o w c a r o t e n e o f carrot r o o t s w a s a l s o t h e p r i n c i p a l c a r o t e n e i n l e a v e s . T h e r e were v a r i o u s i d e a s about t h e r o l e t h i s pigment might p l a y i n n a t u r e . A t t h a t t i m e - o v e r 50 y e a r s ago! - t h e c a r o t e n e of y e l l o w c a r r o t s had been r e p o r t e d t o b e a m i x t u r e of y e l l o w pigments, b u t t h e r e w a s l i t t l e e v i d e n c e f o r t h i s . The e f f i c i e n t chromatographic a d s o r p t i o n method had been used f o r i n v e s t i g a t i o n s , b u t t h e method had i t s s h o r t c o m i n g s . I w a s a n e x p e r i m e n t a l i s t , and I w a s w i l l i n g t o c a r r y o u t new e x p e r i m e n t s and r e v i e w t h e c l a i m s t h a t had been made. T h i s is how my involvement i n chromatography s t a r t e d . F o r t u n a t e l y , w e found some a d s o r p t i o n s y s t e m s t h a t h e l d t h e y e l l o w c a r o t e n e . Tubes f i l l e d w i t h f i b r o u s alumina r e t a i n e d t h e c a r o t e n e and gave e v i d e n c e t h a t t w o y e l l o w pigments were i n d e e d p r e s e n t . F i b r o u s alumina, however, proved d i f f i c u l t t o p r e p a r e , h a n d l e and pack i n t o columns. W e f e l t t h a t p r o g r e s s i n t h i s l i n e depended upon t h e s e l e c t i o n of a b e t t e r a d s o r b e n t f o r u s e i n t h e columns. One d a y , l a r g e l y by a c c i d e n t , I d i s c o v e r e d an u n u s u a l ads o r b e n t . T h i s m a t e r i a l was powdered magnesium o x i d e which w a s a v a i l a b l e commercially. I t had a very s t r o n g a f f i n i t y f o r v a r i o u s c o l o r e d s u b s t a n c e s . I t was a l s o remarkably r e v e r s i b l e . I n d e e d , i t had g r e a t a d s o r p t i o n c a p a c i t y f o r c a r o t e n e , and i t w a s remarkably s e l e c t i v e as w e l l . With these c o n d i t i o n s t h e c a r o t e n e o f c a r r o t r o o t s w a s e s t a b l i s h e d a s a mixture o f t w o isomeric hydrocarbons, each C 4 0 H 5 6 . With chromatographic columns c o n t a i n i n g magnesia, t h e changes of c a r o t e n e i n p l a n t s and a n i m a l s were much e a s i e r t o f o l l o w . I t s h o u l d a l s o be p o i n t e d o u t t h a t o n e o f t h e c a r o t e n e s , B-carotene, had been found t o be a n a t u r a l p r e c u r s o r o f v i t a m i n A. T h i s f a c t focused a g r e a t d e a l o f a t t e n t i o n on t h e c a r o t e n e s and on v i t a m i n A . I t a l s o focused a g r e a t d e a l o f a t t e n t i o n on t h e chromatographic method f o r t h e s t u d y o f c a r o t e n e . I n t h e c h r o m a t o g r a p h i c a n a l y s i s , some of t h e c a r o t e n e w a s d i s s o l v e d i n petroleum e t h e r . Then t h e magnesia w a s packed i n t o a g l a s s t u b e t o form a column. P a s t o f t h e c a r o t e n e s o l u t i o n w a s sucked i n t o t h i s t u b e . The c a r o t e n e components moved a l o n g a t d i f f e r e n t r a t e s so t h a t t h e y s e p a r a t e d from e a c h o t h e r . T h i s was t h e b a s i s o f t h e chromatographic method f o r t h e s e p a r a t i o n o f c a r o t e n e s . I t w a s t h e basis o f t h e chromatographic a d s o r p t i o n method f o r u s e i n v a r i o u s ways. The commercial magnesium o x i d e w a s f i n e l y d i v i d e d o r powdered, and t h u s , t h e column f i l t e r e d t o o s l o w l y . T h i s problem w a s overcome

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F i g . 5 0 . 1 . S t a f f o f t h e new l a b o r a t o r y o f t h e D i v i s i o n o f P l a n t Biology o f t h e C a r n e g i e I n s t i t u t i o n o f Washington, l o c a t e d a t S t a n f o r d U n i v e r s i t y . L e f t t o r i g h t : W. H i e s e y , D. Keck, H.A. Spoehr ( d i r e c t o r ) , J . H . C . S m i t h , J. C l a u s e n , H. M i l n e r , H . H . S t r a i n , W.A. P e s t e l l ( S e c r e t a r y ) . by mixing t h e magnesia w i t h h e a t - t r e a t e d s i l i c e o u s e a r t h . Then t h e p e r c o l a t i o n r a t e o f t h e column was i n c r e a s e d . Much more e f f e c t i v e columns were o b t a i n e d i n t h i s manner. Such columns have been w i d e l y employed f o r s t u d i e s of c a r o t e n e s and v a r i o u s o t h e r s u b s t a n c e s . These columns a r e remarkably a d a p t i v e r e s e a r c h t o o l s . When w e were working w i t h t h e s e columns loaded w i t h zones o f g r e e n and y e l l o w s u b s t a n c e s , w e had d i f f i c u l t y i n showing o u r r e s u l t s , i . e . reproduce t h e c o l o r s f o r o u r own r e c o r d s . I t was s u g g e s t e d t h a t w e have a young a r t i s t draw one o f t h e columns r e p r o d u c i n g c o l o r s , i n t e n s i t i e s , and o v e r l a p p i n g of t h e zones. I t h e n gave t h i s drawing t o a p r i n t e r who managed t o reproduce i t q u i t e w e l l f o r one o f my books. Even t o d a y , a f t e r a l l t h i s t i m e , I am happy about t h e r e s u l t . Now, o f c o u r s e , c o l o r photography is a t a d i f f e r e n t p r o f e s s i o n a l l e v e l ; so t h e r e p r o d u c t i o n o f c o l o r i s s i m p l e r t h a n i t was f o r t y y e a r s ago. There were two o f our men who s p e n t a g r e a t d e a l o f t i m e i n i s o l a t i n g t h e c a r r o t r o o t c a r o t e n e and a l s o t h e c a r o t e n e from t h e g r e e n l e a v e s . They w e r e D r . James H . C . Smith and M r . Harold W . Milner. Both were c h e m i s t s by t r a i n i n g . Mr. Mi'lner was a l s o a s p e c i a l i s t i n

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Fig. 50.2. H.H. Strain filling a large-diameter chromatographic column. Photo taken in 1956.

microanalysis and was able to make the most careful analysis of the carotene and other pigments which I had isolated with our chromatographic tools. Both men died at an early age. Dr. Smith developed some excellent procedures for the determination of the spectral absorption properties of the pigments which I isolated. This was a most helpful tool. I made extensive use of it to study the spectral absorption properties of several new pigments that were isolated in great purity by use of chromatography. One of these was the pigment from the petals of the yellow California poppy. These spectral absorption curves may be found in some of our old publications. Chromatography for carotene was but one application of the technique. These investigations marked the beginning of many modifications in the method. For example the adsorptive agent could be varied as needed. Moreover the wash liquid could also be varied. One of the adsorbents I utilized in our studies was powdered sugar. This is a very mild adsorbent. This could be used to accelerate the separation of mild substances from various basic pigments from tougher structural materials. This opened the way to the separation of complex but labile substances. I was interested in the chlorophylls as possible active agents in photosynthesis, and for their examination, I employed the powdered

441 s u g a r a s t h e s o r p t i v e a g e n t . In t h i s way, w e d i s c o v e r e d new c h l o r o p h y l l s . With t h e chromatographic column c o n t a i n i n g powdered s u g a r these could be recovered unchanged or u n a l t e r e d . These pigments a r e a c t i v e p h o t o s y n t h e s i s . O t h e r examples f o r s u b s t a n c e s w e s t u d i e d a r e t h e marine a l g a e or seaweeds a s t h e y a r e commonly c a l l e d . I n my s t u d i e s , I have developed and used a number o f new chromatographic t e c h n i q u e s . Electrochromatography is one such t e c h n i q u e . I t was developed i n t h e form of one-way, two-way, three-way and continuous procedures. We a l s o used paper chromatography very e a r l y and t h i s proved very u s e f u l w i t h s m a l l q u a n t i t i e s o f m i x t u r e s . The r e s u l t s o f o u r work may have been c o n s i d e r e d t h e r e s u l t of many workers. I n my c a s e , however, t h e bulk o f t h e work t h a t I t u r n e d o u t was t h e product o f my own s t u d i e s . I t was t h e product of my own hands and my own contemplations. From t h i s s t a n d p o i n t my s t u d i e s i n c o r p o r a t e d t h e weaknesses of my own t h o u g h t s a s w e l l as t h e s t a b i l i t y of my independent t h i n k i n g . For a s h o r t time d u r i n g t h e Second World War, I was a s s o c i a t e d w i t h Dr. Winston Manning i n connection w i t h some s t u d i e s on t h e c h l o r o p l a s t pigments. L a t e r , Manning j o i n e d t h e n u c l e a r i n v e s t i g a t i o n s c a r r i e d o u t under t h e a u s p i c e s o f t h e U n i v e r s i t y o f Chicago. H e asked m e t o j o i n him t h e r e b u t I d e c l i n e d , n o t knowing t h e n a t u r e of t h e work. A f t e r t h e War, Dr. Manning became t h e d i r e c t o r of t h e Argonne N a t i o n a l L a b o r a t o r i e s , formed from t h e war-time a c t i v i t i e s . H e asked m e t o r e c o n s i d e r h i s i n v i t a t i o n . When i t was p o i n t e d o u t t o m e t h a t t h e r e would be t i m e f o r r e s e a r c h i n chromatography and f o r s t u d i e s o f p l a n t pigments, I a c c e p t e d t h e i n v i t a t i o n . I t was proven a s a happy and p r o d u c t i v e r e l a t i o n s h i p , and, a s a member of t h e Argonne s t a f f , I could c o n t i n u e my p r o d u c t i v e work u n t i l my retirement. From my a c t i v i t i e s d u r i n g t h i s p e r i o d , t h e p r e p a r a t i o n of f u l l y d e u t e r a t e d c a r o t e n e s and c h l o r o p h y l l s , by growing a l g a e i n heavy w a t e r s h o u l d be mentioned, and I was involved with Katz and o t h e r s i n i s o l a t i n g t h e s e pigments. I o f f i c i a l l y r e t i r e d from Argonne National L a b o r a t o r i e s i n 1969 b u t c o n t i n u e t o s e r v e a s a g u e s t i n v e s t i g a t o r even i n my r e t i r e m e n t .

-0-0-0Chromatographic i n v e s t i g a t i o n s brought t h e s c i e n t i f i c i n t e r e s t i n t o t h e realm of a c t i v i t y of p l a n t s . I t should be noted t h a t some of t h e key a c t i v i t i e s o f t h e p l a n t s l a y i n t h e a c t i v i t y o f t h e pigments. But even h e r e , t h e understanding of t h e r o l e of p l a n t s i n Nature depends upon o u r a n a l y t i c a l p r o c e d u r e s . Some y e a r s ago, t h i s complexity was p r e s e n t e d i n rhyme by m e . I t is p r e s e n t e d here a g a i n because o f i t s c l a r i t y and i t s o b j e c t i v i t y , a s a summary o f my philosophy i n my l i f e ' s a c t i v i t i e s :

442

Capture of S u n l i g h t The i n t e r f a c e o f e a r t h and sky

I s i n t e r l a c e d with green. T h i s is a happy c i r c u m s t a n c e I n N a t u r e ' s p l a n supreme. The meekest mouse, t h e m i g h t i e s t whale Diatom and t a l l e s t t r e e ; Each t h i n g t h a t l i v e s and b r e a t h e s and grows, Consumens much e n e r g y . But man a l o n e i s n o t c o n t e n t , A s other l i v i n g things. H i s n e e d s f o r food and f u e l s u r p a s s H i s c a r e f u l reckonings. Whence comes t h i s power e a c h must have F o r l i f e , f o r b r e a t h , for growth? I t ' s from t h e b l a z i n g sun a f a r : ' T i s l i g h t ; ' T i s f o r c e : I t ' s both.

Sun's s i l e n t s h a f t s o f p o t e n t l i g h t A r e l o s t on b a r r e n ground. But l e t a p l a n t expose i t s g r e e n , And miracles abound.

The f o r c e i n l i g h t is c a p t u r e d t h e n , By pigments i n t h e c e l l . The d r o s s o f b r e a t h i s g a r n e r e d t h e r e ; And b o t h a r e wed a s w e l l .

Most progeny t h i s w i s e b e g o t A r e r i c h i n energy. They a r e t h e carbonaceous p r o d u c t s , From l a n d , from stream, from s e a . That i n t e r f a c i a l , e a r t h y g r e e n , In p l a n t s alone, succeeds To s t o r e s u n l i g h t upon t h e e a r t h , And t h u s t o f i l l l i f e ' s n e e d s .

443

F.H. STROSS

FRED HELMUT STROSS was born i n 1910, i n A l e x a n d r i a , Egypt where h i s f a t h e r s e r v e d i n t h e d i p l o m a t i c s e r v i c e of post-war Aust r i a . H e a t t e n d e d e l e m e n t a r y and secondary s c h o o l s i n A l e x a n d r i a , London ( E n g l a n d ) , a a v i l l a g e i n Bohemia and i n Vienna ( A u s t r i a ) . A f t e r completing h i g h s c h o o l , he s p e n t a y e a r i n C a i r o working a t t h e A u s t r i a n Cons u l a t e . He t h e n took up u n i v e r s i t y s t u d i e s i n Vienna and a t t h e Case I n s t i t u t e of Technology, i n C l e v e l a n d , Ohio, where he obt a i n e d a B.S. d e g r e e i n 1934, and f i n a l l y a t t h e U n i v e r s i t y o f C a l i f o r n i a , a t Berkel e y , where he o b t a i n e d h i s Ph.D. i n chemist r y i n 1938. Immediately a f t e r g r a d u a t i o n he was employed b y t h e S h e l l Development Company and he was a s s o c i a t e d w i t h t h i s company i n v a r i o u s c a p a c i t i e s u n t i l h i s ret i r e m e n t i n 1970 a s a s u p e r v i s o r i n res e a r c h . S i n c e h i s r e t i r e m e n t , he h a s been a s s o c i a t e d w i t h t h e Lawrence Berkeley L a b o r a t o r y o f t h e U n i v e r s i t y o f C a l i f o r n i a , a s a p a r t i c i p a t i n g g u e s t . R e c e n t l y he was a p p o i n t e d a r e s e a r c h p r o f e s s o r a t t h e Chemi s t r y Department of t h e U n i v e r s i t y o f Washington, i n S e a t t l e . D r . S t r o s s h a s o v e r 150 p u b l i c a t i o n s i n v a r i o u s s u b j e c t s . H i s primary i n t e r e s t h a s been a n a l y t i c a l and p h y s i c a l c h e m i s t r y and chromatography; i n a d d i t i o n , h i s s p e c i a l i t i e s i n c l u d e a r c h e o l o g y and p a r t i c u l a r l y t h e a p p l i c a t i o n of p h y s i c a l s c i e n c e s t o a r c h e o l o g y ; h i s Report for AnalyticaZ Chemists on t h e a u t h e n i c a t i o n o f a n t i q u e s t o n e o b j e c t s by p h y s i c a l and chemical methods (Ana1.Chem. 3 2 ( 3 ) ( 1 9 6 0 ) 17A) i s a d e f i n i t i v e publ i c a t i o n on t h i s s u b j e c t . D r . S t r o s s s e r v e d on v a r i o u s committees d e a l i n g w i t h t h e p r o p e r nom e n c l a t u r e o f g a s chromatography; he was a member of t h e IUPAC Committ e e and chairman o f t h e Subcommittee o r g a n i z e d by t h e N a t i o n a l Academy of S c i e n c e s a s w e l l a s t h e Subcommittee o r g a n i z e d by Committee D-2 o f ASTM. H e h a s a l s o s e r v e d on t h e E d i t o r i a l Board of Analytical Chemistry. D r . S t r o s s ' involvement i n g a s chromatography s t a r t e d i n 1954 and he was a c t i v e i n t h i s f i e l d and i t s outgrowths u n t i l h i s r e t i r e m e n t from Shell.

444

This report discusses the involvement of the scientists at Shell Development Company, at Emeryville, California, in the evolution of gas chromatography. Thus, it is not my personal story - it is the story of the Emeryville collective of which I was part. In the summer of 1954 the Shell Development Company at Emeryville, then a subsidiary of Shell Oil Company, became interested in the remarkable technique developed not long before by A . J . P . Martin and his collaborators. Imperial Chemical Industries in England had already made important industrial application of its analytical potential. The method, written into specifications, was used by laboratories of the Shell Group, including the Amsterdam research laboratories, since they had relevant dealings with ICI. Work on the theory and practice of gas chromatography was begun in the Analytical and Physical Chemistry Departments at Emeryville, headed, respectively, by S . Z . Perry and C. Dunn, and continued for the several years, in parallel with the work that was proceeding apace at the Amsterdam Laboratory. Information between the two laboratories was exchanged on a regular basis at frequent intervals. A classic account of this early work is given by Keulemans in his well-known book (I). The Emeryville departments collaborated intensively on the formulation of the basic relationships involved, and on the use that could be made of the insight gained. The potential of the new technique for making convenient measurements of useful thermodynamic quantities, such as partition coefficients, activity coefficients, heats of vaporization, and related parameters was being recognized, and the Emeryville laboratories as well as their colleagues abroad contributed significantly in this area. It was established (2) that partition coefficients determined by GC were independent of such column quantities as carrier gas flow velocity, column length, ratio of liquid phase to solid support, and pressure drop, and could be determined reliably from GC experiments. It was suggested that the extensive data relating to the non-idealities of solutions could be applied directly to understanding and predicting GLC behavior, and a companion paper (3) elaborated on this point by drawing up a semi-empirical model enabling prediction of yo-values of many homologous series from available constants or by means of a small amount of experimental work. Work was also done to provide a sound experimental set-up for these studies, and for the practical applications anticipated. Studies of basic factors such as the effects of sample charging, including "plug flow" vs. "complete mixing" were made (2) and, similarly, of the performance characteristics of individual features of the apparatus ( 4 ) . Effects of detector design, carrier gas, column packing, and experimental variables were determined, and certain solid supports, and helium as carrier gas were suggested, to be more consistent with availability and practice in the U . S . A . than their European counterparts ( 4 ) . An expression for detector sensitivity was also suggested, and was widely used as the Dimbat-Porter-Stross Sensitivity or DIPS Index. Applications to paradigmatic mixtures were made at Emeryville at an early stage. Use of stationary liquids of different polarities,

445 and o f multi-column systems was made ( 5 ) , and o f m o d i f i e r s i n t h e c a r r i e r g a s i t s e l f ( 6 ) o r by "doping" t h e s o l i d s u p p o r t ( 7 ) t o improve peak shape and s e p a r a t i o n . Apparatus improvements were made ; among t h e s e t h e r e were new t y p e s o f d e t e c t o r s (8) and m o d i f i c a t i o n s t o e x i s t i n g d e t e c t o r s ( 9 ) . Many o f t h e s e f e a t u r e s were i n c o r p o r a t e d i n a h i g h l y v e r s a t i l e i n s t r u m e n t developed i n c o l l a b o r a t i o n w i t h H a l l i k a i n e n I n s t r u m e n t s , and marketed by them d u r i n g t h e l a t e f i f t i e s

