The Science of Chromatography (Journal of Chromatography Library)

JOURNAL OF CHROMATOGRAPHY LIBRARY

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volume32

the science of chromatography lectures presented at the A.J. P. Martin honorary symposium, Urbino, May 27-31, 1985

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JOURNAL OF CHROMATOGRAPHY LIBRARY

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volume 32

the science of chromatography lectures presented at the A J F! Martin honorary symposium, Urbino, May 2 7-37, 7985 edited by Fabrizio Bruner lstituto d i Scienze Chimiche, Universit; d i Urbino, Piazza Rinascimento 6, 6 1029 Urbino, Italy

ELSEVl E R Amsterdam - Oxford - New York - Tokyo 1985

ELSEVIER SCIENCE PUBLISHERS B.V. Molenwerf 1 P.O. Box 21 1,1000 A € Amsterdam, The Netherlands Distributors for the United States and Canada:

ELSEVIER SCIENCE PUBLISHING COMPANY INC. 52, Vanderbilt Avenue New York, N Y 10017

ISBN 044442443-1 (Val. 32) ISBN 0444-41616-1 (Series) 0 Elsevier Science Publishers B.V., 1985 All rights reserved. N o part of this publication may ? repro iced, store in a retrieval system or transmitted in any form or b y any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V:/Science & Technology Division, P.O. Box 330, 1000 A H Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts, Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred t o the publisher. Printed i n The Netherlands.

V

CONTENTS Journal o f Chromatography L i b r a r y ( o t h e r volumes i n t h e s e r i e s ) Foreword

A.J.P.

......

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

XI

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

XV

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

XVII

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

XX

Martin..

Contributors

VII

Acknowledgements

B. Koppenhoefer and E. Bayer, C h i r a l r e c o g n i t i o n i n gas chromatographic a n a l y s i s o f enantiomers on c h i r a l p o l y s i l o x a n e s

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

1

.....

43

K. Biemann, The mass s p e c t r o m e t e r as a d e t e c t o r i n chromatography

P.A.

L e c l e r c q , C.P.M.

S c h u t j e s and C.A.

s e n s i t i v e c a p i l l a r y GC/MS.

Cramers, Roads t o f a s t e r and more

A p p l i c a t i o n o f 50 um columns

........

55

..

67

.

87

.......

111

E. Cremer, H i s t o r y and s p e c i a l A u s t r i a n c o n t r i b u t i o n s t o chromatography

L.S.

E t t r e , The f u l l u t i l i z a t i o n o f t h e v a r i a b l e s o f o p e n - t u b u l a r columns

E. G i l a v , S e l e c t o r s f o r c h i r a l r e c o g n i t i o n i n chromatography

P. Rouchon, M. Schoenauer, P. V a l e n t i n and G. Guiochon, Une n o u v e l l e s i m u l a t i o n numerique de l a p r o p a g a t i o n d ' u n s o l u t e dans une colonne de chromat o g r a p h i e e n regime non l i n 6 a i r e : s c h B a de Godounov e t sch6ma a n t i diffuse

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

131

H. Hatano, H i s t o r i c a l i n t r o d u c t i o n and g e l p a c k i n g m a t e r i a l s f o r HPLC

s e p a r a t i o n o f p r o t e i n s and n u c l e i c a c i d s

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

..

179

..........

205

Cs. HorvBth, Displacement chromatography: y e s t e r d a y , today and tomorrow

E.

sz. KovBts, R e t e n t i o n i n l i q u i d / s o l i d chromatography

165

A. L i b e r t i and P. C i c c i o l i , Chromatography f o r t h e e v a l u a t i o n o f t h e atmospheric environment S.R.

-

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

219

L i p s k y , The f u s e d s i l i c a g l a s s c a p i l l a r y column f o r gas chromatography The anatomy o f a r e v o l u t i o n

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

257

VI

M a r i n i B e t t o l o and C. G a l e f f i , D i s c o n t i n u o u s systems i n t h e c o u n t e r

G.B.

...

283

..........,.....

305

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

333

c u r r e n t d i s t r i b u t i o n (CDD). The use o f d i s c o n t i n u o u s m o b i l e phases

M. Novotny, M i n i a t u r i z e d s e p a r a t i o n systems C.S.G.

P h i l l i p s , Chromatography beyond a n a l y s i s

V. P r e t o r i u s , K. Lawson, E. Rohwer and P. Apps, The s o l v e n t e f f e c t i n gas l i q u i d chromatography

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

347

J.H. P u r n e l l , Window a n a l y s i s : an approach t o t o t a l o p t i m i s a t i o n i n chromatography

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

363

P. Sandra, From widebore v i a narrowbore and u l t r a narrowbore t o widebore columns i n c a p i l l a r y gas chromatography. A p o t p o u r r i ? E.D.

Katz, K. Ogan and R.P.W.

.........

381

.....

403

S c o t t , Chromatography column d e s i g n

M. V e r z e l e and C. Dewaele, M i n i a t u r i z a t i o n o f h i g h performance l i q u i d

chromatography (micro-HPLC)

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

435

A. Z l a t k i s , S. Weisner, L. Ghaoui and H. S h a n f i e l d , Trace gas chromato-

..........

449

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

461

g r a p h i c techniques below t h e p a r t - p e r - b i l l i o n l e v e l F.S. Rowland, E a r t h ' s changing atmosphere

VII

JOURNAL OF CHROMATOGRAPHY LIBRARY A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications Although complementary to the Journal of Chromatography, each volume in the Library Series is an important and independent contribution in the field of chromatography and electrophoresis. The Library contains n o material reprinted from the journal itself.

Other volumes in this series Volume 1

Chromatography of Antibiotics (see also Volume 26) 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 Modern Techniques and Applications edited b y Z. Deyl, K. Macek and J. Janak

Volume 4

Detectors in Gas Chromatography by J. gevrik

Volume 5

Instrumental Liquid Chromatography. A Practical Manual o n High-Performance Liquid Chromatographic Methods (see also Volume 2 7 ) b y N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications b y 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 b y E. Heftmann

Volume 9

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

Volume 10

Gas Chromatography of Polymers

by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya Volume 11

Liquid Chromatography Detectors by R.P.W. Scott

Volume 1 2

Affinity Chromatography by J . Turkova

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

VIII Volume 1 7

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

Volume 18A Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques edited by Z. Deyl Volume 1 8 B Electrophoresis. A Survey of Techniques and Applications. Part B : Applications edited by Z. Deyl Volume 19

Chemical Derivatization in Gas Chromatography by J. Drozd

Volume 20

Electron Capture. Theory and Practice in Chromatography edited by A. Zlatkis and C.F. Poole

Volume 21

Environmental Problem Solving using Gas and Liquid Chromatography by R.L. Grob and M.A. Kaiser

Volume 22A Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part A: Fundamentals edited by E. Heftmann Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part B: Applications edited by E. Heftmann

Volume 23A Chromatography of Alkaloids. Part A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte Volume 23B

Chromatography of Alkaloids. Part B: Gas-Liquid High-Performance Liquid Chromatography by R. Verpoorte and A. Baerheim Svendsen

Volume 24

Chemical Methods in Gas Chromatography by V.G. Berezkin

Volume 25

Modern Liquid Chromatography of Macromolecules by B.G. Belenkii and L.Z. Vilenchik

Volume 26

Chromatography of Antibiotics Second, Completely Revised Edition by G.H. Wagman and M.J. Weinstein

Volume 27

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods Second, Completely Revised Edition by N.A. Parris

Volume 28

Microcolumn High-Performance Liquid Chromatography by P. Kucera

Volume 29

Quantitative Column Liquid Chromatography. A Survey of Chemometric Methods by S.T. Balke

Volume 30

Microcolumn Separations. Columns, Instrumentation and Ancillary Techniques edited by M.V. Novotny and D. Ishii

Chromatography and

IX Volume 31

Gradient Elution in Column Liquid Chromatography. Theory and Practice by P. Jandera and J. Chur6Eek

Volume 32

The Science of Chromatography. Lectures Presented at the A.J.P. Martin Honorary Symposium, Urbino, May 27-31.1985 edited by F. Bruner

X

Urbino i n t h e seventeenth century

XI

FOREWORD

" T r i s t o d quel discepolo che non avanza il suo maestro". ("Wretched i s t h e p u p i l who does n o t b e t t e r h i s master".) Leonard0 da V i n c i

Archer John P o r t e r M a r t i n r e c e i v e d t h e Nobel P r i z e i n Chemistry i n 1952, t o g e t h e r w i t h R.L.M.

Synge, f o r t h e i n v e n t i o n o f p a r t i t i o n chromatography.

Also i n 1952, D r . M a r t i n p u b l i s h e d w i t h A.T.

James t h e f i r s t paper d e s c r i b i n g

t h e r e s u l t s obtained b y u s i n g a gas as t h e mobile phase i n p a r t i t i o n chromatography, demonstrating t h e enormous a n a l y t i c a l p o s s i b i l i t i e s o f vapour-phase p a r t i t i o n chromatography. The s c i e n t i f i c community i s h i g h l y indebted t o D r . M a r t i n who c o n t r i b u t e d i n a d e c i s i v e way n o t o n l y t o advances made i n t h e l a s t f o r t y years i n a n a l y t i c a l chemistry and biochemistry, b u t a l s o t o developments i n t h e e n t i r e f i e l d o f chemical and l i f e sciences. I n 1985, D r . M a r t i n c e l e b r a t e s h i s 75th b i r t h d a y . Such an event should n o t pass w i t h o u t an adequate c e l e b r a t i o n and I am g l a d t o have been a b l e t o propose honouring him w i t h an I n t e r n a t i o n a l Symposium dedicated t o him. The idea o f o r g a n i z i n g such a Symposium came t o me d u r i n g a s e r i e s o f seminars on p h y s i c a l a d s o r p t i o n h e l d i n Les D i a b l e r e t s , Switzerland, i n t h e f a l l o f 1983, and organized by Ervin Kovzts. During those days I had a few l o n g conversations w i t h D r . M a r t i n and so came t o a p p r e c i a t e t o t h e f u l l h i s wonderful s c i e n t i f i c and human c h a r a c t e r i s t i c s . I w i l l never f o r g e t h i s d e s c r i p t i o n o f t h e experiments made t o g e t h e r w i t h A.T. James i n t h e very e a r l y days o f gas chromatography, and o f t h e t r o u b l e s he encountered b e f o r e t h e i n v e n t i o n o f t h e gas d e n s i t y balance d e t e c t o r . I was s u r p r i s e d v e r y o f t e n t o hear him, when r e c o u n t i n g h i s experiments, u t t e r phrases 1ike "we were 1ucky" o r " f o r t u n a t e l y i t happened t h a t

.. . , showing "

t h e t r u e modesty o f a g r e a t s c i e n t i s t and researcher. I a l s o n o t i c e d t h e i n t e r e s t he showed i n f o l l o w i n g a l l t h e l e c t u r e s g i v e n i n t h e course o f t h a t seminar, w i t h the a t t e n t i o n t h a t everybody would l i k e t o r e c e i v e from h i s students. He a l s o explained t o me h i s philosophy about s c i e n t i f i c research, which can be summarized as " t r y , t r y and t r y again". I n t h e f o l l o w i n g months I contacted some o f my f r i e n d s and colleagues i n t h e f i e l d o f chromatography, t o p u t forward t h e i d e a o f o r g a n i z i n g such a Symposium, and t h e i r r e a c t i o n s were so e n t h u s i a s t i c t h a t I came t o t h e d e c i s i o n t o h o l d i t d u r i n g 1985.