(10). Soon t h e need f o r more r i g o r o u s a s s e s s m e n t s o f t h e c h a r a c t e r i s t i c s of t h e a p p a r a t u s became a p p a r e n t . Methods f o r e v a l u a t i n g q u a n t i t a t i v e l y t h e d e t e c t o r s e n s i t i v i t y and column e f f i c i e n c y w e r e proposed (11,12). A t t h e same t i m e i t became a p p a r e n t t h a t i t would be c o n v e n i e n t i f t h o s e t h a t worked i n t h e f i e l d o f GC were t o t a l k t h e same l a n g u a g e , a t l e a s t f i g u r a t i v e l y s p e a k i n g . T h i s c a l l e d f o r a d e f i n i t i o n o f terms and c o n c e p t s , and t h i s became an i n t e r n a t i o n a l , a s w e l l as a n a t i o n a l u n d e r t a k i n g . I n 1958 a s p e c i a l group was formed by t h e I n t e r n a t i o n a l Union o f P u r e and Applied Chemistry to m a k e recommendations a l o n g t h e s e l i n e s . The chairman o f t h i s group was D. Ambrose and i t s members were A.T. James, A . I . M . Keulemans, E . K o v i t s , H. Rock, C . Rouet and m y s e l f . The c o n c l u s i o n s w e r e p u b l i s h e d i n t h e Preziminary Recommendat i o n s ( 1 3 ) , and i n t h e Recommendations on Nomenclature and Presentat i o n of Data i n Gas Chromatography ( 1 4 ) . I n t h i s c o u n t r y , t h e Committee on A n a l y t i c a l Chemistry o f t h e N a t i o n a l Research C o u n c i l , N a t i o n a l Academy o f S c i e n c e s , under t h e chairmanship of I.M. K o l t h o f f , formed a Subcommittee on Gas Chromatography (R.O. C l a r k , G . Matsuyama and myself s e r v i n g a s t h e c h a i r m a n ) , t o f o r m u l a t e r e p r e s e n t a t i v e U.S.A. o p i n i o n , and i n t h e same y e a r a p u b l i c a t i o n o f terms and u n i t s i n g a s chromatography (15) appeared under t h e s p o n s o r s h i p o f t h e Study Group on G a s Chromatography o f Subcommittee R.D.IV., Committee D-2 o f t h e American S o c i e t y f o r T e s t i n g & M a t e r i a l s . Out o f t h e i n f o r m a t i o n g a i n e d i n t h e p r a c t i c e o f g a s chromatog r a p h y , new approaches t o o l d problems w e r e developed. A method f o r measuring s u r f a c e a r e a , based on t h e same t h e o r y a s t h e e s t a b l i s h e d Brunauer-Emmett-Teller method, b u t u s i n g t h e equipment and t e c h n i q u e s t h a t had become f a m i l i a r i n GC e x p e r i m e n t a t i o n , was developed (16,17). I t was l i c e n s e d t o t h e Perkin-Elmer C o r p o r a t i o n and was marketed by t h i s f i r m a s t h e Model 212 Perkin-Elmer/Shell Sorptometer f o r about 1 5 y e a r s (18,19). The a d v a n t a g e s o f t h i s "dynamic" method o v e r t h e o l d e r s t a t i c method were t h e g r e a t l y enhanced r a p i d i t y and s i m p l i c i t y o f measurement and i n s t r u m e n t a t i o n . Another i n t e r e s t i n g outgrowth from e x p e r t i s e g a i n e d i n t h e GC f i e l d was t h e development of a t h e r m a l a n a l y s i s system u s i n g a flame i o n i z a t i o n d e t e c t o r t o detect p y r o l y z e d o r g a n i c m a t e r i a l . T h i s i n s t r u ment can measure t h e v o l a t i l i t y of h i g h - b o i l i n g o r g a n i c compounds and y i e l d vapor p r e s s u r e c u r v e s o v e r t h e range o f 0 . 1 t o 5000 m i l l i t o r r . I n a d d i t i o n , t h e t h e r m a l s t a b i l i t y o f such o r g a n i c compounds, and t h e v o l a t i l i z a t i o n p a t t e r n s and t r a c e v o l a t i l e c o n t e n t of polymers c a n b e determined w i t h g r e a t s i m p l i c i t y and s e n s i t i v i t y (20,21). T h i s i n s t r u ment was l i c e n s e d t o C a r l e I n s t r u m e n t s ( 2 2 ) . By t h e s i x t i e s , g a s chromatography had become a u n i v e r s a l l y used t e c h n i q u e , and a l a b o r a t o r y w i t h o u t some GC equipment was n e a r l y a s

446 d i f f i c u l t t o imagine a s a highway w i t h o u t automobiles. Instrument companies were doing t h e i r own r e s e a r c h . For S h e l l Development, t h e t i m e t o abandon f u r t h e r development e s p e c i a l l y on a p p a r a t u s c o n s t r u c t i o n had come. The dawn of t h e day of h i g h - p r e s s u r e l i q u i d chromatography was appearing on t h e h o r i z o n ; and GC had l a r g e l y become r o u t i n e . REFERENCES 1 A . I . M . Keulemans, Gas Chromatography, Reinhold, N e w York, 1957. 2 P.E. P o r t e r , C . H . Deal and F.H. S t r o s s , J . Arner. Chern. Soe. 78 (1956) 2999. 3 G . J . P i e r o t t i , C . H . Deal, E . L . D e r r and P.E. P o r t e r , J . Arner. Chem. Soe. 78 (1956) 2989. 4 M. Dimbat, P . E . P o r t e r and F.H. S t r o s s , Anal. Chern. 28 (1956) 290. 5 E.M. F r e d e r i c k s and F.R. Brooks, Anal. Chern. 28 (1956) 297. 6 H.S. Knight, Anal. Chem. 30 (1958) 2939. 7 F.T. Eggertsen, H.S. Knight and S. Groennings, AnaZ. Chem. 28 (1956) 303. 8 C . H . Deal, J . W . Otvos, J . W . Smith and V . N . Zucco, Anal. Chern. 28 (1956) 1958. 9 F.T. Eggertsen and S. Groennings, Anal. Chern. 30 (1958) 20. 10 See L.S. E t t r e , J . Chrornatogr. S c i . 15 (1977) 89. 11 H.W. Johnson and F.H. S t r o s s , AnaZ. Chern. 31 (1959) 1206. 1 2 H.W. Johnson and F.H. S t r o s s , Anal. Chern. 31 (1959) 357. 13 Preliminary Reconmendations on PJornencZature and Presentation of Data i n Gas Chromatography, Pure A p p l . Chem. 1 (1960) 177. 14 Kecommendations on NornencZature and Presentation of Data i n Gas Chromutography, Pure Appl. Chern. 6 (1964) 553. 15 H.W. Johnson and F.H. S t r o s s , AnaZ. Chem. 30 (1958) 1586. 16 F.M. Nelsen and F.T. Eggertsen, A w l . Chem. 30 (1958) 1387. 17 H.W. Daeschner and F.H. S t r o s s , AnaZ. Chern. 34 (1962) 1150. 18 L.S. E t t r e and G . C a r o t i , Chim. Ind. (Milano) 4 2 (1960) 864. 19 L.S. E t t r e , Application of the Continuous F l o w Method and the

Model 212-D Sorptometer for Surface S t u d i e s , AppZication Brochure No.SO-AP-002, The Perkin-Elmer Corp., Norwalk. Conn., 1966. 20 F.T. Eggertsen and F.H. S t r o s s , J . A p p l . Polymer S c i . 10 (1966) 1171, 21 F.T. E g g e r t s e n , E . E . S e i b e r t and F.H. S t r o s s , Anal. Chern. 41 (1969) 1175. 22 A.C. Stapp and D.W. C a r l e , Pittsburgh Conf. AnaZ. Chem. and A p p l . Spectroscopy, Cleveland, Ohio, March 1969; C a r l e Instruments Co. , F u l l e r t o n , C a l i f . , R e p r i n t No.TA-369.

447

R.L.M. SYNGE

RICHARD LAURENCE M I U I N G T O N SYNGE was born i n 1914 i n L i v e r p o o l , England. H e a t t e n d e d Winchester C o l l e g e and t h e n e n t e r e d T r i n i t y C o l l e g e , U n i v e r s i t y o f Cambridge. Upon g r a d u a t i n g he remained a t t h e U n i v e r s i t y as a r e s e a r c h s t u d e n t under t h e s u p e r v i s i o n of N.W. P i r i e i n t h e Biochemical L a b o r a t o r y headed by S i r F r e d e r i c k G . Hopk i n s u n t i l 1939 when h i s S t u d e n t s h i p was t r a n s f e r r e d t o t h e Wool I n d u s t r i e s Research L a b o r a t o r i e s , i n Leeds. H e r e c e i v e d h i s Ph.D. a t Cambridge i n 1941 and t h e r e a f t e r , h e j o i n e d t h e Wool I n d u s t r i e s A s s o c i a t i o n , i n Leeds. During 1943-1948 he w a s on t h e s t a f f of t h e L i s t e r I n s t i t u t e o f Prevent i v e Medicine, London, working mainly on a n t i b i o t i c p e p t i d e s and s p e n d i n g 1946-1947 a t Uppsala, Sweden, working w i t h A . T i s e l i u s . That v i s i t i n i t i a t e d a c o l l a b o r a t i o n on v a r i o u s a d s o r p t i o n and m o l e c u l a r s i e v e phenomena. Between 1948 and 1967 he was w i t h t h e Rowett Research I n s t i t u t e , a t Bucksburn, Aberdeen and s i n c e 1967 w i t h t h e A g r i c u l t u r a l Research C o u n c i l ' s Food Research I n s t i t u t e a t Norwich where he r e t i r e d from t h e s t a f f i n 1976. A t t h e s e I n s t i t u t e s h i s work h a s been mainly on e l e c t r o p h o r e t i c and m o l e c u l a r s i e v e t e c h n i q u e s ( p a r t i c u l a r l y u s i n g c h a o t r o p i c s o l v e n t m i x t u r e s ) , on p h y t o c h e m i s t r y o f f r e e and c o n j u g a t e d amino a c i d s , on p r o t e i n n u t r i t i o n of r u m i n a n t s and non-ruminants and on r e a c t i o n s o f p l a n t p o l y p h e n o l s l i k e l y t o a f f e c t p r o t e i n n u t r i t i o n . During 1958-1959 he s p e n t a y e a r a t Ruakura A g r i c u l t u r a l Research S t a t i o n , Hamilton, New Z e a l a n d , working on i s o l a t i o n o f t h e mycotoxin s p o r i d e s m i n . D r . Synge i s t h e a u t h o r and c o a u t h o r o f a number of s c i e n t i f i c p a p e r s d e a l i n g w i t h v a r i o u s a s p e c t s of b i o c h e m i s t r y and chromatography; he a l s o p u b l i s h e d a few s t u d i e s r e l a t i n g t o t h e h i s t o r y and s o c i a l i m p l i c a t i o n s o f s c i e n c e . I n 1952, D r . Synge, j o i n t l y w i t h A.J.P. M a r t i n , r e c e i v e d t h e Nobel P r i z e f o r Chemistry. H e h a s a h o n o r a r y d o c t o r a t e from t h e Univers i t y of E a s t A n g l i a , Norwich where he i s a Honorary P r o f e s s o r . D r . Synge i s a fel'low o f t h e Royal S o c i e t i e s o f London and Edinburgh, h o n o r a r y member of s e v e r a l o t h e r s o c i e t i e s and an Honorary Fellow o f T r i n i t y C o l l e g e , Cambridge. D r . Synge i s t h e c o - i n v e n t o r of t h e method o f p a r t i t i o n chromatography and a p i o n e e r i n t h e u t i l i z a t i o n o f chromatographic methods i n b i o c h e m i s t r y . S i n c e 1945, h i s main i n t e r e s t h a s been i n a n a l y t i c a l probl e m s concerning t h e l a r g e r peptide molecules.

448

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I n t h i s c o n t r i b u t i o n I would l i k e t o summarize how I became i n v o l v e d i n chromatography . While s t i l l a t s c h o o l I h e a r d t e l l o f t h e s c i e n c e o f biochemi s t r y which s h o u l d draw t o g e t h e r two a s p e c t s o f n a t u r e i n t o a common s t u d y . I had l o n g been f a s c i n a t e d by l i v i n g t h i n g s ( p a r t i c u l a r l y t h e w i l d p l a n t s ) and more r e c e n t l y , by c h e m i s t r y . I t was i n r e a d i n g S i r F r e d e r i c k Hopkins' p r e s i d e n t i a l a d d r e s s t o t h e B r i t i s h A s s o c i a t i o n f o r t h e Advancement o f S c i e n c e , i n Leicester, i n a newspaper, i n 1933, t h a t impressed on m e t h e i d e a t h a t l i v i n g t h i n g s must have w o n d e r f u l l y p r e c i s e and c o m p l i c a t e d working p a r t s on t h e m o l e c u l a r s c a l e and t h a t t h e b i o c h e m i s t had t h e b e s t chance o f f i n d i n g o u t how t h e s e a r e p u t t o g e t h e r and do t h e i r work. A s I was t o b e g i n s t u d y i n g n a t u r a l s c i e n c e a t Cambridge U n i v e r s i t y i n t h e same y e a r , t h e a m b i t i o n o f s t u d y i n g i n P r o f e s s o r Hopkins' l a b o r a t o r y c o u l d e a s i l y be r e a l i z e d . E x p e r i e n c e s i n t h e e l e m e n t a r y c o u r s e t a k e n by m e d i c a l s t u d e n t s d i d not give t h e impression o f biochemistry being a r e f i n e d o r p r e c i s e s c i e n c e ; however, when embarking on t h e advanced c o u r s e i n h i s l a b o r a t o r y , I immediately came i n c o n t a c t w i t h e x c i t i n g f a c t s and e x c i t i n g i d e a s . The l a t t e r outnumbered t h e f o r m e r , u s u a l l y . A t an e a r l y s t a g e i n t h e c o u r s e t h e s t u d e n t engaged i n some q u i t e r i g o r o u s i s o l a t i v e work under t h e g u i d a n c e o f N.W. P i r i e . H e u s e d t o e n l i v e n t h e l o n g h o u r s a t t h e bench w i t h c a u s t i c a n e c d o t e s from t h e h i s t o r y of b i o c h e m i s t r y which h e l p e d , q u i t e a s much a s t h e i s o l a t i v e work i t s e l f and h i s comments t h e r e o n , t o d e v e l o p t h e c r i t i c a l f a c u l t y i n t h o s e who p o s s e s s e d a rudiment o f i t . On g r a d u a t i n g i n 1936 I c o n t i n u e d i n t h e l a b o r a t o r y a s a r e s e a r c h s t u d e n t under P i r i e ' s s u p e r v i s i o n , and h e s u g g e s t e d t h a t I s h o u l d make a chemical s t u d y o f t h e g l y c o p r o t e i n s , a c l a s s o f s u b s t a n c e s t h e n o b s c u r e i n c h e m i c a l n a t u r e and o f g r e a t p h y s i o l o g i c a l i n t e r e s t . Soon I found t h a t my knowledge b o t h o f c a r b o h y d r a t e and p r o t e i n c h e m i s t r y w a s i n a d e q u a t e t o t h e t a s k , and began some s y n t h e t i c work w i t h D . J . B e l l which i n v o l v e d p a r t i a l l y s u b s t i t u t e d d e r i v a t i v e s o f g l u c o s e . Among many u s e f u l t h i n g s , I l e a r n t from D r . B e l l t h e power o f l i q u i d - l i q u i d e x t r a c t i o n , w i t h and w i t h o u t s a l t i n g o u t , f o r separ a t i n g methylated sugars according t o t h e e x t e n t of t h e i r methylation. I r e t u r n e d t o work on g l y c o p r o t e i n s w i t h t h e i d e a o f s t u d y i n g them by m e t h y l a t i o n . T h i s b r o u g h t m e i n t o c o n t a c t w i t h A. Neuberger who was t h e n working i n P r o f e s s o r C.R. H a r r i n g t o n ' s l a b o r a t o r y a t Univ e r s i t y C o l l e g e H o s p i t a l , London. D r . Neuberger had developed an i n g e n i o u s method f o r i s o l a t i n g t h e c a r b o h y d r a t e moiety from d i g e s t e d egg albumin by N - a c e t y l a t i n g t h e d i g e s t s and t h e n removing N-acetyl amino a c i d s and p e p t i d e s by e x h a u s t i v e e x t r a c t i o n w i t h c h l o r o f o r m . H e t h e n 0 - a c e t y l a t e d t h e r e s i d u e , whereupon t h e a c e t y l a t e d carboh y d r a t e s became e x t r a c t a b l e . I s h o u l d mention t h a t my f i r s t s i g h t o f a chromatogram was i n Cambridge, i n a b o u t 1937. I t was a v e r y b e a u t i f u l o n e , made by E r n e s t

( I ) as w e l l a s on a l e c t u r e p r e s e n t e d a t a Symposium o r g a n i z e d i n 1969 by t h e Biochemical S o c i e t y ( 2 ) .

+t This c o n t r i b u t i o n is p a r t l y b a s e d on t h e 1952 Nobel L e c t u r e

449 Baldwin. H e a l r e a d y had a b e a r d i n t h o s e d a y s , and was known a s "Biochemical L a b o r a t o r y C h r i s t . " The chromatogram was o b t a i n e d from an e x t r a c t o f s e a - u r c h i n pigments. Everybody s t a o d around and goggled a t t h e p r e t t y c o l o r s b u t no e x p l a n a t i o n was forthcoming from anyone p r e s e n t f o r how t h e undoubted s e p a r a t i o n s had come a b o u t . I n 1937, H . R . Marston, d i r e c t o r of t h e CSIRO N u t r i t i o n L a b o r a t o r y a t A d e l a i d e , A u s t r a l i a , came t o o u r l a b o r a t o r y f o r a y e a r a s a g u e s t . H e was g i v e n bench s p a c e i n t h e room where P i r i e and I w e r e working. H e b r o u g h t w i t h him a p p a r a t u s much more c o m p l i c a t e d t h a n most workers i n t h a t l a b o r a t o r y were accustomed t o , i n c l u d i n g an " a r t i f i c i a l rumen" f o r m i c r o b i a l d i g e s t i o n o f c e l l u l o s e and a l o n g f r a c t i o n a t i n g column f o r d i s t i l l i n g t h e esters o f t h e r e s u l t i n g f a t t y a c i d s . D r . Marston was an a d v i s e r t o t h e I n t e r n a t i o n a l Wool S e c r e t a r i a t . T h i s was g i v e n funds by t h e wool growers o f A u s t r a l i a , New Zealand and South A f r i c a f o r t h e p u r p o s e s o f p u b l i c i t y and r e s e a r c h . D r . M a r s t o n ' s a d v i c e was t o apply some o f t h e i r money t o fundamental s t u d i e s o f t h e n a t u r e o f wool and he s u g g e s t e d t h a t p a r t s h o u l d be g i v e n t o m e a s a S t u d e n t s h i p t o s t u d y i n d e t a i l t h e amino a c i d comp o s i t i o n o f wool, b e g i n n i n g by improving t h e methods o f amino a c i d a n a l y s i s . " I f you work s t e a d i l y a t t h a t f o r f i v e y e a r s , you w i l l r e v o l u t i o n i z e t h e whole o f p r o t e i n c h e m i s t r y " , he s a i d . The S t u d e n t s h i p was on u n u s u a l l y generous f i n a n c i a l terms, and a s I a l s o t h o u g h t i t would f i t w i t h a c q u i r i n g a more d e t a i l e d knowledge o f t h e p r o t e i n s i d e o f t h e g l y c o p r o t e i n problem, I r e a d i l y a g r e e d . I began work i n 1938, by s t u d y i n g t h e d i s t r i b u t i o n o f a c e t y l amino a c i d s between c h l o r o f o r m and w a t e r p h a s e s a s a p o s s i b l e a n a l y t i c a l p r o c e d u r e , d i r e c t l y s u g g e s t e d by N e u b e r g e r ' s e x p e r i m e n t s . The p a r t i t i o n c o e f f i c i e n t s showed v e r y e n c o u r a g i n g d i f f e r e n c e s and t h u s , t h e n e x t s t e p would have been t o c a r r y o u t t h e s e p a r a t i o n b y . l i q u i d l i q u i d p a r t i t i o n . I was a d v i s e d t o g e t i n touch w i t h " N u t r i t i o n L a b o r a t o r y C h r i s t " , as A.J.P. M a r t i n was t h e n known ( h e had j u s t s t o p p e d wearing a b e a r d ) . D r . Martin was unacademically f a m i l i a r w i t h chemical e n g i n e e r i n g and had become n o t o r i o u s a t Cambridge f o r h i s e l a b o r a t e g l a s s a p p a r a t u s i n t h e e n t r a n c e h a l l o f t h e Dunn N u t r i t i o n a l L a b o r a t o r y . T h i s was f o r " s t e a d y - s t a t e " l i q u i d - l i q u i d c o u n t e r c u r r e n t d i s t r i b u t i o n w i t h a view t o c o n c e n t r a t i n g Vitamin E from v a r i o u s e x t r a c t s . U n f o r t u n a t e l y M a r t i n ' s machine was n o t s u i t a b l e f o r chloroform and w a t e r . H e i n t r o d u c e d m e t o s i m p l e c o u n t e r c u r r e n t t h e o r y and t o g e t h e r w e b u i l t a c o n t i n u o u s - f l o w , " s t e a d y - s t a t e " , 4 0 - p l a t e c o u n t e r c u r r e n t machine w i t h c e n t r a l f e e d , f o r h a n d l i n g chloroform-water p h a s e s ( 3 ) . B e f o r e t h e machine was f i n i s h e d , M a r t i n moved t o t h e Wool I n d u s t r i e s Research L a b o r a t o r i e s , a t Leeds. S i r C h a r l e s M a r t i n , who s e r v e d a s a s c i e n t i f i c a d v i s e r t o t h e I n t e r n a t i o n a l Wool S e c r e t a r i a t , h e l p e d t o have my S t u d e n t s h i p t r a n s f e r r e d t o Lbeds so t h a t I c o u l d j o i n Archer M a r t i n . Thus, I moved t h e r e w i t h t h e new machine comp l e t e d a t Cambridge. Archer, i n h i s own s t o r y , d i s c u s s e d o u r c o l l a b o r a t i v e work a t Leeds and how l i q u i d - l i q u i d chromatography was developed, and i t is unnecessary t o r e p e a t i t h e r e . R a t h e r , I w i l l make a few a d d i t i o n s .

While s t i l l working w i t h o u r p a r t i t i o n machine, w e a l s o had o c c a s i o n t o chromatograph d i n i t r o p h e n y l h y d r a z o n e s o f a l d e h y d e s on magnesium o x i d e , and Archer w a s always e x p l i c i t about a p p l y i n g c o u n t e r c u r r e n t t h e o r y t o t h e chromatogram. I remember t h a t , about t h a t t i m e , o u r chloroform-water machine was p l a y i n g up b a d l y , and w e d i s c u s s e d changing i t o v e r so a s t o have one o n l y p h a s e on t h e move, and t o p u t i n t h e c h a r g e b a t c h w i s e a t t h e p o i n t o f e n t r y o f t h e moving p h a s e "as i f i t w e r e a chromatogram". I a m s u r e i t was t h i s v e r b a l t w i s t t h a t prompted u s t o go on t o l i q u i d - l i q u i d chromatograms. I t was when t h e s e had been a c h i e v e d , and a n a l y z e d on t h e b a s i s of c o n v e n t i o n a l c o u n t e r c u r r e n t t h e o r y ( 4 ) t h a t i t became m a n i f e s t how t h e s p e c i a l s e p a r a t o r y v i r t u e o f t h e chromatogram r e s i d e s i n i t s v e r y s m a l l HETP. Although w e were t h e f i r s t t o d e m o n s t r a t e t o t h e Biochemical S o c i e t y ( 5 ) e x p e r i m e n t s i n l i q u i d - l i q u i d chromatography, p r o b a b l y i t would o n l y have been a s h o r t w h i l e b e f o r e o t h e r s d i d so - b o t h Van D i j c k i n t h e N e t h e r l a n d s (see K l i n k e n b e r g ( 6 ) ) and S t e n e i n Norway ( 7 ) were a c t i v e l y e x p e r i m e n t i n g on t h e i d e a . I a g r e e w i t h Archer M a r t i n about t h e r e a s o n why w e have p r e c e d e d them: t h e y had no problems t h e y w e r e immediately a b l e t o s o l v e w i t h t h e a i d t o t h e new t e c h n i q u e w h i l e w e had a problem and w e r e l o o k i n g f o r t h e p r o p e r method t o s o l v e i t ( 8 ) . P a r t i t i o n chromatography is o f g r e a t i m p o r t a n c e f o r a s c e r t a i n i n g t h e sequence o f amino a c i d r e s i d u e s i n t h e p e p t i d e - c h a i n s o f p r o t e i n s . Archer M a r t i n and I had t h i s u s e p a r t i c u l a r l y i n mind t h r o u g h o u t o u r work. I f a p e p t i d e c h a i n i s p a r t i a l l y degraded t o d i p e p t i d e and t r i p e p t i d e f r a g m e n t s , e t c . , i t s h o u l d be p o s s i b l e , by i d e n t i f y i n g t h e s e , t o r e c o g n i z e t h e o r i g i n a l compound from which t h e y a r e der.ived. Martin and I , w i t h Consden and Gordon, were a b l e i n t h i s way, mainly u s i n g p a r t i t i o n chromatographic methods, t o d e t e r m i n e t h e amino a c i d sequence i n g r a m i c i d i n - S ( 9 , l O ) . S u b s e q u e n t l y Sanger and c o l l e a g u e s (11,12) e l u c i d a t e d by s i m i l a r methods t h e e n t i r e p e p t i d e sequences i n t h e s t r u c t u r e of ox i n s u l i n s . Although i t was p o p u l a r i z e d i n c o n n e c t i o n w i t h l i q u i d - l i q u i d chromatography, p a p e r chromatography ( 1 3 , 1 4 ) i s i n no way n e c e s s a r i l y l i n k e d t o i t . The i d e a o f changing o v e r from " c a p i l l a r y a n a l y s i s " t o e l u t i o n development on p a p e r was f i r s t p u t forward by T s w e t t (25) and l a t e r by Schwab and J o c k e r s ( 1 6 ) and t h e two-dimensional a r r a n g e ment was adopted by Liesegang ( 1 7 ) . The i n t r o d u c t i o n o f t h i n - l a y e r chromatography e n a b l e d a v a s t l y g r e a t e r range o f chromatographic systems t o p a r t a k e of t h e a d v a n t a g e s o f p a p e r chromatography, a s w e l l a s o f t e n d o i n g b e t t e r some o f t h e t h i n g s t h a t c o u l d a l r e a d y be done on p a p e r . I t h a s been most e x c i t i n g t o have t a k e n p a r t i n s c i e n t i f i c s t u d y i n b i o c h e m i s t r y and n e i g h b o u r i n g f i e l d s r i g h t t h r o u g h t h e " n o n - l a t e n t " p e r i o d o f chromatography, t h a t is s i n c e 1931. Chromatography, a s a group o f methods, h a s i n d e e d been a most f a i t h f u l s e r v a n t (sometimes more l i k e a genie o u t o f t h e Arabian N i g h t s ) b o t h f o r r e v e a l i n g and i s o l a t i n g new s u b s t a n c e s and f o r a n a l y z i n g f o r known o n e s . Seventyf i v e y e a r s a f t e r i t s i n c e p t i o n by T s w e t t , i t is now t h e most widely used a n a l y t i c a l t e c h n i q u e . W e do n o t know what l i e s ahead i n t h e

451 f u t u r e and p r o b a b l y , new methods w i l l e v e n t u a l l y r e p l a c e chromatog r a p h y . However, one t h i n g i s s u r e : chromatography w i l l b e remembered f o r i t s c o n t r i b u t i o n t o s c i e n c e l o n g a f t e r i t i s q u i t e o b s o l e t e as an a n a l y t i c a l method. REFERENCES Synge, Applications of P a r t i t i o n Chromatography, i n Nobel Lectures - Chemistry 1942-1962, E l s e v i e r , Amsterdam, 1 9 6 4 , pp. 374-387 2 R.L.M. Synge, i n B r i t i s h Biochemistry Past and Present (Biochem. 1 R.L.M.

SOC. Symposium N 0 . 3 0 ) ~ T.W. Goodwin, e d . , Academic P r e s s , London, 1970, pp. 175-182. 3 A . J . P . M a r t i n and R.L.M. Synge, Biochem. J . 35 (1941) 9 1 . 4 A . J . P . M a r t i n and R.L.M. Synge, Biochem. J . 35 (1941) 1358. 5 A . J . P . M a r t i n and R.L.M. Synge, Chem. Ind. 19 (1941) 487; Meeting

of t h e Biochemical Society a t t h e National I n s t i t u t e f o r Medical Research, London, June 7 , 1941. 6 A . K l i n k e n b e r g , Disc. Faraday Soc. 7 (1949) 151. 7 S . S t e n e , A r k . Kemi Mineral. Geol. 18A ( 1 9 4 4 ) . 8 A . J . P . M a r t i n , The Development of P a r t i t i o n Chromatography, i n Nobel Lectures - Chemistry 1942-1962, E l s e v i e r , Amsterdam, 1964, pp. 359-371. 9 R.L.M. Synge, Biochem. J . 39 (1945) 363. 10 R . Consden, A . H . Gordon, A . J . P . M a r t i n and R.L.M. Synge, Biochem. J. 4 1 (1947) 596. 11 F. S a n g e r , Biochem. J. 39 (1945) 507. 12 H . Brown, F. S a n g e r and R . K i t a i , Biochem. J . 60 (1955) 556. 13 A . H . Gordon, A . J . P . M a r t i n and R.L.M. Synge, Biochem. J . 37 (1943) P r o c . XI11 1 4 R . Consden, A . H . Gordon and A.J.P. M a r t i n , Biochem. J . 38 (1944) 224. 1 5 M.S. T s v e t , KhromofilZy v RastitZ'nom i Zhivotnom Mire (Chromop h y l l s i n t h e P l a n t and Animal Kingdom), Warsaw, 1910. The book w a s p a r t i a l l y r e p r i n t e d i n 1946: A . A . R i k h t e r and T.A. Krasnosel ' s k a y a ( e d s ) , M. S . Tsvet - Khromatograficheskii Adsorptsionnyi AnaZiz - Izbrannye Raboty, Izd. Akad. Nauk S . S . S . R . , Moscow, 1946; see p . 164. 16 G.-M. Schwab and K . J o c k e r s , Angew. Chem. 50 (1937) 546. 17 R . E . L i e s e g a n g , 2. Anal. Chem. 126 (1943) 1 7 2 , 334; N a t u m i s s . 31 (1943) 348.

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ROY'TERANISHI

ROY TERANISHI was born i n 1922, i n Stockt o n , C a l i f o r n i a , During World War 11, he w a s i n t e r n e d i n A r i z o n a . A f t e r two semesters a t Colorado S t a t e U n i v e r s i t y , F o r t C o l l i n s , C o l o r a d o , he was d r a f t e d , and he s e r v e d i n t h e U.S. Army from 1945 t o 1947. H e r e c e i v e d h i s B.S. d e g r e e i n Chemistry i n 1950 from t h e U n i v e r s i t y of C a l i f o r n i a , B e r k e l e y , and h i s Ph. D. i n O r g a n i c Chemistry from Oregon S t a t e U n i v e r s i t y , C o r n v a l l i s . H e h a s worked a t t h e U.S. Department o f A g r i c u l t u r e , Weste r n R e g i o n a l R e s e a r c h C e n t e r , Albany , C a l i f o r n i a , as a r e s e a r c h c h e m i s t s i n c e 1954. D r . T e r a n i s h i i s t h e a u t h o r and c o a u t h o r o f o v e r 70 s c i e n t i f i c p u b l i c a t i o n s , which a r e mainly on g a s chromatography and f l a v o r chemistry. H e , with Hornstein, Issenberg and Wick, p u b l i s h e d a book on Flavor Research: Principles and Techniques, i n which a p p l i c a t i o n s o f g a s chromatography i n f l a v o r r e s e a r c h are p r e s e n t e d . H e h a s s e r v e d i n 1969 a s t h e Chairman o f t h e F l a v o r S u b d i v i s i o n o f t h e D i v i s i o n o f A g r i c u l t u r a l and Food C h e m i s t r y , American Chemical S o c i e t y and i n 1976 as t h e chairman of t h i s ACS D i v i s i o n . I n 1967, he r e c e i v e d - t o g e t h e r w i t h R . B u t t e r y - t h e S u p e r i o r S e r v i c e Award o f t h e U.S. Department o f A g r i c u l t u r e . D r . T e r a n i s h i ' s work i n g a s chromatography began i n 1958. H e p i o n e e r e d i n t h e a p p l i c a t i o n o f h i g h - r e s o l u t i o n g a s chromatography t o t h e a n a l y s i s o f f l a v o r compounds. H i s c o n t r i b u t i o n s i n c l u d e programmed-tempera t u r e a n a l y s i s w i t h o p e n - t u b u l a r columns, u s e of s u c h columns w i t h mass s p e c t r o m e t e r s , t h e d i r e c t vapor a n a l y s i s of food and a p p l i c a t i o n s of l a r g e - b o r e open t u b u l a r columns.

My i n t r o d u c t i o n t o g a s chromatography came when I r e p l a c e d Keene P . Dimick i n J o e C o r s e ' s F r u i t F l a v o r s I n v e s t i g a t i o n s Group i n 1958. Keene had j u s t l e f t government s e r v i c e t o s t a r t a g a s chromatography company. W e a l l l a u g h e d and s a i d t h a t Keene would be back soon a g a i n i n government s e r v i c e b e c a u s e he had t r i e d once b e f o r e t o s t a r t a company and had f a i l e d . Keene had t h e l a s t l a u g h ; a s everyone knows h i s very s u c c e s s f u l Aerograph Company was s o l d t o Varian f o r m i l l i o n s . Among t h e r e c o r d s I i n h e r i t e d was a diagram o f t h e w h e a t s t o n e b r i d g e , used i n t h e f i r s t g a s chromatographs b u i t a t t h e Western Regional Research C e n t e r , Albany, C a l i f o r n i a . Keene may have f o r g o t t e n t h i s diagram b u t I k e p t i t a s an h i s t o r i c a l memento of t h e b e g i n n i n g of t h e f a b u l o u s Dimick f i n a n c i a l s u c c e s s s t o r y . J o e Corse r e c a l l s t h e u n s e l f i s h h e l p r e c e i v e d from many f r i e n d s a t S h e l l Development Company t h a t was a t E m e r y v i l l e , C a l i f o r n i a . I n t h e 1 9 5 0 ' 9 , o u r b e s t l i n k t o t h e s c i e n t i f i c community engaged i n t h e e x c i t i n g b e g i n n i n g o f g a s chromatography was o u r f r i e n d s a t S h e l l , who were making fundamental c o n t r i b u t i o n s and a l s o who were i n d i r e c t c o n t a c t w i t h D e s t y , Keulemans, M a r t i n , and o t h e r s . One o f t h e many c o n t r i b u t i o n s by t h e S h e l l p e o p l e w a s t h e u s e o f p u l v e r i z e d diatomaceous e a r t h f i r e b r i c k , s o r t e d f o r p a r t i c l e s i z e r a n g e , f o r s o l i d s u p p o r t . The g r e a t power o f g a s chromatography t o s e p a r a t e c l o s e l y r e l a t e d compounds was r e c o g n i z e d e a r l y by p e t r o l e u m c h e m i s t s , b u t J o e Corse r e c o g n i z e d immediately t h e i m p o r t a n c e o f t h i s a n a l y t i c a l method i n f l a v o r c h e m i s t r y t o u n r a v e l t h e complex m i x t u r e s which a r e a v a i l a b l e only i n very small q u a n t i t i e s . Events i n subsequent y e a r s have proved how p e r c e p t i v e J o e was i n 1953. One o f t h e e a r l i e s t a p p l i c a t i o n s o f g a s chromatography was by Dimick and Corse ( 1 ) . I n t h e l a t e f i f t i e s , w e h e a r d o f many e x c i t i n g developments. One of these was of t h e tremendous s e p a r a t i o n s Desty was g e t t i n g w i t h copper c a p i l l a r y columns, t h e c o n c e p t o f c o a t i n g open t u b u l a r w a l l s h a v i n g been i n t r o d u c e d by Golay i n 1957-1958. Because w e d i d n o t want t h e i n t e r a c t i o n s o f v a r i o u s o r g a n i c compounds w i t h c o p p e r , w e bought 1 0 - f o o t s e c t i o n s o f s t a i n l e s s s t e e l hypodermic n e e d l e s t o c k and s i l v e r - s o l d e r e d them t o g e t h e r t o make o u r f i r s t s t a i n l e s s s t e e l , 0 . 0 1 i n c h I . D . , c a p i l l a r y columns. L a t e r , w e were a b l e t o p u r c h a s e such t u b i n g i n one p i e c e up t o s e v e r a l hundred f e e t l o n g . W e improvised s t r e a m s p l i t t e r s t o l i m i t s i z e s t o micrograms o r less o f samples i n t r o d u c e d i n t o t h e s e c a p i l l a r y columns. W e b u i l t a s m a l l volume d e t e c t o r t o f i t i n s i d e t h e s t r o n t i u m - 9 0 argon d e t e c t o r o r i g i n a l l y d e s i g n e d by Lovelock for packed columns. The s m a l l mass o f t h e s e c a p i l l a r y columns wound on s m a l l s p o o l and h e a t e r u n i t s made i t e a s y t o program t h e t e m p e r a t u r e o f t h e s e columns. What a t h r i l l i t was t o o b t a i n more t h a n a hundred p e a k s when w e f i r s t a n a l y z e d s t r a w b e r r y o i l ( 2 ) , w i t h s u c h columns: I t was even a g r e a t e r t h r i l l t o be a b l e t o r e p e a t s u c h a n a l y s e s w i t h many o t h e r complex f l a v o r e x t r a c t s . I n t h o s e d a y s , most commercially a v a i l a b l e g a s chromatographs were equipped o n l y w i t h s i n g l e , packed columns, i s o t h e r m a l l y c o n t r o l l e d , and o n l y w i t h t h e r m a l c o n d u c t i v i t y d e t e c t o r s . T h e r e f o r e , w e had t o d e s i g n and b u i l d o u r f i r s t i n s t r u m e n t s which w e used f o r o u r e a r l y

455

Fig. 53.1. Original Wheatstone-bridge used by K.P. Dimick in the first gas chromatographs built at the Western Regional Research Center.

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F i g . 53.2. GC s e p a r a t i o n o f s t r a w b e r r y o i l , w i t h a 240 f t . x 0.01 i n . 1 . D . s t a i n l e s s s t e e l c a p i l l a r y column, c o a t e d w i t h TWEEN-20 and t e m p e r a t u r e programmed from 80 t o 225OC. Sample s i z e : 50 ug. work on c a p i l l a r y columns used w i t h t e m p e r a t u r e programming. S e v e r a l y e a r s a f t e r w e c o n s t r u c t e d t h i s i n s t r u m e n t , t h e e l e g a n t Perkin-Elmer Model 226 became commercially a v a i l a b l e . As soon a s w e r e a l i z e d t h e g o a l o f s e p a r a t i n g and d e t e c t i n g hundreds od compounds p e r s i n g l e r u n , w e t u r n e d o u r a t t e n t i o n t o t h e t a s k o f i d e n t i f y i n g t h e microgram o r less q u a n t i t i e s o f m a t e r i a l s s e p a r a t e d by t h e s e c a p i l l a r y columns. R . C . Gohlke, i n 1959, had shown t h e a d v a n t a g e o f c o u p l i n g t h e t i m e - o f - l i g h t mass s p e c t r o m e t e r t o a g a s chromatograph. B i l l McFadden and I w e r e a b l e t o c o u l p e t h e 0 . 0 1 - i n c h I . D . c a p i l l a r y column t o t h e t i m e - o f - f l i g h t mass s p e c t r o m e t e r , and w e w e r e a b l e t o a p p l y t h i s p o w e r f u l c o m b i n a t i o n t o i d e n t i f y compounds a s t h e y were e l u t e d from t h e programmed-temperat u r e c a p i l l a r y columns (
457

F i g . 53.3. Gas chromatograph b u i l t a t Western Regional Research C e n t e r i n 1959. Note t h e 1 - i n . t h i c k w a l l l e a d box h o u s i n g t h e "Sr argon det e c t o r . The 1 - i n . t h i c k l e a d t o p h a s been removed t o show t h e microvolume d e t e c t o r going i n s i d e t h e u s u a l l a r g e volume "Sr argon d e t e c t o r . I n t h i s p i c t u r e , t h e two v a l v e d s t r e a m s p l i t t e r i n j e c t o r s and t h e two c a p i l l a r y columns wound on aluminium s p o o l s and c o v e r e d w i t h a s b e s t o s c o r d a r e shown. Temperature o f columns, i n j e c t o r s , and d e t e c t o r was c o n t r o l l e d by v a r i a b l e t r a n s f o r m e r s which a r e s i t u a t e d below t h e column and detector area. o f t h e danger o f r a d i a t i o n from "Sr, and because o f t h e s a f e t y , s i m p l i c i t y , r u g g e d n e s s , and h i g h s e n s i t i v i t y t o o r g a n i c compounds b u t n o t t o water and oxygen, e t c . , o f t h e FID, w e soon abandoned t h e "Sr d e t e c t o r and now u s e t h e FID e x c l u s i v e l y , e x c e p t f o r p r e p a r a t i v e work, i n which t h e r m a l c o n d u c t i v i t y d e t e c t o r s are used. The e l e g a n t l y s i m p l e d e s i g n o f t h e McWilliam and Dewar FID system was most welcome, e s p e c i a l l y a f t e r t r y i n g t o work w i t h "touchy" e a r l y FIDs. I can remember Ron B u t t e r y and myself s t a n d i n g i n f r o n t o f h i s f i r s t FID i n s t r u m e n t , and b e c a u s e t h e d e t e c t o r 'was exposed, w e c o u l d make t h e r e c o r d e r pen swing o f f - s c a l e simply by waving o u r