XI1

U r b i n o was an obvious c h o i c e f o r t h e Symposium because I have been honoured t o teach a t t h i s U n i v e r s i t y f o r t h e l a s t t e n y e a r s . Furthermore, U r b i n o i s a d e l i g h t f u l town where some of t h e most i m p o r t a n t m a s t e r p i e c e s o f t h e I t a l i a n Renaissance a r e l o c a t e d . The U n i v e r s i t y i t s e l f i s a v e r y o l d one f o r t h e Humanities; s t u d i e s and s c i e n t i f i c r e s e a r c h i n b i o c h e m i s t r y and c h e m i s t r y have been e s t a b l i s h e d r e l a t i v e l y r e c e n t l y , b u t t h e r e a r e some v e r y a c t i v e groups i n these f i e l d . The o l d t r a d i t i o n o f h o s p i t a l i t y o f t h e l o c a l p o p u l a t i o n and of t h e U n i v e r s i t y i s one o f t h e f a c t o r s t h a t guarantee t h e success o f t h e Symposium. I n t h i s r e s p e c t , we must be p r o f o u n d l y g r a t e f u l t o Sen. P r o f e s s o r C a r l o Bo, Rector o f t h e U n i v e r s i t y o f U r b i n o , who encouraged me i n t h i s i n i t i a t i v e and s t r o n g l y supported my e f f o r t s towards t h e o r g a n i z a t i o n o f t h e meeting. We a r e a l s o h i g h l y i n d e b t e d t o G i o r g i o F o r n a i n i , P r o f e s s o r o f B i o c h e m i s t r y and Dean of t h e F a c u l t y o f Pharmacy, who f i r s t promoted s t u d i e s and s c i e n t i f i c r e s e a r c h i n b i o c h e m i s t r y and 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 U r b i n o . W i t h o u t h i s a c t i v i t y t h i s Symposium would n o t have been p o s s i b l e . It i s a l s o worth n o t i n g t h a t t h e U n i v e r s i t y o f Urbino, f o l l o w i n g t h e proposal

o f t h e F a c u l t y o f Sciences and a p p r o v a l b y t h e M i n i s t r y o f P u b l i c Education, w i l l honour A r c h e r M a r t i n w i t h a "Laurea H o n o r i s Causal' i n B i o l o g i c a l Sciences, t h a t

w i l l be bestowed a t a ceremony d u r i n g t h e Symposium. Because o f i t s s p e c i a l n a t u r e , t h e Symposium i s o r g a n i z e d i n a q u i t e s p e c i a l way, w i t h a l a r g e number o f p l e n a r y l e c t u r e s by some o f t h e most famous and a c t i v e r e s e a r c h e r s i n b o t h l i q u i d and gas chromatography. The aim o f t h e s e l e c t u r e s i s m a i n l y t o show t h e enormous p r o g r e s s t h a t t h e s e branches o f s c i e n c e have made around t h e w o r l d more t h a n t h i r t y y e a r s a f t e r t h e o r i g i n a l works b y M a r t i n and coworkers. T h i s seems t o me one o f t h e b e s t ways t o honour Archer. T h i s book c o n t a i n s t h e t e x t s o f t h e p l e n a r y l e c t u r e s d e l i v e r e d a t t h e A.J.P. M a r t i n Symposium t o g e t h e r w i t h some d i s c u s s i o n s a b o u t p a s t a c t i v i t y . I t c o n t a i n s many o r i g i n a l t h e o r e t i c a l and p r a c t i c a l r e s u l t s , T h i s i s , i n my o p i n i o n , t h e b e s t way t o show "what i s new" i n chromatographic r e s e a r c h and t o demonstrate t h e new f r o n t i e r s of t h i s s e c t o r o f science. A c t u a l l y , a l t h o u g h chromatography has o f t e n been c o n s i d e r e d t o have become a r o u t i n e technique, i t s t i l l shows an enormous v i t a l i t y , w i t h c o n t i n u o u s improvements and new d i s c o v e r i e s .

I w i s h t o thank a l l t h e e x c e l l e n t s c i e n t i s t s who accepted my i n v i t a t i o n t o d e l i v e r a l e c t u r e and t o submit t h e m a n u s c r i p t i n due t i m e t o a l l o w t h i s book t o be p u b l i s h e d j u s t b e f o r e t h e Symposium. T h e i r r e s e a r c h a c t i v i t i e s made p o s s i b l e most o f t h e progress i n chromatographic science. Because o f t h e i r e f f o r t s , b o t h t h e Symposium and t h e book w i l l have t h e success and importance t h a t t h e y deserve. However, I must a p o l o g i z e t o t h o s e s c i e n t i s t s , and t h e r e a r e many o f them, who have a l s o made v e r y i m p o r t a n t c o n t r i b u t i o n s t o t h e development o f chromatography and whom I c o u l d n o t i n v i t e . T h e i r work s h o u l d a l s o be remembered.

XI11 I t h i n k t h a t t h e p a r t i c i p a t i o n o f two g r e a t s c i e n t i s t s , Klaus Biemann, f r o m t h e Massachusetts I n s t i t u t e o f Technology, and F r a n c i s Sherwood Rowland, f r o m 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 , I r v i n e , makes t h i s book a s p e c i a l one. I n f a c t , n e i t h e r o f them i s c o n s i d e r e d a "chromatographer", b u t , i n t h e i r e x c e l l e n t works i n mass s p e c t r o m e t r y and h o t atom and atmospheric c h e m i s t r y , t h e y have shown t h e g r e a t p o t e n t i a l o f chromatography as a t o o l i n chemical and b i o c h e m i c a l r e s e a r c h . T h i s book seems t o me p a r t i c u l a r l y u s e f u l f o r young r e s e a r c h e r s s i n c e i t shows how t h e new developments i n chromatographic s c i e n c e a r e due t o t h e e f f o r t s o f a few p i o n e e r s who s t a r t e d t h e i r i n v e s t i g a t i o n s soon a f t e r t h e f i n d i n g s o f M a r t i n and h i s coworkers.

January 1 5 , 1985

FABRIZIO BRUNER

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xv A.J.P.

MARTIN

A r c h e r J.P. M a r t i n was b o r n i n London, England i n 1910. Cambridge U n i v e r s i t y , where he r e c e i v e d h i s Ph.D.

i n 1936.

He s t u d i e d a t He was a s s o c i a t e d

f o r s i x y e a r s 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 a t Cambridge U n i v e r s i t y and then, f r o m 1938 t o 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 .

There,

i n 1941, t o g e t h e r w i t h Synge, he 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 Consden and Gordon, paper chromatography.

I t i s worth n o t i n g t h a t

p a r t i t i o n chromatography was i n v e n t e d w i t h t h e aim o f d i s c o v e r i n g t h e s t r u c t u r e of p r o t e i n s , -and b i o c h e m i c a l problems have always r e c e i v e d a t t e n t i o n i n M a r t i n ' s work. From 1946 t o 1948 D r . M a r t i n worked f o r a pharmaceutical company. 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 C o u n c i l .

Then he

I n t h i s p e r i o d he

made t h e f i r s t experiments on gas chromatography, t o g e t h e r w i t h A.T. James.

In

1952 Dr. d a r t i n t o o k up a r e s e a r c h p o s t a t 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 i n London. S i n c e 1956 he has worked as a c o n s u l t a n t i n s e v e r a l l a b o r a t o r i e s .

Between

1964 and 1974 D r . M a r t i n t a u g h t a t t h e Technische Hogeschool i n Eindhoven, The Netherlands, and i n 1973-1974 he was a p r o f e s s o r a t t h e U n i v e r s i t y o f Sussex, B r i g h t o n , England.

I n 1974 he was a p p o i n t e d P r o f e s s o r o f 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 Houston, Houston, Texas, U.S.A.

I n 1980 he j o i n e d t h e

L a b o r a t o i r e de Chimie Technique a t t h e E c o l e P o l y t e c h n i q u e F e d e r a l e de Lausanne, and t i l l 1984 t a u g h t t h e r e as an i n v i t e d p r o f e s s o r and l e d a r e s e a r c h team. 1985 D r . i d a r t i n r e t i r e d and he i s now an Honorary P r o f e s s o r a t t h e f c o l e P o l y t e c h n i q u e de Lausanne where he s t i l l has a r e s e a r c h l a b o r a t o r y .

In

XVI

D r . M a r t i n r e c e i v e d t h e Nobel P r i z e i n 1952 f o r t h e i n v e n t i o n o f p a r t i t i o n chromatography.

He has honorary d o c t o r a t e s f r o m t h e U n i v e r s i t i e s of Leeds and

Glasgow; he i s a member o f t h e Royal S o c i e t y and honorary member o f s e v e r a l other s c i e n t i f i c societies,

He has r e c e i v e d t h e M.S. T s w e t t Chromatography

Medal, t h e American Chemical S o c i e t y Award i n Chromatography and many o t h e r awards and medals f r o m 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 .

XVII

CONTRIBUTORS P. Apps, I n s t i t u t e f o r Chromatography, U n i v e r s i t y o f P r e t o r i a , 0002 P r e t o r i a ,

South A f r i c a E. Bayer, I n s t i t u t f u r Organische Chemie, U n i v e r s i t a t Tubingen, Auf d e r Morgen-

s t e l l e 18, 0-7400 Tubingen, G.F.R. K. Biemann, Department o f Chemistry, Massachusetts I n s t i t u t e o f Technology,

Cambridge, MA 02139, U.S.A.

P. C i c c i o l i , I s t i t u t o Inquinamento Atmosferico d e l C.N.R., Roma, Via S a l a r i a Km 29,300,

C.A.

C.P.

Area d e l l a Ricerca d i

10, Monterotondo Stazione (Roma), I t a l y

Cramers, Laboratory o f Instrumental A n a l y s i s , Department o f Chemical Engineering, Eindhoven U n i v e r s i t y o f Technology, P.O.