458

F i g . 5 3 . 4 . E x t e r i o r o f t h e g a s chromatograph shown i n F i g . 53.3. D i f f e r e n t stream s p l i t t i n g r a t i o s were o b t a i n e d by p l a c i n g hypodermic n e e d l e s o n t o f i t t i n g . The electrometer w a s housed i n s e c t i o n below t h e v a r i a b l e t r a n s f o r m e r s . Temperatures of v a r i o u s p a r t s o f t h e chromatograph w e r e d e t e r m i n e d by u s e o f m u l t i - p o s i t i o n s w i t c h and by r e a d i n g t h e thermocouple v o l t a g e s . Chromatograms were o b t a i n e d w i t h t h e slow, l o w impedance r e c o r d i n g p o t e n t i o meter a v a i l a b l e i n t h o s e d a y s . arms. Also, t h e i n s t r u m e n t r e c o r d e d e v e r y s t r o k e o f a comb t h r o u g h a head of h a i r o f a p e r s o n anywhere i n t h e same room. Before we shock-mounted o u r i n s t r u m e n t s , w e r e c o r d e d e v e r y slam o f drawers o r d o o r s i n t h e room. W e even event-marked e v e r y door slam o f walk-in r e f r i g e r a t o r s s i t u a t e d a f l o o r below u s u n t i l w e i n s e r t e d s o f t r u b b e r s t o p p e r s under o u r i n s t r u m e n t s . T o p i c s l i k e improvement o f s i g n a l - t o - n o i s e r a t i o , s h i e l d i n g , r e d u c i n g e l e c t r i c a l n o i s e by r e d u c i n g v i b r a t i o n o f e l e c t r o d e s i n t h e vacuum t u b e s , p i e z o e l e c t r i c e f f e c t from b e n d i n g w i r e s , p i c k u p o f e l e c t r i c a l n o i s e from l o n g l e a d s , e t c . , t o p i c s r e l a t i v e l y u n f a m i l i a r t o o r g a n i c c h e m i s t s , w e r e d i s c u s s e d w i t h Ron B u t t e r y and Bob Lundin o v e r many cups of c o f f e e . Drawings on n a p k i n s w e r e re-drawn, n o t much b e t t e r t h a n t h o s e on t h e n a p k i n s a c c o r d i n g t o o u r m a c h i n i s t s ,

and s u b m i t t e d t o t h e machine shop. A few days l a t e r , o u r i d e a s would be e a g e r l y checked o u t i n t h e l a b o r a t o r y . Although some o f t h e e l e c t r o m e t e r t u b e s have been r e p l a c e d w i t h s o l i d s t a t e e l e c t r o m e t e r s , and t h e c o l l e c t o r e l e c t r o d e s have been exchanged f o r l a r g e r o n e s t h a n t h e very f i r s t o n e s u s e d , some of t h e o r i g i n a l FIDs a r e s t i l l i n o p e r a t i o n a t t h e Western Regional Research C e n t e r a f t e r about 15 y e a r s o f use. A c t u a l l y , t h e b a t t e r y - o p e r a t e d systems a r e s t i l l more s t a b l e t h a n t h e s o l i d s t a t e , l i n e - o p e r a t e d systems. Direct vapor a n a l y s e s have proved u s e f u l i n f l a v o r c h e m i s t r y , e s p e c i a l l y i n f o l l o w i n g development o f r a n c i d i t y d u r i n g s t o r a g e o f v a r i o u s food p r o d u c t s , r i p e n i n g o f f r u i t s , d i f f e r e n t i a t i n g t h e v a r i e t i e s o f f r u i t s and v e g e t a b l e s , e t c . Ron B u t t e r y and I r e c e i v e d t h e U.S. Department o f A g r i c u l t u r e S u p e r i o r S e r v i c e Award i n 1967 f o r o u r work i n d i r e c t vapor a n a l y s e s o f food p r o d u c t s and o t h e r a p p l i c a t i o n s o f gas chromatography i n f l a v o r c h e m i s t r y . The f i r s t d i r e c t vapor a n a l y s e s w e r e w i t h packed columns. Attempts t o u s e t h e 0.01-inch I . D . c a p i l l a r y columns d i d n o t succeed very w e l l because o f t h e s m a l l flow t h r o u g h t h e s e columns. The d r i v e t o have as much r e s o l u t i o n w i t h vapor a n a l y s e s a s w i t h l i q u i d i n j e c t i o n s i n t o t h e 0.01-inch I.D. c a p i l l a r y columns made us i n v e s t i g a t e t h e p o s s i b i l i t i e s o f t h e l a r g e b o r e open t u b u l a r columns ( 5 ) . Dick Mon j o i n e d o u r l i t t l e group i n t h e e a r l y s i x t i e s , and he soon became o u r e x p e r t open t u b u l a r column maker. Through h i s e f f o r t s , t h e 0.03-inch I . D . columns, 500- and 1000-feet l o n g , were developed and became s t a n d a r d equipment i n o u r l a b o r a t o r y . These columns have n e v e r become commercial i t e m s , b u t w e have found t h e s e columns a b s o l u t e l y e s s e n t i a l f o r o u r f l a v o r r e s e a r c h work, p e r m i t t i n g t h e h i g h - r e s o l u t i o n chromatographic a n a l y s i s o f d i r e c t vapor samples. These l a r g e b o r e open t u b u l a r columns have proved t o b e v e r y u s e f u l i n GC-MS a n a l y s e s , a l s o . The l a r g e sample s i z e t o l e r a n c e p e r m i t s a w i d e r range o f amounts t o be a n a l y z e d p e r r u n t h a n w i t h t h e v e r y s m a l l sample s i z e t o l e r a t e d by t h e 0.01-inch I.D. columns. T h i s c a p a b i l i t y is v e r y i m p o r t a n t i n f l a v o r c h e m i s t r y because o f t h e extreme sample s i z e l i m i t a t i o n s , which n e c e s s i t a t e o b t a i n i n g a s much i n f o r m a t i o n a s p o s s i b l e p e r r u n . These l a r g e b o r e open t u b u l a r columns are now r o u t i n e l y used f o r d i r e c t vapor a n a l y s e s , GC-MS a n a l y s e s , and even f o r p r e p a r a t i v e work. For such work, non-destruct i v e t h e r m i s t o r d e t e c t o r s a r e u s e d , and v e r y c l o s e l y r e l a t e d compounds can be p u r i f i e d : compounds which c a n n o t b e s e p a r a t e d w i t h packed columns. Thus, r e t e n t i o n d a t a from a l l t h r e e t y p e s o f s e p a r a t i o n s d i r e c t v a p o r s , GC-MS, and p r e p - a r e now e a s i l y c o r r e l a t e d w i t h s i m i l a r o r i d e n t i c a l columns. I n t h e 1 9 6 0 ' s Bob F l a t h j o i n e d o u r r e s e a r c h team. Bob d e v i s e d a micro c e l l f o r n u c l e a r magnetic resonance a n a l y s e s o f samples p u r i f i e d by t h e l a r g e b o r e open t u b u l a r columns. H e d e s i g n e d and c o n s t r u c t e d a membrane s e p a r a t o r i n t e r f a c e between t h e l a r g e b o r e open t u b u l a r columns and mass s p e c t r o m e t e r s . Our s e p a r a t o r s have been i n o p e r a t i o n f o r about t e n y e a r s w i t h v e r y l i t t l e down t i m e . Bob d e v i s e d t h e vapor sampling systems u t i l i z i n g C a r l e v a l v e s and o b t a i n e d h i g h r e s o l u t i o n s e p a r a t i o n s o f v o l a t i l e s from v a r i o u s food products.

460

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Fig. 53.5. High-resolution vapor analysis of red delicious apple, with a 900-ft. x 0.03 in. I.D. stainless steel open tubular columns coated with SF-96(50) methylsilicone oil. Sample of 20 ml vapors was trapped, then introduced into the column by a Carle switching valve system. I look back to the early years of the development of gas chromatography and recall many happy moments shared with those mentioned, and also with others too numerous to mention. It was great to live in an era in which a new idea was being developed and applied to break open an area of research, flavor research in our case. The late 1960's and 1970's were wonderful years in which many interesting applications of gas chromatography were made. It has been an interesting and satisfying experience to have been a small part of this exciting development.

REFERENCES 1 K.P. Dimick and J. Corse, Food TechnoZ. 10 (1956) 360. J. Corse, Anal. Chem. 32 (1960) 1384. 3 W.H. McFadden, R. Teranishi, D.R. Black and J.C. Day, J . Food Sci. 28 (1963) 316. 4 R. Teranishi, R.C. Buttery and R.E. Lundin, Anal. Chem. 34 (1962) 1033. 5 R. Teranishi and R.R. Mon, Anal. Chem. 36 (1964) 1490. 2 R. Teranishi, C.C. Nimmo and

461

J.J. VAN DEEMTER

J A N JOSEF VAN DEEMTER was born i n 1918, i n

K e l l e n , Germany ( n e a r t h e Dutch b o r d e r ) o f Dutch p a r e n t s . H e f i r s t s t u d i e d a t t h e Univ e r s i t y o f Groningen, t h e N e t h e r l a n d s , r e c e i v i n g a d o c t o r a t e i n p h y s i c s i n 1945. After short periods a t Philips physical l a b o r a t o r y , i n Eindhoven, and t h e I n s t i t u t e f o r S o i l R e s e a r c h , i n Groningen, he j o i n e d t h e Koninklijke/Shell-Laboratorium, i n Amsterdam (KSLA, The Royal Dutch/Shell l a b o r a t o r y ) i n 1947. H i s work a t t h e S o i l Research I n s t i t u t e led t o a doctor's t i t l e i n a p p l i e d mathematics a t t h e Municipal U n i v e r s i t y of Amsterdam. A t S h e l l he s t a r t e d i n t h e T h e o r e t i c a l Department and t r a n s f e r r e d t o t h e Chemical E n g i n e e r i n g Department f o u r y e a r s l a t e r . From 1955 t o 1957 he had an assignment a t t h e S h e l l O i l Company i n Houston, Texas (U.S.A.). A f t e r r e t u r n i n g t o KSLA he became department head, s u c c e s s i v e l y , o f o i l p r o c e s s development and o f bitumen r e s e a r c h . I n 1968-1969 he f i l l e d t h e p o s t of department head Chemical E n g i n e e r i n g Research i n t h e E m e r y v i l l e l a b o r a t o r y o f S h e l l Development Company ( C a l i f o r n i a , U.S.A.). I n 1969 he Became d i r e c t o r o f p r o d u c t i o n . r e s e a r c h a t t h e S h e l l Exp l o r a t i o n and P r o d u c t i o n L a b o r a t o r y , i n R i j s w i j k , t h e Net-herlands; and i n 1972 d i r e c t o r o f e n g i n e e r i n g s c i e n c e s a t KSLA. I n 1978 he r e t i r e d from S h e l l . D r . Van D e e m t e r i s t h e a u t h o r and c o a u t h o r o f some 25 p a p e r s and rep o r t s i n a p p l i e d mathematics and mechanics, f l u i d f l o w , r e a c t o r e n g i n e e r i n g and chromatography. D r . Van Deemter's e a r l i e r i n t e r e s t was mainly i n chemical e n g i n e e r i n g r e s e a r c h b u t d u r i n g t h e y e a r s of 1952-1957 he was a c t i v e i n s t u d i e s o f t h e t h e o r y o f chromatography and r e l a t e d m a t t e r s . I n 1956, t o g e t h e r w i t h M r . Zuiderweg and D r . Klinkenberg he p u b l i s h e d t h e b a s i c p a p e r r e p r e s e n t i n g t h e f o u n d a t i o n o f t h e r e l a t i o n s h i p between column e f f i c i e n c y and v a r i o u s o p e r a t i o n a l p a r a m e t e r s .

462 I n 1949 F . van de C r a a t s , who w a s head o f t h e gas a n a l y s i s group i n t h e S h e l l l a b o r a t o r i e s i n Amsterdam, remembered from o l d l i t e r a t u r e a s e p a r a t i o n p r o c e d u r e i n which a series o f s m a l l g a s a b s o r p t i o n vessels were f i r s t p a r t l y l o a d e d w i t h t h e g a s m i x t u r e t o be a n a l y s e d and t h e n s t r i p p e d by i n e r t g a s t o o b t a i n s e p a r a t i o n o f components. H e mentioned t h e i d e a t o F . J . Zuiderweg, a t t h a t t i m e head of d i s t i l l a t i o n r e s e a r c h . Z u i d e m e g , w i t h t h e h e l p o f t h e Theor e t i c a l Department, worked o u t a t h e o r y which can b e found i n r e f . 1 ( p . 110). T h i s s e p a r a t i o n p r o c e d u r e i s s o c l o s e l y r e l a t e d t o g a s chromatography t h a t i t i s s t i l l amazing t h a t none o f t h e S h e l l e x p e r t s came upon t h e i d e a o f f i x i n g t h e a b s o r p t i o n s o l v e n t i n a column r a t h e r t h a n f i l l i n g a series of b o t t l e s . I myself was working i n t h e T h e o r e t i c a l Department and d i d n o t even pay a t t e n t i o n t o what my c o l l e a g u e H . A . Lauwerier was d o i n g f o r my good f r i e n d Zuiderweg. L a t e r , by t h e end o f 1952, t h e b a s i c work o f M a r t i n and James w a s a p p r e c i a t e d i n a number o f r e s e a r c h d e p a r t m e n t s a t t h e K o n i n k l i j k e / S h e l l L a b o r a t o r i u m , i n Amsterdam. Names t h a t came t o my mind i n t h i s c o n n e c t i o n a r e : H . Boer, F. van de C r a a t s , A.I.M. Keulemans, A . Kwantes and G . W . A . R i j n d e r s . Keulemans i n p a r t i c u l a r f o l l o w e d up M a r t i n ' s work w i t h g r e a t v i g o u r . H i s r e s p o n s i b i l i t y a t t h a t t i m e was t h e e x e c u t i o n of e x p l o r a t o r y and b a s i c s t u d i e s r e l a t e d t o p r o c e s s development work. H e had c o n s i d e r a b l e freedom and u s e d i t to p i c k up a n y t h i n g which looked i n t e r e s t i n g t o him. H i s e a r l y e x p e r i m e n t s s t i m u l a t e d many c o l l e a g u e s around him i n c l u d i n g Zuiderweg and m y s e l f . Zuiderweg r e a l i z e d immediately t h a t t h e t h e o r y of t h e s t r i p p i n g o f a b s o r p t i o n v e s s e l s c o u l d b e a p p l i e d t o g a s chromatography and p o i n t e d t o t h e u s e o f t h e s t a g e c o n c e p t (which was l a t e r t o be named: h e i g h t e q u i v a l e n t t o a t h e o r e t i c a l p l a t e , HETP). I n t h e meantime my own i n t e r e s t i n c r e a s e d , mainly b e c a u s e I had t h e f e e l i n g t h a t c e r t a i n chemical e n g i n e e r i n g c o n c e p t s , which had a proven u s e i n packed column p r o c e s s e s , c o u l d p o s s i b l y be a p p l i c a b l e i n g a s chromatography - a l t h o u g h I d i d n o t immediately see any s i m p l e means o f approach. By t h a t t i m e I was working a s head o f a s m a l l f l u i d flow group i n t h e Chemical E n g i n e e r i n g Department. Zuiderweg and I s t a r t e d t o work on chromatography more o r l e s s i n d e p e n d e n t l y . H e c o n s i d e r e d t h e e f f e c t s o f sample s i z e and looked i n t o t h e p o s s i b i l i t i e s o f s c a l i n g up t h e p r o c e d u r e f o r p r e p a r a t i v e s e p a r a t i o n s . I began t o s t u d y t h e l i t e r a t u r e on t h e s u b j e c t and had a look a t t h e v a r i o u s d i f f e r e n t i a l e q u a t i o n s i n v o l v e d i n d e s c r i b i n g t h e p r o c e s s . I n t h e c o u r s e o f t h e s e a c t i v i t i e s I found a Company r e p o r t from 1945 by A . K l i n k e n b e r g which p r e s e n t e d a comprehensive t h e o r y of l i q u i d - l i q u i d chromatography. T h i s t h e o r y c o u l d , a f t e r s l i g h t m o d i f i c a t i o n , a l s o b e a p p l i e d t o g a s - l i q u i d chromatography, a l t h o u g h t h e r e s u l t i n g formulae w e r e s t i l l q u i t e c o m p l i c a t e d . E x a c t l y t h e same t h e o r y had i n t h e meantime a l s o been d e r i v e d by Amundson and Lapidus who p u b l i s h e d i t i n 1952 ( 2 ) . The n e x t s t e p was a comparison of t h e HETP c o n c e p t and t h e rate t h e o r y . T h i s l e d f o r t h e f i r s t t i m e t o t h e u n r a v e l l i n g o f t h e two main n o n - i d e a l i t y f a c t o r s f o r a p r a c t i c a l c a s e . A t t h e same t i m e t h e well-known s i m p l e e q u a t i o n f o r t h e HETP was o b t a i n e d .

463 HEIGHT OF A THEORETICAL STAGE, m m

-

10 8 6-

CO?

/

Nz

4-

2-

--t-HEIGHT

I -

0.6 -

1

STABLISHMENT F EOUlLlBRlUY

/t”

0.8 0.4

OF YI/CIW/pLETE

-

2

F i g . 5 4 . 1 . F i g u r e from an i n t e r n a l S h e l l r e p o r t (1954) p r e p a r e d by Zuiderweg and Van Deemter showing t h e e f f e c t o f g a s v e l o c i t y on s t a g e h e i g h t . S o l u t e : propene; s t a t i o n a r y l i q u i d : isooctadecane.

4 6 810 20 406080 SUPERFICIAL GAS VELOCITY, m m / r

An i n t e r n a l r e p o r t was p r e p a r e d ’ b y Zuiderweg and myself and i s s u e d i n 1954. One o f t h e f i g u r e s of t h e r e p o r t showed such a breakdown d e r i v e d from t h e very l i m i t e d amount o f d a t a t h e n a v a i l a b l e t o u s . L a t e r , more e x p e r i m e n t a l work was done t o check t h e t h e o r e t i c a l c o n s i d e r a t i o n s . I worked t h i s o u t d u r i n g 1954-1955 and p r e p a r e d t h e 1956 a r t i c l e f o r ChemicaZ Engineering Science ( 3 ) . Zuiderweg and Klinkenberg were, f o r obvious r e a s o n s , t h e c o a u t h o r s . I n t h e summer o f 1955 I t r a n s f e r r e d t o S h e l l ’ s American a s s o c i a t e s i n Houston, Texas, t o work i n t h e a r e a o f c a t a l y t i c c r a c k i n g and f l u i d i z a t i o n . I n c i d e n t a l l y , t h e development o f o u r two-phase flow model o f f l u i d i z a t i o n was p a r t l y s u g g e s t e d by t h e two-phase c o n c e p t s used i n g a s - l i q u i d chromatography. I n t h e U . S . A . I had t h e o p p o r t u n i t y t o p r e s e n t t h e t h e o r y o f g a s chromatography on v a r i o u s o c c a s i o n s , among which w a s t h e 1956 Gordon Research Conference on p h y s i c a l s e p a r a t i o n s . Keulemans was by t h e n s p e n d i n g most o f h i s t i m e w r i t i n g a book on g a s chromatography ( I ) . When he p r e s e n t e d m e w i t h a copy I found o u t t h a t h e had a s s o c i a t e d my name w i t h t h e column e f f i c i e n c y e q u a t i o n . H e had n o t c o n s u l t e d m e i n advance, a s he wanted t o s u r p r i s e m e . H e succeeded A f t e r my r e t u r n i n 1957 t o S h e l l ’ s Amsterdam l a b o r a t o r y I g r a d u a l l y phased o u t r e s e a r c h on chromatography. During 1957 I p r e p a r e d a l a s t i n t e r n a l r e p o r t on “Peak s h a r p n e s s and column e f f i c i e n c y i n l i n e a r chromatography”, i n which t h e more s i m p l e t h e o r e t i c a l concept of t h e second moment o f a d i s t r i b u t i o n f u n c t i o n was i n t r o d u c e d a s a measure of peak s h a r p n e s s . The c o r r e s p o n d i n g formula is g i v e n i n t h e Appendix below. My l a s t a c t i v i t y i n t h e a r e a w a s an i n v i t e d a d d r e s s to t h e second g a s chromatography symposium, i n Amsterdam, May 1958.