Box 513, 5600 MB Eind-

hoven, The Netherlands

E. Cremer, I n s t i t u t e o f Physical Chemistry, U n i v e r s i t y o f Innsbruck, Innsbruck, Austria C. Dewaele, Laboratory o f Organic Chemistry, S t a t e U n i v e r s i t y o f Ghent, K r i j g s l a a n 281 (S.4),

8-9000 Ghent, Belgium

L.S. E t t r e , Chromatography D i v i s i o n , The Perkin-Elmer Corporation, Norwalk, CT 06856, U.S.A. C. G a l e f f i , L a b o r a t o r i o d i Chimica d e l Farmaco, I s t i t u t o Superiore d i Sanita, Roma, I t a l y L. Ghaoui, Chemistry Department, U n i v e r s i t y o f Houston, Houston, TX 77004, U.S.A. E. G i l a v , Department o f Organic Chemistry, The Weizmann I n s t i t u t e o f Science,

Rehovot 76100, I s r a e l G. Guiochon, L a b o r a t o i r e de Chimie A n a l y t i q u e Physique, Ecole Polytechnique, F-91128 Palaiseau Cedex, France H. Hatano, Department o f Chemistry, F a c u l t y o f Science, Kyoto U n i v e r s i t y ,

Kyoto 606, Japan Cs. H o r v i t h , Department o f Chemical Engineering, Yale U n i v e r s i t y , New Haven, CT 06520, U.S.A.

XVIII

E.D.

Katz, The Perkin-Elmer C o r p o r a t i o n , Norwalk, CT 06856, U.S.A.

B. Koppenhoefer, I n s t i t u t f i r Organische Chemie, U n i v e r s i t a t Tubingen, A u f d e r M o r g e n s t e l l e 18, D-7400 Tubingen, G.F.R. E. sz. Kovats, L a b o r a t o i r e de Chimie-technique,

i c o l e P o l y t e c h n i q u e F e d e r a l e de

Lausanne, CH-1015 Lausanne, S w i t z e r l a n d K. Lawson, I n s t i t u t e f o r Chromatography, U n i v e r s i t y o f P r e t o r i a , 0002 P r e t o r i a ,

South A f r i c a L e c l e r c q , 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 , Department o f Chemical

P.A.

Engineering, Eindhoven U n i v e r s i t y o f Technology, P.O.

Box 513, 5600 MB Eind-

hoven, The Netherlands

A. L i b e r t i , I s t i t u t o Inquinamento A t m o s f e r i c o d e l C.N.R., Roma, V i a S a l a r i a Km 29,300,

C.P.

Area d e l l a R i c e r c a d i

10, Monterotondo S t a z i o n e (Roma), I t a l y

L i p s k y , S e c t i o n o f P h y s i c a l Sciences, Y a l e U n i v e r s i t y School o f Medicine,

S.R.

New Haven, CT 06510, U.S.A. M a r i n i B e t t o l o , D i p a r t i m e n t o d i B i o l o g i a Vegetale, U n i v e r s i t d d i Roma "La

G.B.

Sapienza"; C e n t r o Chimica d e i R e c e t t o r i e d e l l e M o l e c o l e B i o l o g i c a m e n t e A t t i v e , I s t i t u t o d i Chimica, U n i v e r s i t d C a t t o l i c a d e l Sacro Cuore, Roma, I t a l y

M. Novotny, Department of Chemistry, I n d i a n a U n i v e r s i t y , Bloomington,

I N 47405,

U.S.A.

K. Ogan, The Perkin-Elmer C o r p o r a t i o n , Norwalk, CT 06856, U.S.A. C.S.G.

P h i l l i p s , I n o r g a n i c Chemistry L a b o r a t o r y , O x f o r d U n i v e r s i t y , South Parks

Road, O x f o r d O X 1 3QR, U.K.

V. P r e t o r i u s , I n s t i t u t e f o r Chromatography, U n i v e r s i t y o f P r e t o r i a , 0002 P r e t o r i a , South A f r i c a J.H. P u r n e l l , Department o f Chemistry, U n i v e r s i t y C o l l e g e of Swansea, S i n g l e t o n

Park, Swansea, Wales, U.K. E. Rohwer, I n s t i t u t e f o r Chromatography, U n i v e r s i t y o f P r e t o r i a , 0002 P r e t o r i a ,

South A f r i c a P. Rouchon, i c o l e P o l y t e c h n i q u e , F-91128 P a l a i s e a u Cedex, France

F.S. Rowland, Department o f Chemistry, U n i v e r s i t y o f C a l i f o r n i a , I r v i n e , CA 92717, U.S.A.

XIX P. Sandra, L a b o r a t o r y o f Organic Chemistry, S t a t e U n i v e r s i t y o f Ghent, K r i j g s -

l a a n 281 ( S . 4 ) ,

8-9000 Ghent, Belgium

M. Schoenauer, Centre de Mathematiques Appliquees, f c o l e P o l y t e c h n i q u e , F-91128 P a l a i s e a u Cedex, France

C.P.M.

S c h u t j e s , 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 , Department o f Chemical

E n g i n e e r i n g , Eindhoven U n i v e r s i t y o f Technology, P.O. hoven, The N e t h e r l a n d s R.P.W.

Box 513, 5600 MB E i n d -

S c o t t , The P e r k i n - E l m e r C o r p o r a t i o n , Norwalk, CT 06856, U.S.A.

H. S h a n f i e l d , Chemistry Department, U n i v e r s i t y o f Houston, Houston, TX 77004,

U.S.A. P. V a l e n t i n , Centre de Recherche de S o l a i z e , ELF/ERAP, B.P.

22, F-69360 S t .

Symphorien d'ozon, France

M. Verzele, L a b o r a t o r y o f Organic Chemistry, S t a t e U n i v e r s i t y o f Ghent, K r i j g s l a a n 281 ( S . 4 ) ,

8-9000 Ghent, Belgium

S. Weisner, C h e m i s t r y Department, U n i v e r s i t y o f Houston, Houston, TX 77004,

U.S.A.

A. Z l a t k i s , Chemistry Department, U n i v e r s i t y o f Houston, Houston, TX 77004, U.S.A.

xx

AC KNOWL EDGEMEPITS

We w i s h t o thank t h e I t a l i a n M i n i s t r y o f Education, t h e M i n i s t r y f o r S c i e n t i f i c Research, and t h e I t a l i a n Research C o u n c i l f o r s u p p o r t i n g t h e Symposium f i n a n c i a l l y . The s u p p o r t o f t h e Group o f Chromatography and o f t h e A n a l y t i c a l Chemistry D i v i s i o n o f t h e I t a l i a n Chemical S o c i e t y i s g r e a t l y a p p r e c i a t e d .

Gratitude i s

a l s o extended t o t h e S o c i e t a Chimica I t a l i a n a , t h e Chromatographic S o c i e t y , t h e Groupement pour 1 'Avancement des M6thodes Spectroscopiques e t Physico-Chimiques d'Analyse, t h e A r b e i t s k r e i s Chromatographie d e r Fachgruppe A n a l y t i s c h e n Chemie d e r G e s e l l s c h a f t Deutscher Chemiker, and t h e S u b d i v i s i o n o f Chromatography and S e p a r a t i o n Chemistry o f t h e A n a l y t i c a l D i v i s i o n o f t h e American Chemical S o c i e t y . These o r g a n i z a t i o n s have sponsored t h e Symposium.

I w i s h t o express my deep a p p r e c i a t i o n t o a l l t h e members o f t h e O r g a n i z i n g and S c i e n t i f i c Committees f o r t h e i r h e l p and a d v i c e . F i n a l l y , I w i s h t o thank my c o l l e a g u e s and coworkers o f t h e I s t i t u t o d i Scienze Chimiche f o r t h e i r c o o p e r a t i o n i n t h e o r g a n i z a t i o n o f t h e Symposium. S p e c i a l thanks a r e due t o Miss P i e r a n g e l a Donnanno f o r h a n d l i n g t h e m a n u s c r i p t s and t h e correspondence.

FABRIZIO BRUNER

1

Chiral Recognition in Gas Chromatographic Analysis of Enantiomers on Chiral Polysiloxanes

Bernhard Koppenhoefer and Ernst Bayer*

Institut fur Organische Chemie der Universitat Tubingen, Auf der Morgenstelle 18, 7 4 0 0 Tubingen (GFR)

Summary

The present article is a critical review on enantiomer resolution by gas chromatography on chiral amides, with emphasis

on the thermostable polysiloxane Chirasil-Val. Thermodynamic studies demonstrate the significance of hydrogen bonding. Chiral recognition factors can describe chiral recognition quantitatively. Further insight is given by considering the conformations of both solute and solvent.

New methods for the chemical and biotechnological synthesis of chiral biologically active substances ’’*) have led to extraordinarily high enantiomeric purity (eel, which can be accurately determined only by sensitive, direct methods. Gas chromatography on chiral stationary phases

’‘)

such as chiral

metal complexes5 e 6 1 1 0 ) or chiral a m i d e ~ ” ~ )is suitable to solve these problems. Liquid chromatography9) is superior to

2

gas chromatography only if preparative amounts of the pure enantiomers are required. For analytical applications, gas chromatography is the method of choice, providing the substances are sufficiently volatile and have favorable chromatographic properties (retention time, resolution factor c( , peak shape).

From an economical point of view, a unique stationary phase allowing the resolution of a large variety of enantiomeric pairs is preferable. Originally, only nitrogen-containing molecules were amenable to enantiomer resolution on amides of amino acids as the chiral stationary phase3). In 1 9 6 6 , the resolution of N-acyl amino acid esters on N-TFA-L-leucine dodecylester by Gil-Av et a1.l’ ) was the first reported successful resolution of enantiomers by gas chromatography. Diamides of amino acids of low molecular weight have been investigated in the last two decades to resolve enantiomeric pairs of & - , 0-,and f-amino acid derivatives, acylated amines or amino alcohols’ ) and amides of halocarboxylic acids’3,

. Amides

of 1 ( 1 -naphthyl Iethylamine have been used

to resolve enantiomers of alkylcarboxylic acid amides’

*

)

and

halocarboxylic acid amides”) , and recently reported by 0”i et al., the enantiomers of menthol, nitriles, chrysanthemates

’

I ’

7,

and macrolides’ 8 ,

. Amino

acids linked to triazine

were also investigated as a chiral stationary phase 19)

.

Finally, (S)-mandelic acid-(S)-’(1-pheny1)ethylamide was synthesized by Konig et a1.”)

to separate 0-acylated hydroxy

acid acid esters under carefully optimized conditions.

From Fig. 1 we can conclude that almost all these compounds and many other solutes have been separated into enantiomers

3

3 -

-2

-1

%ox NHX

OX

RY R' NHX

6 -

"R' H

0

-7

OH ,&Ope

R q o P e

0

RYYoPe HO 0 HO 0 11

12 -

4

0 15

13 -

3

0

0

7

0

%

OH

0 17

+- 4

Hal *OX

ox

OH

HO

19

21 -

20 -

NHX

W O *

22 -

s+OR' II 0

23

0 2L -

5

2 6 -

NHX

Q

\o-L+OR. II 0

0-

I S-P-Nx SII

0 20 -

Figure 1 .

27 -

y(o-k+II

0

29

Selected solutes resolved into enantiomers o n Chirasil-Val I ( X = CZF5-CO- , Pe = 3-pentyl).

on a single stationary phase,

5Chirasil-Val

I (Fig. 2 ) *

Though the resolution factors may be inferior in particular c a s e s , as compared to some applications referenced above,

*

Fused silica columns coated with L- or D-Chirasil-Val commercially available from Chrompack, Middelburg, The Netherlands.

are

6

I

I

-0-s

I

I

I

i-0-5ik0-S i- 0-5 i -

Figure 2.

Polysiloxanes acting as a chiral stationary phase.

there is no stationary phase reported in literature covering a similar range of molecular structures to be successfully resolved into enantiomers. Attachment of a chiral diamide residue,

& of

L-valine-t-butylamide2' ) or D-valine-

t-butylamide2*) to a polysiloxane backbone led to a unique combination of two basic principles in biomimetic chemistry:

The imitation of chiral recognition in natural systems by hydrogen bonding between suitable amino acid moieties, originating from Gil-Av et a1.3) has been transferred by Frank, Nicholson and Bayer2') to a multifunctional polymer 7 )

.

The polysiloxane does not only render its favorable physical properties to Chirasil-Val, that is low volatility, low melting point, suitable polarity and good wettability, but also its flexibility, probably giving rise to cooperative effects (induced fit).

Owing to the improved thermostability and polarity, all enantiomers of common protein amino acids (e.9. - as N,O,S-penta-

fluoropropionyl-isopropylesters 1 ) have been completely separated in a single run2'

).

Various deactivation proce-

dures for glass columns and different derivatives of amino acids have been studied in

permitting not only 24,25) the control of racemization during peptide synthesis

but also the quantitative analysis of amino acids using the methodology of "enantiomer labelling"26)

.

Amino alcohols

acting as sympathomimetica, such as ephedrine or synephrine

2,

have been resolved as the N,O-perfluoropropionates 2 and respectively2",

the differences in interaction of the

stationary phase - A A H reveiling an intriguing correlation with the adrenergic effect of the different drugs 28)

.

The enantiomeric purity of derivatives of amino alcohols 4 and 5 formed in high selectivity by ring opening of -

oxiranesZ9) such as styrene oxide with ammonia has been studied using Chirasil-Va13').

Simple amines are also re-

solved in analytical scale as perfluoroacyl derivatives

5,

thus allowing to control the efficiency of preparative resolution of enantiomers via diastereomeric salts3').

Similarly,

8

lactames

2

.

are separated without deri~atization~l ) Many

different derivatives of 2-hydroxy acids (e.g. been investigated3’).

E, 9 )

have

Some of them suffer from byproducts

formed during the multi-step procedures, particularly in the preparation of urethanes

S

from i s ~ c y a n a t e s ~ ~that ) , have

been studied also by other authors34). Racemization of mandelic acid was observed, if traces of amines are present. Therefore a clean-cut soliltion for mandelic acid became necessary, excluding nitrogen compounds as far as possible. The easily accessible free hydroxy acid esters

lo

are resolved

on fused-silica columns coated with Chirasil-Val without significant peak tailing3’).

Many different alcohols have been

tested for esterification, 3-pentanol giving the best results with respect to resolution factors, peak tailing and volatility. According to Koppenhoefer, Allmendinger and Nicholson36 ) , the 3-pentyl esters

are also the most suitable derivatives for other

hydroxy carboxylic compounds, such as 3-hydroxy acids 2,3-dihydroxy acids acids

13. The

12 and

fi,

lactones from 2-hydroxy dicarboxylic

preparative useful enantiomers of tartaric

acid are most conveniently analyzed after conversion to the bisacetonide

14 using

2 , 2 I -dimeth~xypropane~~). Consequently,

several lactones are resolved without derivatization. These compounds are increasingly important,

e.g. in metabolism

E, mevalolactone 5) and (fluoromevalolactone E ) .Contradictory

(pantolactone

pest control to tentative

assumptions, a three-point attraction model is not a prerequisite for a successful resolution. The clean separation of simple alcohols36) (primary

20)

’8,

scondary 19 and tertiary

exemplifies that one strong attraction is sufficient

in many cases on a diamide phase, significant enantiomer discrimination coming from additional weak van der Waals attrac-

9

tions and repulsions. The one-point attraction has been demonstrated also in complexation chromatography 29,37). The first successful resolutions of nitrogen-free enantiomers on diamide phases 27'30) have been carried out with diesters of arylethane-l,2-diols 21 and of the atropic isomer 2,2'-dihydroxy-1,l'-binaphthyl 22. The latter entry, used in asymmetric synthesis (enantiomeric yield "100

demonstrates the

stereochemical integrity of the atropic isomer even at 180OC. Esters of polyols such as sugars3')

or sugar alcohols40) have

been resolved by Konig et al., again on polymeric stationary phases of the Chirasil-Val type4). In contrast to the multifunctional esters, cyclic carbonates obtained from diols and phosgene have only one carbonyl group and therefore are only separated under carefully optimized conditions41

)

.

Simple

carbonyl compounds such as 3,4-diphenyl-2,5-hexanedion 23 are resolved completely if the structure is well-designed 3 0 ) This promising field has been investigated only briefly

.

42)

.

In alicyclic ketones, complexation gas ~ h r o m a t o g r a p h y) ~ is ~ superior to chiral p o l y s i l ~ x a n e s ~ ~though ), conversion to oximes has been proposed 4 5 )

.

Chiral nitrogen has been investigated using complexation chrom a t o g r a p h ~ ~and ~ ) chiral sulfur and phosphorus on Chirasil-Val Derivatives of amino acids containing an additional "chiral sulfur" (i.e., 24) are resolved into - methionine-S-oxide four peaks47). Consequently, simple chiral sulfoxides

26 are

25,

separated into the e n a n t i ~ m e r s ~ ~In ) . contrast

to thioethers like methionine, thiol groups in amino acids such as cysteine form chiral disulfide bridges on oxidation, which are limited in stability. If replaced by a thioether moiety, stable dimers are formed in various stereoisomers

.

10

(i.e., 27) that are resolved on Chirasil-Val - lanthionine after suitable derivatization4*)

.

"Chiral phosphorus" occurs

in various molecules. The antibiotic amino acid phosphinotrycine, bearing a phosphinic acid moiety, is' configurationally stable as the methyl ester

S

only, and resolved after the

typical derivatization of the amino acid part into all four

stereo isomer^^^)

.

Following this line, simple phosphorus com-

pounds acting as pestizides (e.9. 29) are separated into the e n a n t i o r n e r ~ ~ ~and ) , toxic nerve agents like soman 30 into four stereoisomers due to an additional chiral carbon atom 51 )

.

Though the valine-t-butylamide residue in Chirasil-Val gave optimized resolution factors for 2-amino acid derivatives and some other compounds, a variety of similar stationary phases has been synthesized by T h ~ m m ~in~ 1)9 7 8 , already. Variation of the amino acid residue (i.e., - L-leu-n-butylamide, L-leu-cyclohexylamide, L-leu-t-butylamide, L-ile-t-butylamide, L-phe-cyclohexylamide) 49) gave no substantial improvement, but alteration of the amino component led to increased resolution factors for various solutes. Besides the well known L-valine-t-butylamide, amides of L-valine with aliphatic amines such as n-butylamine, cyclohexylamine, cyclooctylamine, 1-adamantylamine and (-)menthylamine have been studied4').

Particularly promising were amides con-

taining an aromatic residue, such as S-valine-R-l(1-naphthy1)ethylamide I11 (Fig. 2 ) and the diastereomers S-valine-Rl(1-pheny1)-ethylamide and S-valine-S-l(1-pheny1)ethylamide I1 (Fig. 2 ) , respectively. The homochiral (like) combination S,S-I1 gave better resolution factors with derivatives of amino acids and amines, whereas the heterochiral (unlike) combination S,R-I1 was the better tool for special amino-

11

alcohols and glycols4’).

Recently, KOnig3’)

reported reso-

lutions on S,R-111, too. The stationary phase is mos

conven-

iently prepared by equilibration between polydimethy siloxane and poly(2-carboxypropyl)methylsiloxane and coupling of the amino component to the copolymer with dicyclohexylcarbodiimine DCC)28) or carbonyldiimidazole (CDI)49). Alternative pathways have been also investigated thoroughly, but with less promising results4’)

.

For instance, the saponification of the

nitrile group in polysiloxanes like XE-60 or OV-225 to the carboxylic group gives rise to some degradation of the polymer backbone. Nevertheless, this route was also followed by VerzeleS3 ) and others. Notably, the poly,siloxanes

always

contain some functionalized c y c l ~ o l i g o m e r s ~ ~besides ), the linear polymer chains expected.

With increasing number of enantiomers resolved on amide phases it becomes more and more difficult to understand the mechanism of action. The three-point-attraction models proposed by Feibush and G ~ ~ - A were v ~ ~insignificant ) in terms of peptide conformations and later withdrawn56). The initial assumption that only nitrogen containing solutes are efficiently resolved into enantiomers on a peptide phase was also disproved by many examples. S o it becomes necessary to look at the problem more deeply.

The enantiomers of the solute are discriminated by the stationary phase due to differences in interaction with the chiral solvent. But the incorporation of the solute molecules into the chiral liquid is also influenced by the forces between the solvent molecules. From this point of

12

Figure 3.

0

cu

in c-

F

0

Left: Relative frequency f ( x ) of distances x between the chiral side-chains in Chirasil-Val for different loadings l/a of the polymer. Right: Con-

tx

.o c

0

tribution of the distances x to the mean distance a.

13

view it is preferable to insulate the diamide moieties from each other by means of the polysiloxane backbone, simultaneously decreasing the melting point and the viscosity of the stationary phase. Polymers in which more than a quarter of the silicon atoms are functionalized with chiral groups are hard and high-melting owing to formation of a D-plated sheet. However the amide groups are random distributed at the polysiloxane backbone (Fig. 3). Shorter distances are preferred. Only the first momentum (contribution of the distances x to the mean distance a) shows a maximum around the mean distance a. From these

consideration^^^)

it follows

that complexes between the solute and two or more diamide groups play an important role. In simple d i a ~ n i d e s ~ ~the ), situation is likewise complicated by the different orientations of the solvent molecules in the chiral liquid.

In order to get more insight, we investigated a variety of solutes, e.g. simple esters, acting only as a hydrogen bonding acceptor and not as a donor (like N-H- or O-H-containing solut'es). From butane-2,3-diol diperfluoropropionate (not resolved) via the phenylethane-l,2-diol derivative to the 1,2-diphenyl compound

32, the

35

thermodynamic para-

meters indicate a strong increase in the difference of fitting of the enantiorners with the stationary phase. The increasing number of phenyl groups gives not only rise to

?f-y -inter-

actions, but even more important to a stabilization of the preferred conformations. The perfluoropropionyl groups are useful in terms of retention time, but the diacetates are also resolved (Fig. 4 ) .

14 Rs

c.0

R,= CHJ

1

"?i

Figure 4.

Influence of the acyl part on the chromatographic behaviour of esters of 1,2-diphenylethandiol-l,2. Duran glass capillary, 20 m x 0 . 2 5 mm, coated with L-Chirasil-Val, 0.4 bar H2, 1 3OoC.

The different fitting of the enantiomers is not concluded from the resolution factor d at a single temperature, since this as a rule figure is strongly dependent on temperature. Differences in free energy - A A G

0

are calculated directly from

the resolution factors according to equation (I).