.....

464

Fig. 54.2. At the Second Gas Chromatography Symposium, Amsterdam May 19-23, 1958. Left A to right:&.G. Bay16 , A. Glueckauf and J.J. van Shell Laboratorium, Energy Authority, Harvell).

REFERENCES 1 A.I.M. Keulemans, Gas Chromatography, Reinhold, New York, 1957. 2 L. Lapidus and N.R. Amundson, J. Phys. Chem. 56 (1952) 984. 3 J.J. van Deemter, F.J. Zuiderweg and A . Klinkenberg, Chem. Eny. S c i . 5 (1956) 271. APPENDIX The peak sharpness formula reads:

or, in words,

+ terms (relative peak width) = - (rel. eff. sample volume)' 12 representing longitudinal (eddy) diffusion, longitudinal molecular diffusion, diffusional resistance in the mobile phase and diffusional resistance in the stationary phase.

465 thickness of stationary liquid; d = particle diameter; Dm f I-- molecular diffusivity of mobile pgase; D, = molecular

where d

diffusivity of stationary phase; k = partition factor (amount of component in stationary phase/amount in mobile phase, at equilibrium); L = length of column; u = linear interstitial velocity of mobile phase; VR = retention volume; vs = sample volume; B = dimensionless, approcimetely 0.01;y = dimensionless tortuosity, somewhat less then 1; = dimensionless packing factor, in the range of 1-4; 0 = relative standard deviation of concentration peak. For small samples:

where, H is the HETP. The main differences with formula 53 in ref.3 are that the above includes additional terms which can perhaps not be neglected under all conditions, and the replacement of 8/r2 with the more exact 2/3.

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467

A.A. ZHUKHOVITSKII

ALEXANDER ABRAMOVICH ZHUKHOVITSKII was born i n 1908, i n Rostov-on-the-Don, i n Russia. H e s t u d i e d a t t h e Donskoi P o l y t e c h n i c a l I n s t i t u t e and graduated i n 1930 a s a chemi c a l e n g i n e e r . H e t h e n j o i n e d t h e Karpov I n s t i t u t e o f P h y s i c a l Chemistry, i n Moscow. H e was a s s o c i a t e d w i t h t h i s I n s t i t u t e u n t i l 1948; d u r i n g t h i s t i m e he w a s awarded t h e d e g r e e s of a Candidate (1933) and Doctor of Science (1937) and advanced from a grad u a t e s t u d e n t t o a r e s e a r c h s c i e n t i s t and a s s i s t a n t s c i e n t i f i c d i r e c t o r . In 1948 h e was i n v i t e d t o j o i n t h e S t e e l and A l l o y s I n s t i t u t e , i n Moscow, a s t h e Head of t h e C h a i r i n P h y s i c a l Chemistry and h a s been associated with t h i s I n s t i t u t e s i n c e then. P r i o r t o 1975 D r . Z h u k h o v i t s k i i has a l s o been a s s o c i a t e d w i t h t h e All-Union Research I n s t i t u t e f o r Geological Prospecting of Petroleum ( V N I G N I ) and, s i n c e 1975, w i t h t h e All-Union S c i e n t i f i c R e s e a r c h and Design I n s t i t u t e of Automation and C o n t r o l Systems i n t h e O i l and Gas I n d u s t r y (VNIIKANNEFTEGAS). D r . Z h u k h o v i t s k i i i s t h e a u t h o r and c o a u t h o r of more t h a n 300 s c i e n t i f i c a r t i c l e s , f i v e monographs and t e x t b o o k s , and more t h a n 50 c e r t i f i cates of i n v e n t i o n s and p a t e n t s i n v a r i o u s f i e l d s of e n g i n e e r i n g . I n 1968 he r e c e i v e d t h e t i t l e of Honored S c i e n t i s t of t h e Russian S o v i e t F e d e r a t e d S o c i a l i s t Republic and i n 1 9 7 7 , t h e M.S. T s w e t t Chromatography Medal. D r . Z h u k h o v i t s k i i ' s s c i e n t i f i c a c t i v i t i e s e x t e n d t o v a r i o u s branches of p h y s i c a l c h e m i s t r y , f o r example, quantum.chemistry, t h e t h e o r y of l i q u i d and s o l i d s t a t e , t h e t h e o r y of s o l u t i o n s and s u r f a c e phenomena. H e became i n v o l v e d i n g a s chromatography i n t h e l a t e 1 9 4 0 ' s and developed a number o f novel approaches and new methods such as chromathermography, vacancy chromatography, chromatography without c a r r i e r g a s and d i f f e r e n t i a l chromatography.

468

Probably the best way to present my recollections of my involvement in the evolution of chromatography is to survey my activities and particularly the various chromatographic techniques which I helped to develop. It started in 1940. At that time, together with my colleague K.A. Gol'bert, I was concerned with the thermal adsorption separation technique which is now referred to as the thermal desorption technique. We introduced the gas mixture into the column along which a furnace was moved. At that time we did not relate this technique to chromatography which was little known to us. During the war I dealt with the theory of sorption dynamics and with a great interest familiarized myself with chromatography. In 1944 I was engaged in research on the analysis of oil gases in the Institute "Neftegazorazvedka" where, together with K.A. Gol'bert and N.M. Turkel'taub (head of the Laboratory of Gas Analysis), I developed chromatographic methods for the determination of light hydrocarbons in air, utilizing gas adsorption chromatography and a gas interferometer as the detector. However, nonlinearity of the isotherm limited the possibilities of such an analysis and therefore, Turkel'taub and I tried to figure out how the "tailing" could be reduced. It was in this way that chromathermography, the elution analysis employing a moving temperature field with a negative gradient was developed. Later we gained an understanding of the existence of certain characteristic temperatures permitting the enrichment of components at these temperatures even when the isotherm is linear. The first paper on chromathermography was published in 1951 ( I ) . Close to chromathermography was the heat-dynamic method in which the furnace with temperature gradient rotated continuously around an unclosed annular tube. The mixture was supplied continuously while the impurity content was registered periodically (2). Such devices are being produced in the U.S.S.R. and the German Federal Republic. At the same time we devised a circulation apparatus for the enrichment of impurities which consisted of two columns connected in series. The mixture was introduced alternately either into the first or into the second column. This apparatus was described in our book, published in 1962 (3). At that time we failed to understand the existence of the enrichment limit and it was not until twenty years after the discovery of chromathermography that we finally established the theory of this phenomenon when developing chromatography without carrier gas. While in the early 1950's there were only a few scientists who were interested in chromatography, the First All-Union Conference on Chromatography held in 1959, in Moscow, attracted about a thousand participants. Much attention was given by us to the development and applications of chromatographic techniques in various fields of science and engineering. Thus, in collaboration with E.V. Vagin, we adapted the thermal desorption method for the air separation industry, and, together with B.A. Kazanskii and O.D. Sterligov, we developed a method for the determination of the individual components in complex hydrocarbon mixtures.

469

Fig. 55.1. Presidium of the First All-Union Conference on Gas Chromatography, held in 1959. Left to right: K.A. Gol'bert, D.A. Vyakhirev, A.A. Zhukhovitskii and N . M . Turkel'taub.

Fig. 55.2. Presidium of the Second All-Union Conference of Gas Chromatography, held in 1962. In the first row, the second from left is A . A . Zhukhovitskii followed by D.A. Vyakhirev, N . M . Turkel'taub, E.A. Vagin and K.A. Gol'bert. In the second row, the second from the left is M.I. Yanovskii.

470

During 1960-1962, together with N.M. Turkel'taub, we developed four new methods. In vacancy chromatography the sample mixture is fed continuously into the column while the carrier gas is injected periodically ( 4 ) . In d i f f e r e n t i a l chromatography the mixture to be analyzed is injected into a continuously flowing standard mixture having a fixed composition. The peaks obtained characterize the difference in the composition of the sample and the standard mixture (5). In i t e r a t i o n chromatography the composition of the standard mixture is adjusted until no signal is produced when injecting this into the mixture to be analyzed (6). In s t e p chromatography, owing to an increase in the sample volume, steps are obtained instead of peaks, the height of each being related to the concentration of a single component. To determine the composition of the mixture there is no need to separate these steps from each other. This method has recently found an application in high-speed analysis (7). Since 1965, together with S.M. Yanovskii, V.P. Shvartsman, L.G. Novikova, A.F. Shlyakhov and M . L . Sazonov, I was engaged in the development of the technique chromatography without c a r r i e r gas (CWCG) ( 8 ) . The distinguishing feature of CWCG is that the flow-rate in the zones depends appreciably on the composition. As a result, zone boundaries are stationary, which substantially reduces zone broadening. Based on the fact that the retention volumes are also concentration dependent we were able to develop the e l u t i o n version of CWCG. According to this version, any substance can be introduced into the continuously flowing mixture to be analyzed. The composition is determined from the retention volumes of the peaks. In CWCG, separation is accompanied by an enrichment. Utilizing this phenomenon, we have developed two variants of CWCG, both aimed at the determination of impurities. In the case of light impurities, the sample mixture is passed through the column which is either evacuated or has previously been filled with a substance which is less strongly adsorbed than any of the sample components. Some devices based on this principle have been manufactured in the U.S.S.R. In the case of heavy impurities, a substance which is more strongly adsorbed than any of the sample components is introduced into the column previously filled with the sample mixture. D i f f e r e n t i a l CWCG represents another variant of this technique. Here, the sample and a standard mixture are supplied alternately into the sorbent bed. The step heights produced are related to the differences in the concentrations of the individual components. Within the scope of CWCG, frontal adsorption and frontal desorption analyses can be employed as well. In the latter case the so-called "gas-piston" is used. If its Henry's coefficient is less than those of n most strongly adsorbed components, then this "gaspiston" will be present in the zones of these n components. Such a specific phenomenon characterized by us as the "substitution" can be utilized for the analysis of mixtures or impurities. The chromathermographic and heat-dynamic versions of CWCG (8,9) as well as the so-called method of fixed concentrations (10) belong to methods employing a temperature field. In the latter method, as well as in some other versions of CWCG, the sample com-

471 position is determined from the step widths, the detector being used as a null-point instrument. My chromatographic investigations before 1975 were all performed in the All-Union Research Institute for Geological Prospecting of Petroleum (VNIGNI) while since 1975 they have been carried out in the All-Union Scientific Research and Design Institute of Automation and Control Systems in Oil and Gas Industry (VNIIKANNEFTEGAS). In this Institute, together with. V.S. Yusfin we developed a technique for the analysis of impurities; this method is usually called multiple thermal adsorption concentrating. In this technique which can also be referred to as the "jumping furnace technique", the furnace is moved rapidly and repeatedly. This method permits one to obtain a high enrichment which is limited by the broadening of the sorbate zone during the heating of a given part of the sorbent bed. In the method chromarheography developed in collaboration with S.M. Yanovskii, V.P. Shwartsman, L.G. Novikova and others ( 2 1 ) , there is a negative flow velocity gradient moving along the bed. In a sense this method is the velocity analogue of chromathermography. Enrichment of bands for some characteristic velocity values also takes place in this method. Four ways of achieving such a velocity field have been developed, each of them using the sorption of the carrier gas in the column. This method provides a means for enriching and determining impurities which are.adsorbed less strongly than the carrier gas. For example, we have demonstrated the possibility of determining the helium and hydrogen concentration of air with a sensitivity of (ppm) when using a thermal conductivity detector. Together with V.I. Kalmanovskii, we have also developed a technique for the determination of both light and heavy impurities, based on a cyclic variation of the column temperature. The mixture to be analyzed is passed continuously through the column. Enrichment and determination are achieved during cooling for light impurities, and during heating for heavy impurities. The basic advances of chromatography are related to the use of small concentrations and samples. In some specific problems, however, such as increasing the productivity of preparative separation, determination of impurities and investigation of the physico-chemical properties of concentrated solutions, etc., chromatography is faced with the opposite: high concentrations and large sample volumes. In the last few years together with S.M. Yanovskii and in collaboration with V.P. Shwartsman, L.G. Novikova, M.O. Burova, O.N. Alksnis and I.A.Silayeva, I have been working on chromatography of vapours close t o saturated conditions (CVCS) and on its special case, chromad i s t i l l a t i o n (12). Since in the concentration region considered, the isotherms are concave, CVCS can be termed as the "chromatography of concave isotherms". If no liquid phase is used and the inert support serves as a filler (usually in the form of beads), CVCS transforms to chromadistillation, i.e., distillation in the chromatographic regime. Both methods - CVCS and chromadistillation - have two versions: thermal and restrictive. In the first method, the column is exposed to the constant temperature field with a negative gradient while in

472 the second version, being an antipode of the displacement analysis, a more volatile component is located ahead of the mixture to be separated. Both versions of both methods result in the complete resolution of the sample components. Using these methods as a basis, we have developed several techniques for the analysis of mixtures, the determination and enrichment of impurities, fractionation of complex mixtures according to the boiling points (including a micromethod employing a capillary column), determination of the thermodynamic activity etc. While summarizing my past activities, I must also mention that during all my work, I was fortunate to be also able to collaborate closely with Czechoslovak chromatographers, particully with Eva Smolkovl and Jaroslav Jan&. I have visited Czechoslovakia five times, and discussions and lectures on our new techniques helped me considerably in my own work. When considering our efforts and the work of many years I experience a great satisfaction. The beautiful idea of Tswett, and the new field of knowledge, chromatography, resulting from it, provides many possibilities for scientific invention, development of science, and satisfaction of the needs of engineering. These possibilities are inexhaustible. Chromatography has a promising future: the requirements imposed by science and engineering upon the analysis of multicomponent systems are limitless and most of these can be resolved by chromatography. Closely allied with physics, chemistry and cybernetics, chromatography will go through further golden ages, for the benefit of mankind. REFERENCES 1 A . A . Zhukhovitskii, O.V. Zolotareva, V . A . Sokolov and N.M. Turkel'taub, Dokl. Akad. Nauk SSSR 77 (1951) 435. 2 A . A . Zhukhovitskii, N.M. Turkel'taub and T.V. Georgiyesvskaya, Dokl. Akad. Nauk SSSR 9 2 (1953) 987. 3 A . A . Zhukhovitskii and N.M. Turkel'taub, Gazovaya Khromatografiya, Gostoptekhizdat, Moscow, 1962, p. 441. 4 A . A . Zhukhovitskii and N.M. Turkel'taub, Dokl. Akad. Nauk SSSR 143 (1962) 646. 5 A . A . Zhukhovitskii, V . E . Shevtsov, V . P . Shwartsman, L.G. Gel'man and M.L. Sazonov, Zavod. Lab. 32 (1966) 402. 6 A . A . Zhukhovitskii and N.M. Turkel'taub, Dokl. Akad. Nauk SSSR 150 (1963) 113. 7 E . A . Myagkov and A . A . Zhukhovitskii, Zavod. Lab. 41 (1975) 763. 8 A . A . Zhukhovitskii and M.L. Sazonov, J . Chromatogr. 49 (1970) 153. 9 A . A . Zhukhovitskii, M.L. Sazonov, L . G . Gel'man and V.P. Shwartsman, Chromatographia 4 (1971) 547. 10 A . A . Zhukhovitskii, M.L. Sazonov, L.G. Gel'man and V.P. Shwartsman, Zh. Fiz. Khim. 45 (1975) 655. 11 A . A . Zhukhovitskii, E . V . Kanunnikova, L.G. Novikova, M.L. Sazonov, V . P . Shwartsman and S.M. Yanovskii, Chromatographia 8 (1975) 369. 12 A . A . Zhukhovitskii, S.M. Yanovskii and V . P . Shwartsman, J . Chromatogr. 119 (1976) 591.

473

ALBERT ZLATKIS

ALBERT ZLATKIS was born i n 1924 i n Pomorzany, Poland and e m i g r a t e d t o Canada a t t h e age o f t h r e e . H e r e c e i v e d a B.A.Sc. d e g r e e i n Chemi c a l E n g i n e e r i n g from t h e U n i v e r s i t y of Toronto i n 1947 and a M.A.Sc. a t t h e same s c h o o l i n 1948. H i s Ph.D. was o b t a i n e d from Wayne S t a t e U n i v e r s i t y i n 1952. A f t e r one y e a r of p o s t - d o c t o r a l work on a Michigan Heart A s s o c i a t i o n F e l l o w s h i p he j o i n e d t h e S h e l l O i l Company i n Houston, Texas a s a research chemist. D r . Z l a t k i s joined t h e f a c u l t y o f t h e U n i v e r s i t y o f Houston i n 1955 a s a s s i s t a n t p r o f e s s o r o f c h e m i s t r y . H e became chairman o f t h e department i n 1958 f o r f o u r y e a r s and was a p p o i n t e d p r o f e s s o r i n 1963. D r . Z l a t k i s i s t h e a u t h o r o f more t h a n 150 s c i e n t i f i c p a p e r s . H i s r e s e a r c h i n t e r ests include c l i n i c a l chemistry, c a p i l l a r y chromatography, f l a v o r a n a l y s i s , i o n i z a t i o n d e t e c t o r s , c o n c e n t r a t i o n t e c h n i q u e s , environmental a n a l y s i s and g a s chromatography-mass s p e c t r o metry s t u d i e s o f p r o f i l e s o f v o l a t i l e m e t a b o l i t e s o f b i o l o g i c o r i g i n . H e i s t h e co-author o f A Concise Introduction t o Organic Chemistry and h a s c o e d i t e d s e v e r a l books such a s The Practice of Gas Chromatography, Preparative Gas Chromatography and High Performance Thin-Layer Chromatography. D r . Z l a t k i s h a s been e d i t o r of Advances i n Chromatography s i n c e i t s i n c e p t i o n i n 1963. H e i s on t h e e d i t o r i a l board of JournaZ

of Chromatography, Chromatographia, Journal of and J o m a 2 of High Resolution Chromatography.

Chromatographic Science

D r . Z l a t k i s r e c e i v e d t h e American Chemi'cal S o c i e t y Award i n Chromatography i n 1973. H e w a s a l s o honored w i t h t h e NASA Technology Award i n 1975 and 1978. H e h a s l e c t u r e d i n t h e U.S.S.R. and Poland under t h e a u s p i c e s of t h e i r Academies o f S c i e n c e s and h a s been a t o u r s p e a k e r f o r t h e South A f r i c a n Chemical S o c i e t y . D r . Z l a t k i s has been a c t i v e l y engaged i n chromatography s i n c e 1954. H e o r g a n i z e d t h e I n t e r n a t i o n a l Symposium on Advances i n Chromatography i n 1963 and h a s been i t s chairman f o r t h e twelve m e e t i n g s t o d a t e .

474 During my senior year as a student in chemical engineering at the University of Toronto, I was introduced to an area of research which was to have a deep impact on both my technical and social life. In the fall of 1947 I was assigned to carry out a research problem under Don McArthur who was studying the oxidation of trans decalin for his doctoral research. The location of the laboratory seemed a return to nineteenth century conditions since it was in a sub-basement in the Mining Building and was affectionately called the “coal hole“. It was here that I started my journey into separation science. The problem involved isomerization of commercial decalin, a mixture of cis-tr4uns isomers, into the predominately trans form and then the fractionation of the pure isomers by the use of Stedman distillation columns. Although the literature indicated that the isomerization proceeded easily with anhydrous aluminium chloride, I was unable to effect this simple conversion. It was only after I had tried my sixth brand of aluminium chloride that any success was evident. This was from an old bottle which had its origin in Germany. Fortunately the label was still readable and I noted the presence of ferric chloride as an impurity. Indeed it was this extra trace compound which was promoting the isomerization. Although a minor achievement it was this experiment which turned my interest from engineering to chemistry. After obtaining my master’s degree I decided to accept a fellowship at Wayne State University in Detroit and proceeded to work in

Fig. 56.1. A. Zlatkis, at the University of Toronto, Canada, in 1947.

475 organic synthesis under A.J. Boyle. It was here that I met Bennie Zak with whom I collaborated to develop a procedure for the determination of cholesterol in blood serum ( 1 ) and this interest in clinical chemistry has continued to the present. Graduation led to a position as research chemist with the Shell Oil Company in Houston, Texas, and my introduction to chromatography. At that time I joined the analytical group to become familiar with all of the techniques used and involved in analyzing the products of hydroprocessing. In 1954 Lloyd Snyder joined this group and shortly thereafter Mr. Zuiderweg of Shell in Amsterdam was visiting Houston and excited us with the news of the development of gas chromatography by Martin and James. Our group immediately started building an instrument and in a few months we were making hydrocarbon separations on 50-ft. packed columns. I had been teaching organic chemistry on a part-time basis at the University of Houston and enjoyed this academic contact although I must say that my time spent at the University and some long evenings in the library made it difficult for my wife Esther. She has indeed been patient for a long period - since our early days in Canada. In August of 1955 I was invited t o join the chemistry department of the University of Houston as an assistant professor and I readily accepted. Since I was interested in stereochemistry and had an experimental background in gas chromatography, I decided to attempt to solve a classical problem, i.e., to separate the enantiomers of a compound which contained an asymmetric nitrogen atom. This seemed feasible for an aziridine molecule from an energetic viewpoint, but low temperature would be necessary. Gas chromatography should provide the answer using an optically active stationary phase. 2-Brucine, an alkaloid was dissolved in quinoline and the chiral column was ready. Although the procedure seems rather simple we had to make our own chromatographic supports and this involved obtaining one firebrick from Johns-Manville, crushing, washing and sieving this material. Hamilton syringes had not yet been invented and micro-dippers were used. Three months were spent in attempting the resolution of amines without success I continued to work with the same chromatographic column since it showed some interesting selectivity in making hydrocarbon separations. After submitting my first manuscript ( 2 ) on this topic to Analytical Chemistry, I attended an I.S.A. symposium on Gas Chromatography at Lansing, Michigan in August 1957. Here I met two outstanding young men who were already pioneers in the field Tony James and Courtenay Phillips. The third of these symposia was in 1961 and several of my colleagues urged me to organize a meeting

-

*.

*In collaboration with Dr. V. Schurig of the University of Tiibingen we have just recently solved this problem. Resolution of l-chloro2,2-dimethylaziridine was resolved using a 100 m x 0.5 mm nickel capillary column coated with an optically active nickel complex of 3-trifluoroacetyl camphorate. The separation factor of 1.43 was indeed large.

4 76

i n Houston which would improve t h e e x i s t i n g symposia, i . e . by h a v i n g more s p e a k e r s from Europe where m o s t o f t h e developments were b e i n g made, p a p e r s which w e r e reviewed b e f o r e b e i n g p r e s e n t e d and p u b l i c a t i o n of t h e s e p a p e r s i n a j o u r n a l . Sandy Lipsky o f t h e Yale U n i v e r s i t y School of Medicine w a s a l r e a d y e s t a b l i s h e d i n g a s chromatography,

F i g . 56.2. M i a m i Beach Symposium, J u n e 2 , 1970. Standing: ( l e f t - t o - r i g h t ) A. Z l a t k i s , I . Halhsz, A . I . M . Keulemans, C . G u i l l e m i n , L.S. E t t r e . S e a t e d : K i t t y E t t r e , Agnes Halhsz and E s t h e r Zlatkis. p r i m a r i l y i n t h e area o f l i p i d s e p a r a t i o n s , and i t w a s h e who f i n a l l y convinced m e t o f o r m a l i z e t h e I n t e r n a t i o n a l Symposium on Advances i n G a s Chromatography. The f i r s t m e e t i n g h e l d i n Houston, J a n u a r y 21-24, 1963 was i n d e e d a memorable one. I t was a l s o t h e b e g i n n i n g o f c l o s e f r i e n d s h i p s w i t h many of t h e l e a d i n g s c i e n t i s t s i n t h i s f i e l d . A l l 23 p a p e r s p r e s e n t e d a t t h e m e e t i n g w e r e reviewed and p u b l i s h e d i n t h e A p r i l i s s u e o f AnaZyticaZ Chemistry, a r a t h e r new e x p e r i e n c e f o r t h i s j o u r n a l and i t s e d i t o r L a r r y H a l l e t t . The Houston symposium had a n unusual g e n e s i s . A f i e r c e w i n t e r storm had c l o s e d many o f t h e major a i r p o r t s and t r a v e l was n e x t t o i m p o s s i b l e . Houston i t s e l f had one o f i t s rare snow and i c e s p e c t a c u l a r s . On t h e e v e o f t h e meeting o n l y 50 of t h e d e l e g a t e s had a r r i v e d a t t h e S h e r a t o n Hotel and s e r i o u s c o n s i d e r a t i o n w a s g i v e n t o a one-day postponement. By s i x o ' c l o c k i n t h e morning a l l 600 had a r r i v e d and t h e symposium was i t s way t h r e e h o u r s l a t e r .

477

F i g . 56.3. Symposium a t L a s Vegas, Caesars' P a l a c e , J a n u a r y 2 0 , 1969. L e f t t o r i g h t : P e a r l L i p s k y , S.R. Lipsky, C . G . S c o t t , A. Z l a t k i s , R.S. Evans and Vicki S c o t t . Each symposium seems t o have had unusual moments which c a n b e r e a d i l y r e c a l l e d . A t t h e morning s e s s i o n o f o u r f i r s t meeting, D r s . Bayer, Schomburg and Halksz had j u s t a r r i v e d from Germany, t h e f i r s t two v i s i t i n g t h e U.S.A. f o r t h e f i r s t t i m e . They s a t i n amazement a s Herman F e l t o n opened his l e c t u r e w i t h a song about g a s chromatography. The second symposium was a l s o h e l d i n Houston i n A p r i l 1964 and d u r i n g a r e c e p t i o n a t t h e home o f Marjorie and Evan H o m i n g one o f t h e Dutch s c i e n t i s t s , J . Pypker, walked t h r o u g h a g l a s s w a l l . O t h e r i n c i d e n t s come t o mind such a s t h e meeting i n New York where one e v e n i n g a t L i n d y ' s , P r o f e s s o r Bayer o r d e r e d a l l o f t h e food f o r a group o f 12 and f o r g o t t o p l a c e h i s own o r d e r ; however, h e d i d manage t o r e c e i v e t h e b i l l . I n M i a m i Beach, one o f t h e s p e a k e r s from F i n l a n d gave h i s l e c t u r e wearing a t e r r y c l o t h r o b e and s h o r t s and p r e s e n t e d r o s e s t o two of h i s f r i e n d s p r i o r t o t h e t a l k . A r t h u r Karmen o f t h e A l b e r t E i n s t e i n C o l l e g e o f Medicine, a c o n s t a n t cont r i b u t o r t o t h e s e symposia, h a s always r e l a x e d h i s a u d i e n c e w i t h some w e l l chosen s t o r i e s . Two meetings were h e l d i n Las Vegas and i t was h e r e t h a t Sandy Lipsky showed a s k i l l a t t h e t a b l e s t h a t w a s matched o n l y by h i s s c i e n t i f i c endeavors. I t w a s a l s o a t C a e s a r s ' P a l a c e i n L a s Vegas where a t h e s i s w a s p r e s e n t e d and defended by P a t Howard - i t seemed i t w a s t h e o n l y p l a c e t h a t a l l o f t h e p r o f e s s o r s on h i s committee c o u l d b e g a t h e r e d t o g e t h e r . P r o f e s s o r Bayer and I had p r e v i o u s l y v i s i t e d L a s Vegas i n 1966. On t h i s o c c a s i o n w e were accorded a r o y a l welcome a t t h e T o u r i s t Bureau s i n c e w e were t h e 1 , 0 0 0 , 0 0 0 t h v i s i t o r .

478

F i g . 5 6 . 4 . P a t Howard d e f e n d i n g h i s t h e s i s d u r i n g t h e symposium a t L a s Vegas, C a e s a r s ' P a l a c e , on December 2 , 1971. S t a n d i n g : P.Y. Howard, s e a t e d : J . O r o , E. Gil-Av, W.E. Wentworth, J . L . Bear, W . P a r r , A . Z l a t k i s , and B. H a l p e r n . O t h e r symposia h a v e been h e l d i n T o r o n t o , Munich and Amsterdam. The l a t t e r had a r e c e p t i o n a t t h e Rijksmuseum amongst t h e Rembrandts, Vermeers and F r a n s H a l s ' . A p e r s o n a l t r i b u t e from some of my c o l l e a g u e s a t t h e Munich m e e t i n g w i l l always b e remembered. A s I l a t e r found o u t , t h e o r i g i n a l s c r o l l they had prepared w a s l o s t i n t h e o v e r s e a s m a i l and i t had t o b e r e p l a c e d a t t h e l a s t m i n u t e . Among t h e f o r e i g n s c i e n t i s t s who have c o n t r i b u t e d h e a v i l y t o t h e s u c c e s s o f t h e s e i n t e r n a t i o n a l symposia a r e V i c t o r P r e t o r i u s o f South A f r i c a , Denis D e s t y , Howard P u r n e l l , Tony James, C h a r l e s Brooks and John Knox o f t h e U n i t e d Kingdom, George Guiochon o f F r a n c e , E r n s t B a y e r , G e r h a r d Schomburg and Rudolf Kaiser o f Germany, J o s e f Huber of A u s t r i a and p a r t i c u l a r m e n t i o n s h o u l d b e made o f a "Hungarian I s t v h n H a l h s z (Germany), Csaba q u a r t e t " : L e s l i e E t t r e (U.S.A.) Horvkth (U.S.A.) and E r v i n KovP s ( S w i t z e r l a n d ) who are e s t a b l i s h e d i n v e s t i g a t o r s i n chromatography Many o f t h e s e h a v e been honored w i t h t h e T s w e t t Chromatography medal which w a s i n t r o d u c e d a t t h e 1974 Symposium i n Houston. The r e n n a i s s a n c e o f l i q u i d chromatography i n 1966 h a s b r o u g h t many o f t h e g a s chromatography s p e c i a l i s t s i n t o new f r o n t i e r s . Among o t h e r s , D r s . H a l i s z , H o r v h t h , Knox, K i r k l a n d , Huber and S c o t t have made t h i s t r a n s i t i o n e f f o r t l e s s l y . I t now a p p e a r s t h a t t h i n - l a y e r chromatography w i l l a l s o h a v e a s e c o n d g e n e r a t i o n of r e s e a r c h and development. T h e r e i s p r o b a b l y no one i n d i v i d u a l who i n f l u e n c e d t h e c o u r s e o f my career more t h a n t h e E n g l i s h s c i e n t i s t J i m Lovelock. My f i r s t a w a r e n e s s o f t h i s g e n t l e man w a s t h r o u g h h i s p a p e r on t h e a r g o n i o n i z a t i o n d e t e c t o r which w a s p u b l i s h e d i n t h e f i r s t i s s u e o f t h e Jouu*na2 of Chromatography i n 1958. I n t h e f a l l of t h a t y e a r I l e a r n e d

479

Fig. 56.5. Las Vegas, 1966. A. Zlatkis and E. Bayer at the tourist bureau. that he was planning to spend some time in Dr. Lipsky's laboratory at Yale. Shortly after his arrival in the U.S.A. I called New Haven and asked him to visit me in Houston. I was pleasantly surprised when he accepted and it was only much later that I learned that Jim has always had difficulty refusing invitations by telephone. We spent an exciting week working to assemble a capillary chromatographic system and then published the first American paper on this subject (3). This was the beginning of a long personal and professional relationship with Jim Lovelock, much of our collaboration involving ionization detectors. In 1975 Jim was awarded a Tswett Medal for his outstanding contributions to chromatography. Special mention should be made of Professor A.J.P. Martin whose genius has been responsible for the direction of many careers. He has made profound contributions to all fields of chemistry over the past 45 years. His work has ranged from research in vitamin deficiencies, factors controlling insulin secretion and the production of penicillin to the development of liquid partition chromatography, the invention of paper chromatography, the development of displacement electrophoresis and above all the invention of gas chromatography. If one considers the problems scientists would face working in the field of chemistry without the techniques of paper chromatography, liquid partition chromatography, and gas chromatography, one begins to realize the tremendous impact of this man's work in science today. Very few laboratories indeed do not possess the gas chromatograph, an instrument which is an ever present monument to Dr. Martin's innovative achievements and scientific contributions.

480

F i g . 56.6. Symposium a t Houston, O c t o b e r 30, 1976. S t a n d i n g ( l e f t - t o - r i g h t ) U t e H e z e l , C . M e r r i t t , C. G u i l l e m i n , M . J . E . Golay, G . D i j k s t r a , R.E. Sievers. Seated: Esther Zlatkis, A. Zlatkis. Although a volume c o u l d be w r i t t e n p e r t a i n i n g t o my p e r s o n a l r e m i n i s c e n c e s of D r . Archer M a r t i n , one i n c i d e n t may b e o f i n t e r e s t . A f t e r c o n v i n c i n g him t o j o i n t h e U n i v e r s i t y o f Houston i n 1974, a f o r m a l i t y o f o b t a i n i n g a l i s t o f h i s p u b l i c a t i o n s w a s r e q u i r e d . When I v i s i t e d Archer a t Abbotsbury - a medieval c a s t l e i n London, - I asked him f o r t h i s l i s t . S i n c e one d i d n o t e x i s t h e s u g g e s t e d he c o u l d s u p p l y some of t h e r e p r i n t s . An a n c i e n t c a b i n e t d i s c l o s e d a l a r g e number o f unopened packages c o n t a i n i n g r e p r i n t s i n c l u d i n g h i s f i r s t p u b l i c a t i o n i n 1931 e n t i t l e d "On a New Method o f D e t e c t i n g P y r o e l e c t r i c i t y " . I c o l l e c t e d t h e n e c e s s a r y r e p r i n t s and w a s a b l e t o assemble t h e r e q u i r e d l i s t . I t may b e o f i n t e r e s t t o n o t e t h a t h i s n i n t h p a p e r p u b l i s h e d i n 1941 on p a r t i t i o n chromatography a t t h e age o f 30 paved t h e way f o r h i s Nobel P r i z e . Twenty y e a r s have p a s s e d s i n c e my f i r s t chromatography p a p e r and d u r i n g t h i s t i m e my e f f o r t s have been d i r e c t e d i n t o some f a i r l y d i v e r s e areas. These i n c l u d e c a p i l l a r y columns, i o n i z a t i o n d e t e c t o r s , flow programming, a n a l y s i s o f lunar m a t e r i a l s , and f l a v o r c o n s t i t u e n t s o f c o f f e e , c h e e s e s and mushrooms. I n r e c e n t y e a r s t h e environment and b i o l o g i c a l f l u i d s have a t t r a c t e d o u r a t t e n t i o n . N e w t e c h n i q u e s have been developed f o r t h e c o n c e n t r a t i o n o f o r g a n i c v o l a t i l e s by u s i n g a hydrophobic porous polymer Tenax ( 2 , g - d i p h e n y l polyphenylene o x i d e ) ( 4 ) . The a n a l y s i s of t r a c e o r g a n i c c o n s t i t u e n t s ( 5 , 6 ) i n a i r ,

481

Fig. 56.7. At the University of Houston, in 1961: J.E. Lovelock and A . Zlatkis examining new ionization detector designs.

Fig. 56.8. Presentation of the Tswett Chromatography Medal to J.E. Lovelock at Munich, November 3, 1975. L.S. Ettre, A. Zlatkis and J.E. Lovelock.

482 water, u r i n e , serum, s a l i v a and c e r e b r o s p i n a l f l u i d are now r e a d i l y o b t a i n e d . I n c o l l a b o r a t i o n w i t h D r . Hartmut L i e b i c h o f Tubingen, Germany, w e have b e e n a b l e t o d e t e r m i n e a number o f s i g n i f i c a n t m e t a b o l i t e s which a r e i m p o r t a n t i n t h e d i a g n o s i s o f diabetes m e l l i t u s M e t a b o l i c p r o f i l e s are p r e s e n t l y b e i n g d e v e l o p e d f o r s e v e r a l o t h e r diseases ( 7 ) . These y e a r s h a v e been most e v e n t f u l and e x c i t i n g , y i e l d i n g many f r i e n d s h i p s from a l l p a r t s o f t h e g l o b e . I n p a r t i c u l a r , my c o l l a b o r a t i o n w i t h L e s l i e E t t r e , o u r r e s i d e n t h i s t o r i a n o f chromatography, i n e d i t i n g many books and o r g a n i z i n g t h e i n t e r n a t i o n a l symposia h a s i n d e e d been a r e w a r d i n g o n e . REFERENCES 1 A . Z l a t k i s , B. Zak and A . J . Boyle, J. Lab. CZin. Med. 4 1 (1953) 486. 2 A . Z l a t k i s , Anal. Chem. 30 (1958) 332. 3 A . Z l a t k i s and J . E . Lovelock, Anal. Chem. 31 (1959) 620. 4 A . Z l a t k i s , H . A . L i c h t e n s t e i n and A . T i s h b e e , Chromatographia 6 (1973) 6 7 . 5 K . Y . L e e , D. Nurok and A . Z l a t k i s , J. Chromatogr. 158 (1978) 377 6 G . H o l z e r , H . S h a n f i e l d , A . Z l a t k i s , W. B e r t s c h , P . J u a r e z , H. Mayfield and H.M. L i e b i c h , J. Chromatogr. 1 4 2 (1977) 755. 7 H . M . L i e b i c h , O . A l - B a b b i l i , A . Z l a t k i s and K . K i m , CZin. Chem. 212 (1975) 294.

483

THOSE WHO ARE NO LONGER WITH US by Leslie S. Erne

This book would not be complete without remembering, at least briefly,those pioneers who are no longer with us. A large number of scientists from the past contributed significantly to the evolution of chromatography and their lives and activities should be part of any "historical dialogue" on chromatography. We should remember CHARLES DHdRf of Switzerland who was the first to realize the importance of Tswett's discovery, ALFRED WINTERSTEIN who, while in KUHN's laboratory, did much to help other scientists in learning the intricacies of column chromatography, and HARRY WILLSTAEDT who worked in Germany and Sweden and published a pioneering book (the second after Zechmeister's) on chromatography, to mention but a few. Unfortunately, due to both lack of space and shortage of information on their lives and activities, it is impossible to cover them all. For this reason, I am restricting this last chapter of our book to seven scientists. The first is, naturally, Tswett, the inventor of the technique; since our present title is "a historical dialogue", I will concentrate on his controversy with his contemporary scientists and the question of why his invention was not immediately adapted by others. Further pioneers to be discussed here are L . S . Palmer, A. Tiselius, L. Zechmeister, F.H. Pollard, A.I.M. Keulemans and S. Dal Nogare. M.S.

Tswett (1872-1919)

MICHAIL SEMENOVICH TSWETT*, the inventor of column adsorption chromatography, was born in 1872, in Asti, Italy, in a hotel where his parents happened to stay while passing through the town. He grew up in Switzerland, and studied at Geneva University receiving

* For a long time, details of Tswett's life and activities were not known at all and e.g. Zechmeister, although he tried, could not obtain enough information for the introduction of his book on chromatography (see later). Dh6r6 was the first who gave biographical details(1) and he was then followed by Zechmeister (see, e.g., ref. 2 ) . Recently, Sakodynskii (3) and Senchenkova ( 4 ) published detailed accounts on the life and activities of this remarkable scientist while it was the special merit of Robinson ( 5 ) who presented a balanced discussion of Tswett's entire scientific output, to place it in the proper historical and scientific context and demonstrate the controversies with his contemporaries. In my work, I not only utilized Robinson's discussion but studied in original form the most important papers.