15

R

T

R

Equation ( I ) i n combination w i t h t h e Gibbs-Helmholtz s h i p g i v e s e q u a t i o n ( I1 )

'*

)

. Differences

relation-

i n e n t h a l p y and

entropy between t h e i n t e r a c t i o n of b o t h enantiomers with t h e s t a t i o n a r y phase are c o n v e n i e n t l y obtained by p l o t t i n g I n U v e r s u s 1 / T . The d i f f e r e n c e i n f i t t i n g b e t w e e n p a i r s o f e n a n t i o m e r s i s r e f l e c t e d i n d i f f e r e n t o r d e r o f c o m p l e x e s . The higher ordered complexes g i v e rise to higher ( u n f a v o r a b l e ) d i f f e r e n c e s i n e n t r o p y of t h e i n t e r a c t i o n w i t h t h e s t a t i o n a r y p h a s e . T h e r e f o r e most o f t h e g a i n i n e n e r g y i s c a n c e l l e d o u t . S i n c e t h e e n t r o p y term i s i n c r e a s i n g l y i m p o r t a n t a t e l e v a t e d temperature, a c e r t a i n temperature T

may e x i s t , w h e r e t h e

e n t r o p y term a n d t h e e n t h a l p y term a r e b a l a n c e d , a n d t h e r e s o l u t i o n f a c t o r OC

In%

=

0

of t h e enantiomers drops t o one.

a t T = T

Ts

=

-

AA

(111)

AA S

S i n c e t h e e n t r o p y term h a s n o r m a l l y t h e o p p o s i t e s i g n o f t h e enthalpy term, positive T Above T

S'

values a r e observed a s a r u l e .

i t would b e r e a s o n a b l e t o p o s t u l a t e a r e v e r s e i n

o r d e r o f e l u t i o n o f t h e t w o e n a n t i o m e r s . The c r o s s s e c t i o n point T

i s i n h e r e n t t o e q u a t i o n (11). I f i t s e x i s t e n c e i s

d i s p r o v e d , a fundamental i d e a of thermodynamics i s no l o n g e r a p p l i c a b l e i n t h i s m a n n e r . However i t i s d i f f i c u l t t o i m a g i n e

a s t r o n g e r r e t e n t i o n o f t h e worse f i t t i n g e n a n t i o m e r d u e t o a s m a l l e r ( u n f a v o r a b l e ) e n t r o p y term. I n a l l e x a m p l e s shown i n

16 20OY

1oo.t

25'C 5

31

32

33 4

34

35

Figure 5 .

Temperature dependence of resolution factors

57)

.

Fig. 5 , the resolution factors decrease at increasing temperature. But the slopes of the straight lines are different, and similarly the resolution factors of various enantiomeric pairs can change their relative magnitude. Any statement on the resolution of one enantiomeric pair as compared to another one is always confined to the particular temperature of measurement. Yet the difference in fitting is roughly given by the differences in enthalpy and entropy, respectively. These two figures are losely correlated3')

.

Comparing isomeric

k.the perfluoropropionates of 1-amino-2-hydroxy1-phenylethane 2 and 1-hydroxy-2-amino-I-phenylethane 4,the

compounds,

latter is resolved even better at higher temperature, due

17 t o t h e smaller d i f f e r e n c e i n entropy. That i s ,

4exhibits

a

smaller difference i n f i t t i n g , since the stronger interacting a m i d e g r o u p i s f a r from t h e a s y m m e t r i c c e n t e r . I n a n o t h e r example, t h e amino g r o u p i n

4 (line

B i n F i g . 5 ) i s re-

p l a c e d by a second hydroxy g r o u p t o g i v e t h e g l y c o l d e r i v a tive

( l i n e A i n F i g . 5 ) . The two l i n e s A and B a r e a l -

most p a r a l l e l o v e r a w i d e r a n g e o f t e m p e r a t u r e ,

ter r e s o l v e d t h a n

%.

4being

bet-

Y e t t h e reason is not t h e s t r o n g e r

i n t e r a c t i o n o f b o t h e n a n t i o m e r s o f t h e a m i n o compound

4 with

t h e s t a t i o n a r y p h a s e . O n l y t h e d i f f e r e n c e - A a H w o u l d be i m p o r t a n t i n t h e s e t e r m s , b u t t h e s l o p e o f l i n e A is e v e n

steeper t h a n t h e s l o p e of B. Again, t h e r e a s o n is t h e smaller d i f f e r e n c e i n f i t t i n g a l r e a d y mentioned.

We h a v e p o i n t e d o u t 5 ' )

t h a t f i g u r e s i n -AAG calculated

a c c o r d i n g t o e q u a t i o n ( I ) are d i f f e r e n t from t h o s e d e r i v e d by S c h u r i g e t a 1 . 5 8 ) . I n t h e l a t t e r a p p r o a c h , a " p h y s i c a l " ( a c h i r a l ) p a r t , given by t h e s o l v e n t ( e . g . ,

squalane) is

c a l c u l a t e d f r o m r e f e r e n c e m e a s u r e m e n t s and t h e n u s e d f o r corr e c t i o n of t h e r e l a t i v e r e t e n t i o n of t h e donor molecules w i t h t h e s o l u t i o n o f a c h i r a l metal c o m p l e x i n t h e same solv e n t . By t h i s m e t h o d , h i g h e r v a l u e s i n - L A G

result, since

o n l y t h e "chemical" p a r t of i n t e r a c t i o n i s used f o r t h e f i n a l c a l c u l a t i o n . Such a s e p a r a t i o n o f t h e t o t a l i n t e r a c t i o n i n t o " a c h i r a l " a n d " c h i r a l " p a r t s may h e l p t o i n t e r p r e t e t h e r m o d y n a m i c d a t a i n terms of c h i r a l r e c o g n i t i o n , b u t i n p r a c t i c e , t h e t w o c o n t r i b u t i o n s are always c o e x i s t e n t ( e x c l u s i o n o f t h e a c h i r a l p a r t as f a r a s p o s s i b l e would i n d e e d enhance t h e r e s o l u t i o n f a c t o r s s i g n i f i c a n t l y ) . T h e r e is no d o u b t t h a t t h e difference i n f r e e enthalpy

-

AGO o f

a particular enantio-

mer i s d e r i v e d f o r t h e t r a n s i t i o n o f o n e mole o f t h e s o l u t e

ACHIRAL PHASE -AHsE~o

CHIRAL RECOGNITION PAR9METER

Figure 6 .

I

X

-(

=

-1

AHR -AHS 1 -AH'

~

S p e c i f i c i n t e r a c t i o n of t h e s o l u t e w i t h t h e c h i r a l d i a m i d e c o r e of L - C h i r a s i l - V a l .

19

from the gas phase to the liquid stationary phase, unregarded of its chemical structure. Furthermore, the same is true for the antipode and for the difference - M G o ,

as well.

Therefore we try to improve the significance of thermodynamic data by correlation of the -&H-values

actually measured

with the specific interaction - AHO of the solute with the chiral diamide moieties of Chirasil-Val (Fig. 6 ) . It has been - A H ' is composed of the interstated p r e v i ~ u s l y ~ ~that ),

actions of the solute Ei with the chiral solvent ChirasilVal as compared to the polydimethylsiloxane SE 3 0 , and of the molar heats - b H 0 h of formation of the hole, that is occupied by the solute molecule, equation (IV).

-

AH'

=

-

Ei (Chirasil-Val!

+

Ei (polydimethylsiloxane) (IV)

0

- AHh(Chirasil-Val)

+

~H~(polydimethyl~~loxane)

The interactions between the solvent molecules surrounding the solute are disturbed by the solute molecule. For instance,

a strong hydrogen bonding solute may disrupt the hydrogen bonds initially present between the diamide units in Chirasil-Val. Therefore

-

4 H '

is only in first approximation a

-

measure for the interaction of the functional groups, e.g.

hydrogen bonding. The chiral recognition factor 1( gives an answer to the question: Which fraction of the specific interaction is used for chiral recognition? According to the mode of calculation, the maximum value of

?( is 2 (if one enantio-

mer shows no specific interaction). Often we observe only 2 % to 6 % of the binding to the chiral selector being used for enantiomer discrimination. Only in well-designed molecules,

N

0

k:l' 4

3

2

1

0

-AAH xi==

054191

.OW61

.01912/

.040/51

~ H C H ~ * P I FHP 6PFP

o < HNHPFP CH2-OPFP

~~~~~

Figure 7 .

.034/51

.2931%01

0:HCHjOPFP OPFP

O W H 3 NHPFP

@H.CH

Specific interactions - A H ' (bars indicating the errors), differences in enthalpy between the enantiomers

-

A A H , and chiral recognition factors

(errors given in parantheses for the last d i g i t ) 5 7 )

0

BF9FP

.

21

INCREMENT - SYSTEM: HYDROGEN BONDS INCREMENT 1,~’ [-IKCAL ROL

BUHBEE OF H-B,

FUNCTION

1 -lo0

CA,

1

H

-rN-

2

0

-c-0-

/j

(HINDERED)

=1

(HINDERED)

<2

H

- C- N-

bl

Figure 8.

Incrementation of contributions of hydrogen bonding moieties for the specific interaction

%-values of 0 . 3 and more are obtained, e.g. acid esters or in the diester

32. In

- A HI5’).

in N-acyl amino

such cases, model

building is meaningful. The main contribution to the specific interaction comes from hydrogen bonding (Fig. 8 ) , though systems without hydrogen bonds have also led to enantiomer resolution,

&.

resolution of proline derivatives as solu-

tes on proline derivatives acting as the stationary phase 59) N,N-disubstituted arnides are well known to form strong ciates via dipolar forces

60)

asso-

.

Fig. 9 shows the molecular parameters used for model building3’).

The trans-arnide bond is observed exclusively in

secondary amides6’ ) , since the cis-amide structure is less stable by approximately 7 to 8 kcal/Mo16*). Hence the two

.

22

H

Figure 9.

Parameters for modelling Chirasil-Val.

23

l3-plated sheet

LFigure 1 0 .

I1

I

I

I

a-helix

R-

Conformations in proteins.

important degrees of freedom left are the angles of torsion

0 and

w.

In proteins, the diami.de unit usually adopts

three distinct conformations, *,

the D-plated sheet, and

the left-handed or right-handed helix (Fig. 1 0 ) . In these structures, the maximum number of hydrogen bonds between the protein backbones is formed. The D-plated sheet can act with a hydrogen bond-accepting and hydrogen bond-donating counterpart at either the C - 7 side or at the C-5 side. Similar models for the resolution of derivatives of amino acids on peptide stationary phases have been developped by Beitler and Fei-

24

bush56)

.

The h e l i x , h o w e v e r , h a s b e e n g i v e n o n l y l i t t l e

attention3').

The t w o c a r b o n y l g r o u p s c a n a c t b i d e n t a t e l y

a s a hydrogen bond a c c e p t o r , t h e t w o amino g r o u p s a s a h y d r o gen donor. I n n a t u r a l p r o t e i n s , t h e v a l i n e moiety appears

t o b e c o n s i d e r a b l y r e s t r i c t e d i n c o n f o r m a t i o n space d u e t o t h e b u l k y i s o p r o p y l g r o u p . Out o f 3 5 3 e x a m p l e s a n a l y z e d b y x - r a y d i f f r a ~ t i o n ~ 1~ 6)3, e n t r i e s ( 4 6 % ) a d o p t t h e f3-con-

c-Region

I

\a-Region

-180' -1 80

I I

0

180

B

180

~

,Q

@@'

Yo.

Q

&$

-180 Figure 1 1 .

Ramachandran-Plot f o r t h e L - v a l i n e u n i t . A ) Nat u r a l occurence i n p r o t e i n s 6 3 ) . B) Description of s e l e c t e d c o n f o r m a t i o n s . C ) Energy c a l c u l a t i o n s

on N - a c e t y l - L - v a l i n e

m e t h y l amide a c c . t o Pullman 6 4 1

and D ) a c c . t o Popov 6 5 )

.

25

f o r m a t i o n ( i n more g e n e r a l t e r m s , a n e x t e n d e d c o n f o r m a t i o n ) , (31 % ) a d o p t t h e R - & - h e l i x

111 entries

and o n l y a s i n g l e

e x a m p l e h a s b e e n f o u n d f o r t h e L- % - h e l i x

(see F i g .

1 1 , A and

B ) . These f i n d i n g s a r e r e p r o d u c e d by e n e r g y c a l c u l a t i o n s u s i n g PCILO ( F i g . 11, C ) 6 4 ) o r e m p i r i c a l p o t e n t i a l f u n c t i o n s

11, D ) 6 5 ) ,

(Fig.

methylamide. Scheraga e t

f o r N-acetyl-L-valine

a l . 66 r 6 7 ) c a l c u l a t e d t h e f r a c t i o n of d i s t i n c t c o n f o r m a t i o n s , w h i c h a r e a l s o a s s u m e d t o b e m o s t f r e q u e n t l y o c c u r i n g i n LChirasil-Val

( F i g . 1 2 ) . O€ c o u r s e , i n v a c u o t h e i s o l a t e d d i -

amide i s s e l f - a s s o c i a t e d t o f o r m a seven-membered membered r i n g ( C 7 e q

and C 5 ,

or a five-

r e s p e c t i v e l y ) . This r e s u l t is

c o r r o b o r a t e d b y NMR s p e c t r o s c o p y f o r a n A r - m a t r i x 6 ' )

and f o r

.

d i l u t e s o l u t i o n s i n t e t r a c h l o r ~ m e t h a n e ~ A~ t) e l e v a t e d t e m p e r a t u r e ( e x t r a p o l a t e d up t o 200°C), t h e open-chain c o n f o r m a t i o n s R-h/-helix

and

#

a r e i n c r e a s i n g l y f a v o r e d d u e t o t h e i r lower

e n t r o p i e s o f c o n f o r m a t i o n , a s i n d i c a t e d b y NMR s p e c t r o s c o p y 7 o ) and by

calculation^^^)

.

These conformations a r e a l -

so d e d i c a t e d f o r i n t e r a c t i o n s with v a r i o u s s o l u t e s , i n d i c a t e d

by a n a r r o w ( F i g . 1 2 ,

cf. F i g .

1 0 ) . The m o t i o n s o f p a r t s o f

t h e diamide moiety a r e f a s t ( 1 0 l 3 t o 10l5 s e c - ' ) ,

but the

b r e a k i n g of hydrogen bonds p r e s e n t c a n l i m i t t h e speed o f a s s o c i a t i o n with a c o u n t e r p a r t 7 ' ) , though t h e formation of h y d r o g e n b o n d s i s a l s o slow ( a p p r o x i m a t e l y 10' A s a consequence,

sec-' ) 7 2 )

.

t h e r e i s e n o u g h time f o r t h e d i a m i d e r e s i -

dues i n Chirasil-Val

t o a d o p t t h e most f a v o r a b l e conforma-

t i o n i n order t o f o r m a s u i t a b l e complex w i t h a g i v e n s o l u t e (induced f i t ) . Notably t h e i s o p r o p y l group can r o t a t e around 1,

a s i n d i c a t e d a l s o i n h i g h d i f f e r e n c e s M S f o r hydroxy a c i d

esters b e a r i n g an i s o p r o p y l s i d e c h a i n

35)

.

26

R-aHelix

27

The probability for intercalation of solutes between two diamide residues depends o n the distribution function for the chiral groups (Fig. 3 ) , and on the geometry of the polysiloxane chain (Fig. 9 , bottom). For the distance between two silicon atoms in 1,5-position separated by 4 single bonds the maximum distance has been estimated to 6.9 distance to 4 . 7

8

8,

the minimum

30). Hence it follows that either a R - o l -

helix between two adjacent diamides (requiring a distance of 5.4 6.5

8

8)

or a A-plated sheet (requiring a distance of

or 7

8,

respectively)61) can be formed.

Intercalation needs at least two silicon atoms in 1,9-position, leaving out three or more silicon atoms of the polymer backbone. On the other hand intercalation can also occur between diamide groups of different chains. After all, the solute is surrounded from all sides by the chiral solvent. The tormation of a A-plated sheet by intercalation of a derivative of lactic acid (type

Figure 12.

9)

is shown in Fig. 1 3 .

Main conformations of L-Chirasil-Val side chains. Arrows indicating the interactions of two

N-H-

groups with two C=O-groups of the solute in the R-helix conformation (top) and the interactions of one N-H-group and one C=O group with a suitable derivative of an amino acid or a hydroxy acid in the extended

f

.

-conformation (middle) Bent arrows

indicate rotations around angles of torsion in order to reach the next conformation (from top to bottom), after ref.3 0 )

28

Figure 1 3 .

f3-Plated sheet formed by intercalation of O-pentafluoropropionyl-L-lactic

acid cyclohexylamide between

two diamide moieties of L-Chirasil-Val acc. to Frank et al. 2 7 )

As for the bidentate hydrogen bonding of dicarbonyl compounds to the two N-H-groups depicted in the R-X-helix (Fig. 1 2 , top), we studied a useful principle, called "principle of complementary angles of torsion"30) . For instance in cyclohexane (Fig. 1 4 ) , the chair conformation is balanced due to parallel axes of the two half-parts (Fig. 14, left hand), the angles of torsion being unlike. In the boat conformation, like angles -

of torsion lead to unfavorable interactions bet-

29

C H A I R

B O A T ANGLE

U N L I K E

CONVENIENT

Figure 1 4 .

O F

TORSION

L I K E

INCONVENIENT

Complementary (unlike) angles of torsion are more convenient, exemplified for chair and boat conformation of cyclohexane.

ween parts of the molecule. The same principle governs the resolution of the diester

by L-Chirasil-Val. The R , R -

isomer adopting a positive angle of torsion is fitting better. The counter-part is a diamide residue in an R-&-helix conformation with negative angle of torsion, modelled according to Pauling 61 )

.

30

RR

(C0 R'l

(R"1

yL

I I

2.55

z

I

ld

I \

/

Figure 1 5 .

'KO R'l

Positive angle of torsion between the ester groups in R , R - 32 , negative angle of torsion between the amide groups in L-Chirasil-Val ( R - K helix)

.

The association complex is depicted in Fig. 16. In molecular models, strong repulsions between selector and selectand are observed, if two hydrogen bonds are formed in the same manner with the S,S-enantiomer (not depicted in Fig. 1 6 ) . In the latter case, like angles of torsion are probably omitted by formation of only one hydrogen bond, a s indicated in the high ?(-value (Fig. 7 ) . Unlike angles of torsion would be

CO-PoIy m e r

convenient t-Bu C(CH3)2 H R-Helix

Figure 16.

Principle of complementary angles of torsion, exemplified for the better fitting of R , R - Z with L-Chirasil-Val

.

32

S

10 Figure 17.

Enantiomer resolution of

20 12. on

min L-Chirasil-Val,

Duran glass capillary 20 m x 0.25 rnm, 0 . 4 bar H2, 1 8OoC.

inconvient either in formation of a L-K-helix (ecliptic position of carbonyl and isopropyl group) or in the gauche arrangement of the two bulky phenyl groups of

S , S - z

(the

antiperiplanar position of the phenyl groups in 1,2-diphenylethanediol-1,2 has been demonstrated for a3.1 three stereoisomers by energy calculations and spectra73 )

)

.

33

\

\ \

Figure 1 8 .

Polymer

Two-point-attraction modelled for worse fitting S - 22 with L-Chirasil-Val by computer graphics

42 1

Yet the solute is surrounded from all sides by the chiral solvent, and all the models depicted above show some shortcomings after a long period of research. Particularly solutes with small’)(-values atropic isomer

22

should not be overinterpreted. The

is difficult to resolve, the R-enan-

tiomer being stronger retained at L-Chirasil-Val (Fig. 1 7 ) .

34

RS

S

i S

Figure 19.

I

I

10

20

min

Enantiomer resolution of N,O-perfluoropropionyl-

2,2'-diamino-6,6'-dihydroxymethylen-l,l'-biphenyl, Duran glass capillary 20 m x 0.25 m m , coated with L-Chirasil-Val , 0.4 bar H2, 1 4OoC.