484 h i s Ph. D. i n 1896. H e t h e n f o l lowed h i s f a t h e r t o R u s s i a b u t had d i f f i c u l t i e s i n s e t t l i n g down: t h e R u s s i a n d e g r e e s b e i n g q u i t e d i f f e r e n t (even t o d a y ! ) h i s Swiss d o c t o r a t e w a s n o t acc e p t e d and h e h a d a g a i n t o e a r n h i s M.S. and D o c t o r ' s d e g r e e s . H e d i d t h e f i r s t a t Leningrad and Kazan' r e c e i v i n g t h e M a s t e r ' s d e g r e e i n 1901. H e t h e n moved t o W a r s a w where h e s p e n t t h e n e x t 14 y e a r s a s s o c i a t e d f i r s t w i t h t h e U n i v e r s i t y ' s P l a n t Anatomy and P h y s i o l o g y Department, t h e n with the Veterinary I n s t i t u t e and f i n a l l y , w i t h t h e Chemistry and Mining Department o f t h e Polytechnic I n s t i t u t e . He received h i s R u s s i a n Doctorate i n 1910 and w a s awarded t h e N. Akhamatov Grand P r i z e of t h e R u s s i a n Academy o f S c i e n c e s i n 1911. F i g . 57.1. M.S. T s w e t t . I n 1915, German t r o o p s approached Warsaw and Tswett moved t o Moscow ( l e a v i n g a l l h i s books and a r c h i v e s b e h i n d him) and from t h e r e , t o g e t h e r w i t h t h e rest o f t h e e v a c u a t e d Warsaw P o l y t e c h n i c I n s t i t u t e t o N i z n i i Novgorod ( t o d a y C o r k i i ) . H e w a s a l r e a d y ill t h e n . For some t i m e , h e t r i e d t o r e c u p e r a t e i n Vladikavkaz ( t o d a y : Ordzhon i k i d z e ) , i n t h e C a u c a s i a n Mountains, w i t h h i s s i s t e r ' s f a m i l y and i t w a s t h e r e t h a t h e r e c e i v e d h i s appointment t o f u l l p r o f e s s o r s h i p a t t h e U n i v e r s i t y o f Y u r ' e v ( T a r t u , E s t o n i a ) where h e moved i n t h e autumn of 1917. However, i n a few months, when German t r o o p s approached t h a t c i t y , he f o l l o w e d t h e e v a c u a t e d u n i v e r s i t y t o Voronezh. H e became one of t h e f i r s t p r o f e s s o r s o f Voronezh U n i v e r s i t y . H e w a s v e r y s i c k ( w e are n o t s u r e w h e t h e r i t w a s t u b e r c u l o s i s o r a h e a r t d i s e a s e ) and d i e d on June 26, 1919. H e w a s b u r i e d i n t h e cemetery o f t h e Alekseev monastery b u t f a t e would n o t l e t him rest i n p e a c e f o r e v e r : d u r i n g t h e Second World War t h e cemetery w a s d e s t r o y e d and i t i s now imposs i b l e t o f i n d h i s grave. T s w e t t w a s c o n s i d e r e d t o b e a b o t a n i s t ; however, t o d a y w e would probably c a l l h i m a p h y s i c a l c h e m i s t or even a b i o c h e m i s t ( j u s t as M a r t i n and Synge were b i o c h e m i s t s a l t h o u g h t h e i r most l a s t i n g cont r i b u t i o n is i n t h e f i e l d o f s e p a r a t i o n s c i e n c e ) . H i s whole l i f e ' s a c t i v i t y r e v o l v e d around s e p a r a t i o n and p u r i f i c a t i o n methods and t h e p l a n t p i g m e n t s , t h e i n v e s t i g a t i o n o f which w a s made p o s s i b l e w i t h t h e h e l p o f t h e s e methods. H e f i r s t t r i e d t o u s e s o l v e n t e x t r a c t i o n methods and t h e t i t l e of h i s 1901 Master's t h e s i s i s a l r e a d y i n d i c a t i v e o f h i s i n t e r e s t : "Physicochemical S t r u c t u r e o f t h e C h l o r o p h y l l G r a i n . Experimental and C r i t i c a l S t u d y . " I t i s i n t e r e s t i n g t o n o t e t h a t i n

485

Fig, 57.2. Location of towns important in Tswett's life. this paper, he already observed the phenomenon of selective adsorption on paper: "I could vividly see differently colored rings when filtering petroleum ether extracts of leaves through Swedish paper." It is not surprising that 10 years later, in the foreword of his magnum opus he said that "the source of my chromatographic method lies in my Russian work of 1901" ( 3 ) . In his subsequent work in Warsaw, Tswett developed the adsorption technique which, three years later, he called "chromatography". His first presentation of the new method was in his paper "On a New Category of Adsorption Phenomena and Their Application to Biochemical Analysis" which he presented on March 21, 1903* at the meeting of the Biological Section of the Warsaw Society of Natural Sciences (6). This was then followed three years later by.his two detailed papers "Physico-Chemical Studies of Chlorophyll, The Adsorptions" (7) and "Adsorption Analysis and the Chromatographic Method" (8). In 1907, he personally participated at two meetings (May 31 and June 28, 1907) of the Deutsche Botanische Gesellschaft (the German Botanical Society) of which he was a member; at the first meeting he showed various solutions of pure separated pigments while at the second, he showed a "chromatogram", i .e. a column with the colored "rings" ( 9 ) . Sakodynskii (3) mentions Tswett's various trips to the Botanical Gardens of Berlin and the universities of Kiel, Amsterdam, Leiden, Delft, Brussels and Paris; one can consider it as self-evident that he

* The actual date was March 8 because of the time lag then existing between the Russian and Western calendars. The time lag is the reason why the "Great October Revolution" happened on November 7, 1917.

486 discussed his method with his peers during these visits, particularly since we have evidence that he actually carried out various investigations during these visits Tswett's main interest was the study of the natural pigments and most of his papers - Senchenkova ( 4 ) lists 7 4 titles in the period of 1894-1919 - were devoted to this subject. As mentioned his main work was his Doctorate thesis published in 1910 as a book (12) which unfortunately, has never been translated into other languages (except as a private translation done for Willstatter within about a year of its publication, which he later gave to Richard Kuhn, his former student, professor at the ETH and then director of the Institute of Chemistry at the Kaiser Wilhelm Institute for Medical Research, in Heidelberg*). The organization of this book clearly illustrates Tswett's fields of interest: the first part is devoted to methodology (the chromatographic technique), the second part described his results on the study of natural pigments and, finally, the third part presented his ideas about the mechanism of photosynthesis. The remarkable feature of Tswett's work is the detail in which he studied the separation process and its variables. In fact, he warned future chromatographers to avoid certain pitfalls, e.g. activity of the adsorbent, and the possibility of chemical changes of the substances being chromatographed. Obviously his antagonists during his life did not read his warnings carefully! The basis of Tswett's column chromatography was adsorption. His interest in adsorption was, however, not restricted to this application; he studied adsorption in general and considered it a part of natural processes. The following quotation from Robinson's treatise ( 5 ) summarizes Tswett's thinking: "He not only used (the phenomenon of adsorption) as an analytical t o o l , he also invoked it to explain certain natural systems. For example, he proposed that the chloroplast pigments are adsorbed on the substratum of the granum and this is why nonpolar solvents cannot extract them (except carotene) from leaves although after extraction they are soluble in such solvents as benzene or ligroin The presence of alcohol breaks down the adsorption complex, allowing the pigment to be dissolved. He went so far as to perform

*.

In 1905, Tswett published a paper ( 1 0 ) criticizing the results of Molisch on the pigments of brown algae. One year later, in another paper on the same subject ( 1 1 ) he refers to the previous paper as based on his "investigations in Kiel". It is interesting to note that in this second paper he mentions that his investigations were partly carried out with the help of a new adsorption method he developed; as stated, until then this method was described only in Russian publications but a German publication will soon follow. Lookin at the dates of the publications, papers (11). ( 7 ) and (8) were obviously submitted about the same time. This translation was used by E. Lederer in late 1930 when he started his investigations on the chromatographic separation of xanthophylls (see Dr. E. Lederer's recollections in this volume).

487 model experiments showing that pigment mixtures adsorbed onto various artificial substrata behave just as they do in the leaf with regard to extraction. These models apparently led Tswett to develop his method of column chromatography, although the connection is certainly not very clear."

It is well known that Tswett's work was not appreciated in his lifetime. The reason for this is complicated and even although most of our sympathies lie with him, we have to understand the situation and scientific life of that period. First, it is clear that Tswett utilized techniques which were highly unconventional. As expressed by Zechmeister ( 2 ) , "Evidently this method (i.e. chromatography) was so original that it layed outside the border lines within which the chemists were accustomed to work." In this respect, it is worth quoting Willstatter who called Tswett's method "an odd way" ( 1 3 ) . The problem here, however, is deeper: Tswett represented a thinking which was far ahead of the philosophy of the organic chemists of his time. In the historical review I wrote three years ago with Dr. C . Horvhth of Yale University ( 1 4 ) we tried to follow the development of organic chemistry in the second part of the 19th century and the prewar period of the a0th century (dominated first by synthesis and by isolation and purification) in order to. explain this difference in philosophy, As we stated, "

... at this time, the keywords were still isolation and purification: isolation from the accompanying material and purification from trace impurities - this was done at that time by extraction and crystallization. Tswett's method just did not fit this interest: there was no real interest to isolate all the components present - one only wanted to isolate a few key components and there was really no desire or need to work with very small amounts "

.

It took some time until the interest of the new generation changed. This happened toward the end of the 1920's; as we said, finally

... the activities of the new generation ... brought forth the importance of total separation (i.e. separation of all components present), not just isolation of one or two major constituents, and the possibilities of carrying out this separation with small quantities. The rebirth of chromatography coincided with the great shift in chemical research from synthesis to analysis, a new way of thinking which has been dominant since then."

"

This is certainly one explanation why Tswett's method did not get immediate acceptance. It would, however, be an oversimplification to attribute the lack of acceptance of chromatography to this reason

488 only. Let us also not forget that in spite of his Swiss education and the knowledge of languages, Tswett was an "outsider" ("the botanist of Warsaw") for the close knit community of (mostly German) organic chemists and botanists. Furthermore, it should be clear that Tswett did not publish an "analytical method" although this is the impression one gets from reading the articles dealing with Tswett's activities*. The major subject of Tswett's publication is not chromatography but his studies (and theories) on chlorophylls and other plant pigments; chromatography is subordinate to this work. In this respect, we reach the most important reason why his work was not appreciated: Tswett showed results which, if proved correct,would have meant that prominent Western scientists of his time were wrong in their ideas about the natural pigments. Even more significant, he did not restrict himself to simply stating his result's: he named the others, pointed out the incorrectness in their work and debated their results. To use a colloquial expression, Tswett stepped on the toes of a number of important chemists in the Western scientific establishment. This fact resulted in a feud between Tswett and a number of contemporary scientists which lasted for about a decade. The most articulate among these was L. Marchlewski, at that time Professor of Medicinal Chemistry at Cracow University* and member of the Cracow Academy of Sciences. This controversy was often heated: Marchlewski called Tswett's whole work "volkommen falsch" (absolutely wrong) ( 1 6 ) , stated that the so-called chromatographic method would not last very long and that hopefully Tswett will realize that "one cannot with help of a filtration experiment swing oneself to the height of a reformer of chlorophyll chemistry" ( ' I ... jetzt wird er aber doch wohl eingesehen haben, dass man mit Hilfe eines Filtrationsversuches sich nicht auf die Hohe eines Reformators der Chlorophyllchemie schwingen kann") ( 1 7 ) . On the other hand, Tswett accused Marchlewski of not knowing the literature and even not reading carefully his (Tswett's) papers (18). Reading these polemic papers is like sitting next to the pzage during a fencing bout and watching the attack, parry and r i p o s t e of the fighters ... Other scientists opposed to Tswett included H. Molisch, professor of plant anatomy and physiology and director of the Institute of Plant Physiology at the German University of Prague, and F.G. Kohl, professor at the highly respected University of Marburg an der Lahn, in Cermany. I have already referred to Tswett's paper (10) on the Figments of sea algae; this paper was written to prove that Molisch was wrong in his investigations ( 2 9 ) . Molisch immediately published

* Including also my

earlier writing about Tswett ( 1 5 ) or Sakodynskii's paper ( 3 ) . The more I dig into the literature of the prewar period the more it is clear to me that in the appraisal of Tswett's work "chromatography" played a minor role. The whole controversy lasting for a decade was centered around his results in pigment chemistry which was diametrically opposed to the belief of his contemporaries; even worse, Tswett was proved to be right.

* That part

of Poland where Cracow is located was then part of the Austro-Hungarian Monarchy.

489 a strong rebuttal (20) to which Tswett responded ( 2 1 ) ; his answer was more scientific but no less polemic. By that time Kohl also entered the ring ( 2 2 ) ; four years earlier, he wrote a book on carotenepigments (22) and thus, he now felt himself an authority on plant pigments capable of teaching Tswett the facts of life. After repeating on many pages Molisch' results and calling him a "vorzuglicher Beobachter and Experimentator" (superb observer and experimenter), he simply pushes aside Tswett's critical remarks by saying that there is nothing new in Tswett's methods* and besides this, Tswett should at least quote his (Kohl's) book. We should not underestimate the effect of this heated exchange of papers since Marchlewski, Molisch and Kohl were well known and very active scientists of the prewar period. In addition, Tswett's disagreements also extended to the work of Richard Willstatter, then the "pope" of German organic chemistry. This controversy was much more dignified but no less substantial. Willstatter had just started his painstaking, thorough and very detailed investigations on chlorophylls when Tswett was already well advanced in the separation of these pigments. Tswett actually stated as early as 1901 that the so-called "crystallizable chlorophyll" is an artifact (23) while Willstatter still believed even in 1908 that it was a single, native pigment and conceded to the contrary only in 1912. Of course, finally,Willstatterachieved the same results as Tswett had done years earlier, and the irony of history is that the ultimate credit went to Willstatter receiving the 1915 Nobel Prize in Chemistry for his work on chlorophyll. The situation is best summarized by Robinson (5): "The chemists were unwilling to have their cherished conceptions overturned by data that were not obtained through standard chemical techniques. Willstatter's beautifully detailed, painstaking application of standard procedures to kilogram quantities of chlorophyll finally convinced everyone that Tswett had been right all along - or, rather, everyone accepted Willstatter as the founder of chlorophyll chemistry, adopted Willstatter's nomenclature, and forgot that Tswett had said the same thing years before - indeed, had said them when Willstatter had been arguing the contrary!" The controversy with Willstatter was not restricted to chlorophyll chemistry, he was also negative concerning chromatography as a separation technique. His opinion was based on the observation that sensitive natural substances sometimes undergo chemical changes on the column. This is, naturally, true and Tswett also emphasized it in his papers. The problem here was simple: Willstatter's students simply ignored Tswett's warnings and used too active sorbents.

* This does not

refer to chromatography; Tswett in the first polemic paper against Molisch ( 1 0 ) does not mention separation by adsorption chromatography. This is mentioned only in the second paper ( 1 2 ) but without going into details. Kohl's paper referred only to the first Tswett paper.

Today, almost 70 years later, one may easily draw the conclusion: Tswett, the underdog, was right but pushed aside by the establishment. This is true but represents an oversimplification of the facts. Tswett was simply ahead of his time and thus, he was deemed for nonacceptance. A change of philosophy was needed to have his technique and investigations fully appreciated. This happened at the end of the 1920's when the exponential evolution of chromatography finally began. The best appraisal of the importance of chromatography came from Paul Karrer, the great organic chemist and co-winner of the 1937 Nobel Prize in Chemistry, in his plenary lecture at the 1947 Congress of the International Union of Pure and Applied Chemistry ( 1 4 ) . "No other discovery has exerted as great an influence and widened the field of investigation of the organic chemist as much as Tswett's chromatographic adsorption analysis."

L . S . PaZmer (1887-1944) After Tswett's death his technique was used by only a few scientists. Among these, the activities of L.S. Palmer, an American scientist, are most important. LEROY SHELDON PALMER was born in Rushville, Illinois (U.S.A.) in 1887, but his parents soon moved to St. Louis, Missouri and he grew up there. He studied at the University of Missouri, in Columbia. It is interesting to note that Palmer's undergraduate training was not in a field particularly related to his future research work but in chemical engineering and his first two papers - coauthored with his twin brother dealt with electrochemical analysis and electrolytic preparation of explosive antimony. Soon, however, he switched to dairy chemistry joining in 1909 the Dairy Research Laboratory at the University and starting his graduate research under Dr. C.H. Eckles, head of the Department of Dairy Husbandry. In Fig. 5 7 . 3 . L.S. Palmer. a few years, Palmer became the head of the Laboratory and member of the faculty of the University. In 1919, he followed Eckles to the University of Minnesota, in St. Paul, where he served for almost 25 years in various capacities, the last being professor and head of the Division of Agricultural Biochemistry. He died there in 1944 when only 57 years old. Palmer's major interest was dairy chemistry which was later extended to animal nutrition problems. In these fields, he was con-

491 sidered as a world-renowned authority: he was the vice-president of the 1923 World Dairy Congress and the first recipient of the Borden Award in Dairy Chemistry (1939). Palmer's doctoral thesis was a fundamental study on "Carotin the Principal Natural Yellow Pigment of Milk Fat". He started to work on it in 1911 (and probably even earlier) finishing in 1913; it was published the following year in five papers coauthored with Eckles ( 2 4 ) . This work was followed by a number of papers on yellow pigments in a wide variety of biological substances and animal products and their relationship with the growth, fecundity and reproduction of animals. Palmer based his work on precise measurements of the individual pigments and for this, he used column adsorption chromatography extensively. In the first publication (24a) he described in detail the chromatographic technique of Tswett, stating that "Tswett has made a beautiful application of this discovery (i.e.,chromatography) to a solution of carotin, xanthophylls and chlorophyll pigments". In the early 1920's, the American Chemical Society decided to begin publishing monographs and the first book was written by Palmer on Carotenoids and Related Pigments (25). Besides giving a very thorough summary of his own work, and the work of others in this field, he discussed in detail Tswett's work and technique. Moreover, he emphasized that a single chromatographic separation is not yet an absolute proof of identity since further examination of the fractions is needed, and that often a chromatographic fraction has to be rechromatographed to obtain high purity. Palmer knew Tswett's major papers which appeared in German and French journals; he also gave reference to Tswett's book ( 1 2 ) but he gives its title in French indicating that he took the reference from another source, perhaps Tswett's papers published in French. Palmer's book was the standard reference for many years on carotenoid chemistry until Zechmeister's book (26) replaced it. Without any doubt, it influenced a number of chemists in selecting chromatography as the separation technique and it specifically influenced E. Lederer who learned about the existence of Tswett's method when reading Palmer's book. In this way, Palmer's monograph represents the bridge to the "rebirth" of chromatography, to the evolution we have been experiencing since then.

L. Zechmeister (1889-1972) When in 1931 the papers of E. Lederer, Winterstein and Kuhn on the utilization of column chromatography to the separation of xanthophylls and other pigments were published, the technique was picked up by many laboratories within a short time. Besides the Heidelberg group, the scientist who probably played the most important role in making chromatography widely known and accepted as a scientific tool was L. Zechmeister.

LASZL~ ZECHMEISTER was born in 1889, in Gy&, Hungary; his father was for 19 years the great reforming mayor of that city whd can be credited with its evolution to one of the major industrial centers of Hungary. He studied at the Eidgenossische Technische

492 Hochschule, in Zurich under Richard Willstatter receiving his Dr.-ing. degree in 1913. He followed Willstatter to the Kaiser Wilhelm Institut fiir Chemie, in Berlin-Dahlem. At the outbreak of the World War, he was drafted in the Hungarian Army, captured on the Russian Front and kept in a prisoner-of-war camp in Siberia until 1918 when he rejoined Willstatter in Munich (he took over Baeyer's chair in 1915). For several years he worked at the laboratories of the Danish Agriculture and Veterinary Academy in Copenhagen and finally, in 1923, he was appointed professor and director of the Chemical Laboratory at the University of P ~ c s , in Hungary where he remained until 1940. In that year he emigrated to the U . S . A . (on the last ship Fig. 57.4. L. Zechmeister. leaving Italy) and became professor of organic chemistry at California Institute of Technology, in Pasadena; he retired in 1959 with the rank of professor emeritus. He died in Pasadena in 1972. Zechmeister was an excellent organic chemist, a superb organizer and teacher and an internationally recognized authority on the chemistry of carotenoids and chromatography. His interest in the investigation of natural substances originated from his early work with Willstatter and he never deserted this subject. His first years at P6cs University were mostly devoted to organizational problems, building up the Institute* and establishing the proper curriculum (e.g., Zechmeister wrote the first modern textbook on organic chemistry in Hungarian!) and he could return to his favorite subject only around 1930. It is impossible to establish whether his use of chromatography is practically parallel to the work of Kuhn's group (let us not forget Palmer's book on carotenoids, the same subject as Zechmeister's:) or is due to his contact with them; however, the fact is that his book on carotenoids (26) published in 1934 already dealt in detail with the principles and application of the method. In 1937 he published the book on chromatographic adsorption method (27) coauthored with L. Cholnoky (his collaborator at P6cs University who later took over his chair) and this volume significantly contributed to the expansion of liquid adsorption chromatography in Europe. It became a scientific bestseller and within a year, a second edition

* The University

of P6cs was a successor to the University of Pozsony (Bratislava). Due to the peace treaties after World War I., Northern Hungary became part of the newly formed Czechoslovak Republic and the former Hungarian university was relocated to P6cs. Naturally, everything had to be organized from scratch and it took years until normal research work could be started.

493 had to be prepared. It was soon translated into English (28) and together with H . H . Strain's book published in 1942 - played an important role in the development of the chromatographic methods in the U.S.A. His next book summarizing the development up to 1947 ( 2 9 ) further helped in the widespread application of the technique. However, Zechmeister not only contributed to the development of chromatography; he also successfully applied it in his own field, the investigations of carotenoids, enzymes and other complex organic molecules. He never considered the development of the technique alone as his particular merit but rather the combination of its development with the applications in the investigation of complex natural substances. Chromatography was not the ultimate subject of his work but represented only the means to achieve his goals. In this respect the following quotation (30) from his lecture held on November 3, 1950, at the meeting of the Southern California Section of the American Chemical Society is most characteristic: "Recently chromatography became so popular that the English language has been enriched by a new noun "the chromatographer". I would protest against such a label. In research chromatography should be considered first of all a tool like e.g. fractional distillation; and those of our colleagues who have achieved success by using distillation methods should certainly not be named "distillers". When, in 1962, he received the American Chemical Society Award in Chromatography and Electrophoresis he selected as the subject of his address "Column Chromatography and Geometrical Isomerism" thus combining the development of the technique with one of its most important applications related to his name, the separation of certain cis and trans isomers. Being from the same country, I cannot deny a partiality to Dr. Zechmeister. His integrity and excellence was well known when I studied in Budapest, even although he was no longer in Hungary. Unfortunately, I never met him personally although I had some correspondence with him in the last years of his life, asking him to give a lecture at a symposium, telling his recollections of the early years of chromatography. He could not accept the invitation due to illness and it is sad that we never had the opportunity to hear (or read) his story. In recent years I have tried to collect data on Dr. Zechmeister's life and activities, and a year ago,in connection with this, I visited the Archives of California Institute of Technology, in Pasadena, where his files are kept. There, I found some correspondence which probably illustrates in the best way his character (30). Soon after the publication of the English translation of his book he saw that a trade publication - the newsletter of a laboratory supply house - reproduced a figure from the book (without permission) and changed its caption with the new text being technically incorrect and misleading. He immediately wrote to the publisher requesting that they demand cor-

rection. When the publisher answered that this was a small item not worthy of bother and, besides, the utilization of a figure in a publication with a wide circulation really represents good publicity Dr. Zechmeister immediately replied (his letter of April 20, 1942 to J . Wiley & Sons): "

. . . my duty as a scientist is to fight against any distortion of If you say that the article in question any scientific truth represents "precisely the sort of publicity which we solicit", I may remark that the distortion of scientific facts, established by long years of effort is just the sort of publicity which no scientist can desire."

...

I believe one does not need to add a single word to this statement.

In the early 1940's column chromatography became a generally accepted and used technique and a number of variants had already been tried in several laboratories. Principal credit is due to Tiselius and his school for formulation and characterization of the principles of the various chromatographic techniques and improvements in methodology. He also contributed significantly to biological analysis by developing electrophoresis - a variant of chromatography and its applications in biochemistry. Finally, as an excellent organizer of scientific research, Tiselius can be credited with building up a whole school of researchers in biochemistry and separation science. For his achievements he received the 1948 Nobel Prize in Chemistry. ARNE WILHELM KAURIN TISELIUS was born in 1902, in Stockholm, Sweden. He studied at the University of Uppsala and spent his whole scientific career at this university, beginning in 1925 as a research assistant to The Svedberg. In 1934-1935 he spent a fellowship in the Frick Chemical Laboratories at Princeton, New Jersey (U.S.A.). In 1937 he became the first recipient of the newly created Karin and Herbert Jacobsson Professorship in Biochemistry. In 1946, biochemistry was established as an independent department and in 1952, Tiselius took over a new Institute of Biochemistry which enabled him to engage a large number of researchers to carry out their seemingly different investigations concomitantly. He strongly Fig. 57.5. A W.K. Tiselius. believed in the cross-fertilization of ideas coming from the personal, day-to-

495 day contact of researchers. He died in 1971 while still active in the Institute. Tiselius is generally credited with a number of significant achievements in the fields of adsorption chromatography and electrophoresis. His activities in electrophoresis started in the second part of the 1920's and his doctoral thesis (1930) already dealt with "The Moving-Boundary Method of Studying Electrophoresis of Proteins." He resumed his interest in this subject upon his return from Princeton and by 1937 he could significantly improve the technique (32). The best summary of the importance of Tiselius' work in this field was given by Professor A. Westgren, Chairman of the Nobel Committee for Chemistry of the Royal Swedish Academy of Sciences when introducing him before he received the 1948 Nobel Prize in Chemistry (32): "Tiselius has made many discoveries of far-reaching effect by applying his method of electrophoresis; that globulin, a protein of blood serum, was not an entirely homogeneous substance, had already been supposed: Tiselius succeeded in separating this seroglobulin into three distinct parts, each comprising slightly different groups of molecules. This finding is at the root of research work of the utmost importance for practical medicine, which was carried out in the United States during the last World War - research work aimed at dividing human blood plasma into fractional parts. If American scientists had not had at their disposal Tiselius' method as a control, they would probably have failed when they tried to solve that problem. In the course of their research work on electrophoresis, Tiselius and his collaborators also carried out experiments of great medical value on the antibodies of a protein nature which are formed in the blood during immunization". The second major field to which Tiselius and his coworkers contributed is adsorption chromatography. In the systematic study carried out in the 1940's - mostly with Claesson - the three basic techniques were defined and their theory developed. These are: frontal analysis (without a carrier); elution analysis (with a pure solvent as the carrier); and displacement development (using a more strongly adsorbed displacer). Their work has been published in a number of very detailed papers (33-38) and summarized by Tiselius ( 3 9 ) and Claesson ( 4 0 , 4 2 ) . In addition to improvements in methodology, the instrumentation has also been significantly improved, most notably by continuous monitoring of the column effluent with help of an interferometer ( 4 2 ) . Another major achievement of Tiselius' laboratory was the introduction of gradient elution in liquid chromatography ( 4 3 ) . Without this technique modern high-performance liquid chromatography would be impossible. In the 1950's Tiselius and coworkers started to investigate "molecular sieving" methods for the separation of biologically important substances on gels. These investigations led to the development of a number of gel types for affinity chromatography. This was also

496 the time when more-and-more researchers from all over the world came to Tiselius' laboratory to work there for some time. Tiselius always believed that, by letting his collaborators deal with a self-selected subject, something that fascinated him, the maximum could be achieved. As Dr. Hjerth ( 4 4 ) quotes him: "the best can be accomplished only if it is pleasing and involves a creator's joy." He will always be universally esteemed by scientists working in biochemistry and chromatography.

F.H. Pollard

(2907-1965)

Chromatography began as a technique for separating organic substances. Its application in inorganic chemistry originated in the 1930's by G.-I. Schwab in Munich, and was continued by M. Lederer, at the Radium Institute, in Paris, and by P.H. Pollard, in England, at the University of Bristol. FREDERICK HENRY POLLARD was born in 1907, in Swindon, Wiltshire, England. He studied at the University of Bristol graduating with a special Honours Degree in Chemistry in 1927. After taking his Diploma in Education in 1928, also at Bristol, he was involved in grammar and high-school teaching for 13 years. During this time he finished his Ph.D. with research on the infrared spectra of carbon monoxide flames. In 1941 he accepted a post as a lecturer in inorganic chemistry at Bristol and remained there in increasingly responsible positions until his sudden death in 1965. During the war, he was involved in research on explosives, studying among others the reactions of the oxides of nitrogen with formaldehyde or acetic acid. This interest, together with his experiences in teaching led him into the field of inorganic chemistry research and he soon became convinced that more powerful analytical methods would greatly help this field. He began his activities in analytical chemistry by investigating how paper and ion-exchange chromatography could be applied to the analysis of inorganic compounds and his emphasis has always been on quantitative analysis. Dr. Pollard published with his collaborators a large number of excellent papers opening new avenues in inorganic analysis. The book "Chromatographic Methods of Inorganic Analysis'' coauthored with J . F . W . McOmie represented a relatively early summary of his results ( 4 5 ) . He also utilized chromatography for the study of reaction mechanisms. His investigations on the reactions of various phosphorus compounds should serve as an illustration of this activity; for example, Part I (46) dealt with the hydrolysis of triphosphonitrilic chloride, Part VII ( 4 7 ) with the hydrolysis of phosphorus trihalides and pseudohalides, and Part XI ( 4 8 ) with the reaction of phosphoryl chloride and sodium hydroxide. Dr. Pollard joined at an early stage of the development the group of scientists interested in gas chromatography. The first paper in this field coauthored by him is entitled "The Effect of Temperature of Injection Upon the Separation of Lipid Mixtures by Gas-phase

491 Chromatography" ( 4 9 ) , and is most probably the first systematic study of the effect of injector volume and temperature on column efficiency and the detectable limit. His thoroughness is best demonstrated by the review on the state-of-art of GLC published in 1959, coauthored with C.J. Hardy (50) which was based on 619 references. His training in inorganic and physical chemistry and in the study of reactions also became evident in his various studies utilizing gas chromatography, such as e.g. the application of the technique to obtain activity coefficients of alkanes, alkyl nitrates, nitroalkanes and alcohols ( 5 2 ) or in the study of organometal compounds (Si, Ge, Sn, Pb) (52-54).

Dr. Pollard was always interested in the preparation of novel stationary phases, and his last project was related to fixing the liquid phase chemically to the support. Today, most chromatographers do not realize that the possibility of producing bonded phases which are now universally used in liquid chromatography was first demonstrated in his laboratory, as part of the Ph.D. thesis work of P.C. Uden (now professor at the University of Massachusetts, in Amherst). Their aim was to eliminate the bleeding of the liquid phase and they utilized the reaction of n-hexadecyl trichlorosilane with Celite to create a Cl6-Celite type column packing. The degree of their success is best demonstrated by the comparison of theoretical and actual values: stoichiometric calculations resulted in an expected 14.5% (w/w) organic content of the final column packing while the actual value found was 13.9%. They finished writing the preliminary communication and then Dr. Pollard flew to Athens, to attend the International Symposium organized by the Union of Greek Chemists and the G.A.M.S., the French Analytical-Chemical Society, (September 19-24, 1965) where he presented an excellent review dealing with the "Critical Comparison of ThinLayer and Paper Chromatography for the Separation of Inorganic Compounds" ( 5 5 ) . I met Dr. Pollard there, and during one of the receptions he mentioned to me the great potentials of these packings. Their manuscript was mailed to the editor of the Journal of Chromatography while he was still in Athens, arriving there on September 26 (56). Within one month, Dr. Pollard was dead, leaving a life's work unfinished. -0-o-o-

Until now, I have dealt almost exclusively with pioneers involved in liquid chromatography although Dr. Pollard already represented the transition from this to the rapidly growing field of gas chromatography. The last two scientists I would like to discuss in this final chapter of our book carried out their activities exclusively in gas chromatography. It is not easy for me to discuss them; for while I never met those mentioned earlier (except Dr. Pollard whom I have met once), I was quite close to the last two, both professionally and personally. I am speaking here about Lou Keulemans and Steve Dal Nogare. Both exercised a lasting contribution to the evolution of gas chromatography.

A. 1.M. XeuCemans (1908-1977) ALOYSIUS IGNATIUS MARIA KEULEMANS was b o r n i n 1 9 0 8 , i n R o t t e r d a m , The Netherl a n d s . H e f i r s t s t u d i e d mathematics a t L c i d e n U n i v e r s i t y f i n i s h i n g i n 1932 and t h e n s t a r t e d work f o r a l i f e i n s u r a n c e company. However, a f t e r t h r e e y e a r s h e u r . e x p e c t e d l y became unemployed due t o t h e amalgamation of t h e company w i t h o t h e r s . He t h e n t o o k a t e a c h i n g j o b i n mathemati c . 9 and s t a r t e d t o s t u d y c h e m i c a l e n g i n e e r i n g a t t h e U n i v e r s i t y o f Technology i x . D e l f t . A f t e r r e c e i v i n g h i s M.S. d e g r e e ic. 1938 h e j o i n e d K o n i n k l i j k e / S h e l l Labo r a t o r i e s i n Amsterdam and worked on c o . t a l y t i c problems w h i l e a l s o p r e p a r i n g h i s Ph.D. t h e s i s o n t h e " I s o m e r i z a t i o n and Thermodynamic S t a b i l i t y o f Hydroc a r b o n s " . H e s u b m i t t e d i t i n 1942 j u s t b e f o r e t h e u n i v e r s i t y was c l o s e d due t o ~ ~r e s i s t a n c ~ e a g a i n s t lt h e German ~ o c c u p a t i~ on ~ i ~ , 5 7 . 6A.I.M. . and t h u s , h e c o u l d r e c e i v e t h e d o c t o r ' s d e g r e e o n l y i n 1945. A f t e r t h e War, h e worked on t h e improvement of d e t e r g e n t s and on problems a s s o c i a t e d w i t h t h e OX0 p r o c e s s , and took an a c t i v e i n t e r e s t i n a n a l y t i c a l problems o c c u r r i n g i n u p s c a l i n g chemical processes. I n 1 9 5 2 , D r . Keulemans v i s i t e d A.J.P. M a r t i n , i n London, and immediately r e a l i z e d t h e i m p o r t a n c e o f g a s c h r o m a t o g r a p h y : i n a few weeks h e b u i l t a g a s chromatograph and s t a r t e d h i s a c t i v i t i e s i n promoting g a s chromatography which changed h i s l i f e . I n 1958 he was named p r o f e s s o r a t t h e new U n i v e r s i t y o f Technology a t Eindhoven and t h e head o f t h e L a b o r a t o r y o f I n s t r u m e n t a l A n a l y s i s . Toward t h e middle of t h e 1 9 7 0 ' s h i s h e a l t h s t a r t e d t o d e t e r i o r a t e and t h i s f i n a l l y f o r c e d him t o r e t i r e i n 1977. H e d i e d on F e b r u a r y 1 9 , 1977. Lou Keulemans i s one o f t h e r e a l p i o n e e r s i n g a s chromatography and h i s book p u b l i s h e d i n 1957 (57) was f o r a number of y e a r s t h e b i b l e f o r everybody working i n GC; i t w a s t r a n s l a t e d i n t o s i x l a n g u a g e s , among them R u s s i a n and J a p a n e s e . H i s p r i n c i p a l m e r i t h a s been t h e d i s s e m i n a t i o n o f i n f o r m a t i o n and t h e exchange of i d e a s . H e t r a v e l e d f r e q u e n t l y and t i r e l e s s l y , c o n t i n u o u s l y d i s c u s s i n g t h e newest a c h i e v e m e n t s w i t h s c i e n t i s t s a t v a r i o u s p l a c e s and a d v i s i n g one how o t h e r s had s o l v e d a p a r t i c u l a r p r o b l e m . H e had an e x c e l l e n t a b i l i t y t o s e l e c t and m o t i v a t e c o l l a b o r a t o r s and a t Eindhoven he b u i l t up a s c h o o l o f worldwide fame: 150 M.S. and 20 Ph.D. d e g r e e s w e r e g r a n t e d t o h i s s t u d e n t s and more t h a n 200 p a p e r s o r i g i n a t e d from h i s i n s t i t u t e . Lou Keulemans was a s t r o n g b e l i e v e r t h a t t h e World s h o u l d n o t b e d i v i d e d by n a t i o n a l and p o l i t i c a l b o u n d a r i e s . I n 1969 h e founded

~

499

together with C. Hamilton (of syringe fame) the Scientific Exchange Agreement (S.E.A.). This organization encourages the exchange of young scientists between Eastern and Western Europe and he personally can be credited with its great success. S. DaZ Nogare (1922-1968)

By 1958, gas chromatography reached a certain plateau and its limitations became clear. The introduction of the ionization detectors, capillary columns and temperature programming obviated the limitations and started a new, exponential development which is still continuing. Of these three achievements, the development of temperature programming is usually identified with s. Dal Nogare. To be sure, he did not "invent" the technique; it was first used as early as 1952 by C.S.G. Phillips at Oxford University, and Burrell Corporation of Pittsburgh, Pennsylvania actually had a commercial instrument permitting such an operation by about 1958. Dal Nogare did, however, show for the first time the advantages of linear temperature programming. STEPHEN DAL NOGARE was born in 1922 in Marostica, Vicenza (near Venice), in Italy and was still a child when his parents emigrated to the U.S.A. He studied at Beloit College, in Beloit, Wisconsin, and the University of Wisconsin receiving his Ph.D. in analytical chemistry in 1947. He then joined E.I. du Pont de Nemours, Inc., first at their Research Division, in Arlington, N.J. and then from 1950 at their Experimental Station, in Wilmington, Delaware. In 1949-1950 he spent a postdoctoral year at Princeton University. In the Fall of 1967 he joined the faculty of Virginia Polytechnic Institute, in Blacksburg, Virginia. His tragic death occurred there in January 1968. Dr. Dal Nogare's activities in gas Fig. 57'7' s' Dal chromatography at DuPont started with the development of special instruments for high-temperature operation (58) and high-sensitivity work ( 5 9 ) . As a continuation of these activities he carried out a pioneering work demonstrating the advantages of linear temperature programming (60-63). His coworkers, learning the technique from him, were the founders of F&M Scientific Corporation which was instrumental in making the technique everybody's tool. Steve also made other important contributions to gas chromatography such as the practical interpretation of the theory of gas chromatography, demonstration of the importance of the phase ratio on resolution and efficiency and the optimization of column parameters (64). He was for a number of years the author - together with R.S.

500 Juvet Jr. - of the biannual Analytical Chemistry reviews on gas chromatography. However, probably his most important contribution besides temperature programming is the book on gas-liquid chromatography written in cooperation with R.S. Juvet and published in 1962 (65) which remained the standard reference book fur many years. Steve was probably the best known American gas chromatographer for a decade, a kind of Mr. GC. He was in great demand as a lecturer, and tried to help everybody. His warm personality and friendly manners were known to everybody. His sudden death in 1968 was a great loss to his friends and to the field of gas chromatography, ACKNOWLEDGEMENT

I would like to express my appreciation to Dr. C. Horvhth of Yale University and the Kline Science Library of that University for helping in searching for the originals of the old publications. REFERENCES C. DhBrC, Condollea 10 (1943) 23. L. Zechmeister, I s i s 36 (1946) 108. K.I. Sakodynskii, J . Chromatogr. 7 3 (1972) 303. E . M. Senchenkova , byikhai 2 Semenovich Tsvet, Nauka, Moscow, 1973. T. Robinson, Chymia 6 (1960) 146. M.S. Tswett, T r . f i o t o k . Varshav. Obshch. E s t e s t v o i s p y t . Otd. B i o l . 14 (1903; publ. 1905). Reprinted in English translation in G . Hesse and H. Weil , .Viehael Tswett ' s F i r s t Paper on Chromatography, M. Woelm, Eschwege, 1954. 7 M.S. Tswett, Ber. Dtsch. Bot. Ges. 2 4 (1906) 316. 8 M.S. Tswett, Ber. Dtsch. Bot. Ges. 24 (1906) 384. 9 Ber. Dtsch. S o t . Ges. 25 (1907) 217, 267. 6 3 (I) (1905) 273. 10 M.S. Tswett, Bat. 11 # . S . Tswett, Ber. Dtsch. Bot. Ges. 24 (1906) 235. 12 M . S . Tswett, Khromofilly u R a s t i t e l ' n o m i Zhivotnom Mire (Chromophylls in the Plant and Animal World), Izd. Karbasnikov, Warsaw, 1910, 380 pp. 13 R. Willstatter and A. Stoll, Yntersuchungen iiber Chlorophyll; ?&thoden and Ergebnisse, Springer, Berlin, 1913; American edition: I n v e s t i j a t i o n s on Chlorophy 21; Methods and R e s u l t s translated by F.M. Schwertz and A.R. Merz, Science Press, Lancaster, Pa., 1928; the quotation is on p. 14 of the translation. 14 L.S. Ettre and C. Horvhth, Anal. Chem. 47 (1975) 422A. 15 L.S. Ettre, A m l . Chem. 43 (14) (1971) 20A. 16 L. Marchlewski, Biochem. 2. 3 (1907) 287. 17 L. Marchlewski, Ber. Dtsch. Bot. Ges. 25 (1907) 225. 18 M.S. Tswett, Fer. Ptsch. Rot. Ges. 25 (1907) 71. 19 H. Molisch, B o t . .?. 63 (I) (1905) 131. 20 H. Molisch, Boz. 2 . 6, ( 1 1 ) (1905) 369. 21 F.G. Kohl, o'er. !Xsch. Bot. Ges. 24 (1906) 124. 22 F.G. Kohl, Untersuchungen Uber das Carotih und s e i n e p h y s i o l o g i s c h e B e d e u t m g ir. der Pjlanze, Borntraeger Verlag, Leiuzig, 1902. 1 2 3 4 5 6

z.

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