35

Yet the S-enantiomer forms the better fitting bidentate complex (Fig. 18). Again, a one-point-attraction model, as suggested for the resolution of simple alcohols36) may be also applicable in sterically hindered dicarbonyl compounds. However, the more flexible 2,2'-bis(hydroxy-methylen-1 ,1'-binaphthyl-diperfluoropropionate) is not resolved 30) , as well as the corresponding amino compound, indicating here a pronounced selectivity of the stationary phase. As expected, the 2 , 2 ' - d i a m i n o - l , l ' - b i n a p h t h y l diperfluoropropionate

2 ex-

hibits a large difference in fitting (Fig. 5), and the S-enantiomer is retained stronger on L-Chirasil-Val. To test the hypothesis further, we synthesized N,O-perfluoropropionyl2,2'-diamino-6,6'-dihydroxymethylen-l,l'-biphenyl

from 2 , 2' -dini tro-6,6 -diphenic acid42 )

.

(Fig. 1 9 )

Again the two amide

groups enclosing a positive angle of torsion dominate the resolution, the S-enantiomer fitting better with L-ChirasilVal as proposed. Considering the intercalation of derivatives of hydroxy acids between two diamide groups (cf. Fig. 131, different derivatives led to conflicting results. The carbamoyl derivative of S-methyl lactate (type

8) is

fitting bet-

ter to L-Chirasil-Val. The formation of a parallel I3-plated sheet is depicted in Fig. 20 (right hand)32). But in the 2-hydroxy acid esters with free hydroxyl group (type

lo)

the R-enantiomers are fitting better to L-Chirasil-Val 3 5 )

.

Consequently, the bidentate complex with the C-5 side of the extended conformation bears the two alkyl residues in transposition (Fig. 2 0 , left hand)32). The same unexpected result would be obtained if interaction to the C-I side is considered, similar to the antiparallel I3-plated sheet. Therefore, the reversed order of elution of the 3-hydroxy acid

0

H

n

H

H

1

" CN YC iH s C

I

H

H

0

0

Figure 20.

Association of derivatives of lactic acid with L-Chirasil-Val.

Left: Stronger retention of R -

methyl lactate. Right: Stronger retention of Sisopropylcarbamoyl lactic acid methylester, acc. to Frank et al. 3 2 )

I CH2N O H

0

H

CH3

H

I

H

H

(S)

31

esters (type lJ)is hard to understand. A similar reversion has been observed in going from the derivatives

1 of

2-amino

acids to the corresponding derivatives of 3-amino acids 56)

.

The stereoselective association of simple diamides has been studied by Nee1 in N M R - s p e c t r o ~ c o p y ~ ~ It ) . is interesting to note that in dilute tetrachloromethane solution the heterochiral complex ( S / R ) appeared to be more stable than the homochiral complex, showing the preference of trans-position for the two alkyl groups in simple associates. From this point of view, only extended studies including larger parts of the chiral solvent can give a decisive picture of the resolution mechanism.

Conclusion

What can we learn about chiral recognition from investigations of the interaction of various enantiomers with a chiral stationary phase? Pauling stated75) ; "I think that a great field

....

in which there are possibilities of tremendous pro-

gress, is the field of the explanation of the highly specific interactions between molecules showing up in biological systems. ....p henomena of biological specificity are determined by the rather weak interactions between molecules

...

sitting together over a considerable area so that the forces of attraction are summed up over this area into an effective bond between these molecules. Here we are pretty ignorant still." In contrast to biological systems, model systems like Chirasil-Val are easily varied, especially on the side of the

38

solute, to get some insight into the mechanism of interaction. Thermodynamic studies as well as spectrometric investigations and theoretical calculations are useful tools. On the other hand, the biological selector,

&a

cavity of

an enzyme or a receptor, is better defined in shape, since a longer peptide chain is more restricted in conformation. Yet the low selectivity of chiral stationary phases in gas chromatography is made up by the high efficiency of the capillary columns. This unique combination is the clue for the versatility of enantiomer resolution by gas chromatography.

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'7

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Angew. Chem.

( 1 9 7 8 ) 363.

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B. Koppenhoefer, K. Hintzer, R. Weber, V. Schurig, Angew.

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2

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*

19)

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N.

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6i

N.

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Konig, S .

H.

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Frank,

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THE MASS SPECTROMETER AS A DETECTOR I N CHROMATOGRAPHY K l a u s Biemann Depa r t m e n t o f C hemi s t ry M a s s a c h u s e t t s I n s t i t u t e o f T e c h n o l o g y , Cambridge, MA 02139 (U.S.A.)

INTRODUCTION The a d v e n t of gas c h r o m a t o g r a p h y i n t h e e a r l y 1 9 5 0 ' s r e p r e s e n t e d a b o o n f o r organic chemists--at pounds--because

l e a s t t h o s e d e a l i n g w i t h r e l a t i v e l y v o l a t i l e com-

i t was s u d d e n l y p o s s i b l e t o w o r k w i t h v e r y s m a l l a m o u n t s o f

r e l a t i v e l y complex m i x t u r e s .

I n t h o s e e a r l y days, packed columns c o a t e d w i t h

u n s o p h i s t i c a t e d l i q u i d phases were u s e d i n c o n j u n c t i o n w i t h t h e r m i s t o r s a s a d e t e c t i o n device.

The a p p a r a t u s made i t p o s s i b l e t o a s s e s s t h e p u r i t y o f a

compound, o r t h e c o m p l e x i t y o f a m i x t u r e , t o r e c o g n i z e a known compound by i t s r e t e n t i o n b e h a v i o r o r i s o l a t e s m a l l amounts o f p u r e m a t e r i a l b y t r a p p i n g t h e e f f l u e n t f o r f u r t h e r study. I t was soon r e c o g n i z e d t h a t gas c h r o m a t o g r a p h y i s v e r y u s e f u l f o r t h e

q u a n t i t a t i v e a n a l y s i s o f m i x t u r e s as w e l l as f o r t h e i d e n t i f i c a t i o n o f o r g a n i c compounds by t h e d e t e r m i n a t i o n o f t h e i r r e t e n t i o n b e h a v i o r on t w o o r more d i f f e r e n t l i q u i d phases.

However,

i t was a l s o a p p r e c i a t e d t h a t one needs s u p p l e -

m e n t a r y i n f o r m a t i o n i f t h e compound a t hand i s o f unknown s t r u c t u r e .

For t h a t

purpose, f r a c t i o n s ( p e a k s ) h a d t o be c o l l e c t e d and s u b j e c t e d t o UV o r I R spectroscopy.

F o r t u n a t e l y , t h e l o a d i n g o n packed c o l u m n s was l a r g e e n o u y h t h a t

s u f f i c i e n t m a t e r i a l c o u l d be c o l l e c t e d , a t l e a s t a f t e r r e p e a t e d i n j e c t i o n s . MASS SPECTRA OF FRACTIONS COLLECTED FROM A GAS CHROMATOGRAPH

When we began i n t h e l a t e 1 9 5 0 ' s t o u s e mass s p e c t r o m e t r y f o r t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e o f n a t u r a l p r o d u c t s we soon s t a r t e d t o u s e gas c h r o matography f o r t h e i s o l a t i o n o r p u r i f i c a t i o n o f t h e compounds o f i n t e r e s t . The f i r s t example i n v o l v e d t h e d e t e r m i n a t i o n o f t h e a m i n o a c i d s e q u e n c e o f s m a l l p e p t i d e s p r e s e n t a s a m i x t u r e o f s u i t a b l e d e r i v a t i v e s ( r e f . 1); i t u s e d gas c h r o m a t o g r a p h y f o r t h e s e p a r a t i o n o f t h e components w h i c h w e r e c o l l e c t e d i n d i v i d u a l l y and s e p a r a t e l y i n t r o d u c e d i n t o t h e b a t c h - i n l e t system o f a CEC-21-103C mass s p e c t r o m e t e r .

The g a s c h r o m a t o g r a p h was a home-made c o n t r a p -

t i o n d e s i g n e d i n o u r C h e m i s t r y d e p a r t m e n t a f t e r E.R.H.

Jones i n t h e e a r l y

1 9 5 0 ' s b r o u g h t t o MIT t h e news o f t h e d e v e l o p m e n t o f gas-1 i q u i d c h r o m a t o g r a p h y b y A.T.

James a n d A.J.P.

Martin (ref.

2 ) a n d i t s w i d e s p r e a d and s u c c e s s f u l u s e

a t I C I and o t h e r B r i t i s h r e s e a r c h l a b o r a t o r i e s .

44

A l s o i n o u r w o r k on t h e s t r u c t u r e o f i n d o l e a l k a l o i d s i s o l a t e d f r o m c r u d e p l a n t e x t r a c t s , gas c h r o m a t o g r a p h y p r o v e d t o b e a t i m e - s a v i n g d e v i c e .

Even

t h o u g h t h e s e compounds w e r e a t t h e e d g e o f w h a t c o u l d b e p u s h e d t h r o u g h a packed A p i e z o n

L column and t h e peak shapes a r e n o t h i n g t o b e p r o u d o f ( s e e

F i g . 1) t h e s e p a r a t i o n was good e n o u g h t o a l l o w c o l l e c t i n g f r a c t i o n s w h i c h gave i n t e r p r e t a b l e mass s p e c t r a .

It t u r n e d o u t t h a t t h e r e were tvro s t r u c t u r a l

types o f a l k a l o i d s present, those w i t h t h e aspidospermidine s k e l e t o n (A) and t h o s e w i t h a s p i d o s p e r m a t i d i n e s k e l e t o n (6). l e t t e r s A and alkaloid.

The n u m b e r s p r e c e e d i n g t h e

B i n Fig. 1 r e p r e s e n t t h e m o l e c u l a r w e i g h t s o f t h e p a r t i c u l a r

The mass s p e c t r a a l l o w e d t h e a s s i g n m e n t b o t h o f t h e s t r u c t u r a l t y p e

a n d o f t h e s u b s t i t u e n t s R1-R3 ( r e f , 3).

C l e a r l y , t h e u t i l i z a t i o n o f gas c h r o -

m a t o g r a p h y as a s e p a r a t i o n d e v i c e and o f mass s p e c t r o m e t r y a s a c h a r a c t e r i z a t i o n method made i t p o s s i b l e t o d e t e r m i n e t h e s t r u c t u r e o f a s e r i e s o f new a l k a l o i d s w i t h i n a few weeks, i n c o n t r a s t t o t h e many y e a r s r e q u i r e d p r e v i o u s l y f o r l a r g e s c a l e i s o l a t i o n f o l l o w e d b y p a i n s t a k i n g c h e m i c a l d e g r a d a t i o n o f each compound.

A

B R1, RE, R3 = H, CH3, OCH3 o r CH3C0 8f-I ,265' ( 6 X Aptezon L!

F i y . 1. Gas chroinatogram o f a m i x t u r e o f a l k a l o i d s e x t r a c t e d f r o m t h e b a r k o f Aspidosperma quebracho btanco. F r a c t i o n s were c o l l e c t e d between t h e p o i n t s i n d i c a t e d by t h e s l a n t e d l i n e s and i n t r o d u c e d i n t o t h e mass s p e c t r o m e t e r f o r t h e s t r u c t u r e d e t e r m i n a t i o n ( f o r d e t a i l s see r e f . 3).

45

INTERFACING OF A GAS CHROMATOGRAPH WITH A MASS SPECTROMETER Two f a c t s caused us t o t a k e a n i m p o r t a n t s t e p f o r w a r d .

F i r s t , i t was i n -

c o n v e n i e n t , t i m e consuming and d i f f i c u l t t o c o l l e c t s u b m i l l i g r a m f r a c t i o n s o f a n a l k a l o i d f r o m t h e e x h a u s t o f a gas c h r o m a t o g r a p h , t o t r a n s p o r t t h e m t o t h e mass s p e c t r o m e t e r and t o s u b l i m e one a f t e r t h e o t h e r i n t o t h e mass s p e c t r o m e t e r ; second, f o r t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e o f new n a t u r a l p r o d u c t s we had t u r n e d t o h i g h r e s o l u t i o n mass s p e c t r o m e t r y b e c a u s e i t i s c l e a r l y m o r e h e l p f u l t o know t h e e l e m e n t a l c o m p o s i t i o n and n o t j u s t t h e n o m i n a l mass o f t h e m o l e c u l e o r o f f r a g m e n t i o n s i f one d e a l s w i t h a n unknown compound.

This l e d

t o t h e d e v e l o p m e n t o f t h e d i r e c t , a l l - g l a s s c o n n e c t o r o f t h e gas c h r o m a t o g r a p h w i t h t h e mass s p e c t r o m e t e r ( r e f . 4 ) .

I n fact,

the f i r s t design incorporated

t h e d i r e c t connection of a packed gas c h r o m a t o g r a p h i c column v i a a porous g l a s s t u b e ( t h e c a r r i e r gas s e p a r a t o r ) t o a CEC 21-110 h i g h r e s o l u t i o n m a s s spectrometer (Fig.

Z), i.e.

t h e gas c h r o m a t o g r a p h was r e a l l y t r e a t e d a s a mere

i n l e t system o f t h e spectrometer ( r e f .

5).

H o w e v e r , s i n c e t h i s d e s i g n was

d i f f i c u l t t o o p e r a t e i n a t e m p e r a t u r e p r o g r a m m e d mode, t h e a t t a c h m e n t o f a c o m m e r c i a l , f u l l f l e d g e d gas c h r o m a t o g r a p h t o t h e inass s p e c t r o m e t e r v i a t h e f i t t e d g l a s s t u b e s e p a r a t o r became t h e permanent s o l u t i o n .

,sample injection

electric sector ton

mapnet

/ /

tolibrotion compound

monit

Fig. 2. Schematic o f mass s p e c t r o m e t e r w i t h gas c h r o m a t o g r a p h a t t a c h e d t o m o d i f i e d i o n source housing.

The u s e o f a M a t t a u c h - H e r z o g d o u b l e f o c u s s i n y mass s p e c t r o m e t e r h a d t h e a d d i t i o n a l a d v a n t a g e t h a t t h e mass s p e c t r u m c o u l d be r e c o r d e d o n a p h o t o g r a p h i c p l a t e i n a s t a t i c mode ( w i t h t h e e x c e p t i o n o f t i m e - o f - f l i g h t i n s t r u m e n t s , f a s t s c a n n i n g mass s p e c t r o m e t e r s , p a r t i c u l a r l y o f h i g h r e s o l u t i o n , w e r e n o t y e t available).

F i g u r e 3 shows a t o t a l i o n m o n i t o r t r a c e r e c o r d e d d u r i n y t h e

gas c h r o m a t o g r a p h i c s e p a r a t i o n o f a m i x t u r e o f i n d o l e a l k a l o i d s ( r e f . 5 ) .

The

46

v e r t i c a l s l o t s r e s u l t f r o m t u r n i n g o f f t h e i o n beam w h i l e t h e p h o t o g r a p h i c p l a t e i s advanced f r o m one r e c o r d i n g p o s i t i o n t o t h e n e x t .

A mass s t a n d a r d

( p e r f l u o r o k e r o s e n e ) i s b l e d i n t o t h e i o n s o u r c e c o n t i n u o u s l y and l a t e r a l l o w s t h e a s s i g n m e n t o f t h e e x a c t mass o f a l l l i n e s on t h e p l a t e .

I

Ill 1 1 F i g . 3. Top: T o t a l i o n m o n i t o r t r a c e r e c o r d e d d u r i n g a gas chromatogram o f a n a l k a l o i d e x t r a c t . Bottom: E n l a r g e m e n t o f a s m a l l s e c t i o n o f t h e p h o t o g r a p h i c p l a t e exposed s t e p w i s e d u r i n g t h e gas chromatograln showing a s h o r t r e g i o n (m/z 279-294) o f t h e s p e c t r a r e c o r d e d d u r i n g e x p o s u r e s 2 1 t h r o u g h 24. R e s u l t s 07e x a c t inass measurements ( e r r o r i n p a r e n t h e s e s ) a n d c o r r e s p o n d i n g e l e m e n t a l c o m p o s i t i o n s a r e n o t e d ( f r o m r e f . 5).

W h i l e t h e a d v a n t a g e s o f h i g h r e s o l u t i o n mass s p e c t r o r a e t r y f o r t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e o f n a t u r a l compounds was n o t i n d o u b t , t h e a p p l i c a t i o n s o f t h e c o m b i n a t i o n o f a gas c h r o i n a t o g r a p h w i t h a mass s p e c t r o m e t e r

(GCMS) t o more a n a l y t i c a l t h a n s t r u c t u r a l p r o b l e m s mushroomed i n a r e a s w h e r e u n i t mass r e s o l u t i o n s u f f i c e d o r was i n d e e d p r e f e r a b l e (because o f s i m p l e r i n s t r u m e n t a t i o n and d a t a ) . The LKB 9000 mass s p e c t r o m e t e r w h i c h i n c o r p o r a t e d t h e j e t s e p a r a t o r d e v e l o p e d b y Ryhage ( r e f . 6 ) was t h e f i r s t c o m m e r c i a l systein and soon f o u n d w i d e use.

41 COMPUTER-AIDED DATA ACQUISITION AND PROCESSIIIG F o r s i m i l a r r e a s o n s we i n t e r f a c e d an A e r o y r a p h M o d e l 6 0 0 g a s c h r o m a t o g r a p h t o a H i t a c h i - P e r k i n E l m e r R M U - 6 s i n g l e f o c u s s i n g mass s p e c t r o m e t e r e q u i p p e d w i t h f a s t , r e p e t i t i v e scan c a p a b i l i t y .

B u t we i m m e d i a t e l y e n c o u n -

t e r e d a new p r o b l e m t h a t needed t o b e s o l v e d : t h e a c c u m u l a t i o n o f a n e n o r m o u s amount o f d a t a t h a t had t o be i n t e r p r e t e d .

The c o n v e n t i o n a l o s c i l l o y r a p h i c

r e c o r d i n g method p r o v e d unmanageable, u n l e s s one o n l y r e c o r d e d one s c a n o n t h e t o p o f each gas c h r o m a t o g r a p h i c peak, a mode o f o p e r a t i o n w h i c h was s u s p e c t e d t o lead t o loss o f information.

The s o l u t i o n t o t h a t p r o b l e m was t h e c o n t i n u -

ous, r e p e t i t i v e r e c o r d i n g o f t h e mass s p e c t r a l d a t a , f i r s t o n t o d i g i t a l magn e t i c tape (ref. ( r e f . 8).

7 ) and soon t h e r e a f t e r d i r e c t l y i n t o a n IBM 1800 c o m p u t e r

-

F i g u r e 4 i l l u s t r a t e s t h e d e s i g n w h i c h Has t h e f i r s t g e n e r a l l y u s e -

f u l GC-MS-computer system.

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F i g . 4. S c h e m a t i c o f t h e gas chromatograph-mass s p e c t r o m e t e r - c o m p u t e r s y s t e m d e v e l o p e d i n t h e a u t h o r ' s l a b o r a t o r y i n 1966-7.

T h i s development serves as a reminder t h a t t h e emergence o f mass s p e c t r o m e t e r - c o m p u t e r s y s t e m s , so commonplace t o d a y , was a c t u a l l y t r i g g e r e d b y a need caused by t h e y r o w i n y s o p h i s t i c a t i o n o f gas c h r o m a t o g r a p h s , w h i c h s p e w e d o u t b e t t e r and b e t t e r r e s o l v e d components o f e v e r more complex m i x t u r e s w h i c h needed t o be c h a r a c t e r i z e d .

The c a p a b i l i t y f o r c o n t i n u o u s r e c o r d i n g o f m a s s

s p e c t r a l d a t a d u r i n g a GC e x p e r i m e n t opened t h e way f o r new a p p r o a c h e s t o d a t a

48 p r o c e s s i n g and e v a l u a t i o n because one d e a l t no l o n g e r w i t h i s o l a t e d mass spect r a b u t c o u l d u t i l i z e t h e t i m e domain r e p r e s e n t e d by t h e c o n t i n u o u s changes i n t h e s p e c t r a r e c o r d e d one a f t e r a n o t h e r a t f i x e d t i m e i n t e r v a l s .

In a d d i t i o n

t o t h e d i s p l a y o f t h e d a t a as mass s p e c t r a one c o u l d a l s o d i s p l a y t h e a b u n dance v a r i a t i o n s o f i o n s o f any m a s s - t o - c h a r y e r a t i o a f t e r t h e e x p e r i m e n t , a f o r i n a t c a l l e d "mass chromatogram" ( r e f . 9). Even t o d a y t h i s a p p r o a c h i s p r o b a b l y t h e most p o w e r f u l t e c h n i q u e f o r t h e e v a l u a t i o n o f a c h r o i n a t o y r a p h i c separation.

I t a l l o w s one,

f o r example,

t o display the elution o f certain

compound t y p e s w h i c h have a common mass s p e c t r a l f e a t u r e even i f t h e y a r e o b s c u r e d i n t h e gas chromatogram o f a v e r y c o m p l i c a t e d m i x t u r e ( F i g .

5).

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S P E C T R U M INDEX NUMBER

F i g . 5. ( a ) T o t a l i o n i z a t i o n p l o t ( g a s c h r o m a t o y r a m ) o f a complex m i x t u r e o f c a r b o x y l i c a c i d s ( o r m e t h y l e s t e r s ) o b t a i n e d by o x i d a t i v e d e g r a d a t i o n o f kerogen; ( b ) mass chromatoyram o f IIJ~ 88, c h a r a c t e r i s t i c o f a - m e t h y l s u b s t i t u t e d m e t h y l e s t e r s ; ( c ) mass chromatogram o f m/l 98, c h a r a c t e r i s t i c o f methyl e s t e r s o f l o n g c h a i n a , w d i c a r b o x y l i c acids.

49

A l t e r n a t i v e l y one c a n p i n p o i n t t h e p r e s e n c e - - o r

prove t h e absence--of

i f i c compound by d i s p l a y i n g t h e mass chromatograms o f

f i / ~v a l u e s

a spec-

a t which the

mass s p e c t r u m o f t h e compound s e a r c h e d f o r has l a r g e , c h a r a c t e r i s t i c peaks. Other developments followed q u i c k l y .

One o f them made use o f t h e c o n t i n -

uous changes i n mass s e p a r a t e d i o n abundances as r e p r e s e n t e d by t h e mass c h r o matoyrams t o e f f e c t i v e l y i n c r e a s e t h e c h r o m a t o g r a p h i c s e p a r a t i o n : f o r t w o i n c o m p l e t e l y s e p a r a t e d compounds e x h i b i t i n y d i f f e r e n t mass s p e c t r a one c a n d i s p l a y t h e i r s e p a r a t e e l u t i o n p r o f i l e s by p l o t t i n g t h e a b u n d a n c e v a r i a t i o n s o f

one o r more s p e c i f i c i o n s vs. t i m e .

Conversely,

plotting only data a t the

leak o f e l u t i o n o f each such compound g e n e r a t e s a "mass r e s o l v e d chromatogram" (Fig. 6).

Froin t h e p o i n t o f s p e c t r a l i n t e r p r e t a t i o n a r e l a t e d f o r m o f d a t a

d i s p l a y i s m o s t u s e f u l : a " r e c o n s t r u c t e d mass s p e c t r u m " g e n e r a t e d b y p l o t t i n g only the

f i / l v a l u e s o f t h o s e i o n s w h i c h m a x i m i z e i n t h e same s c a n p r o d u c e s a

mass s p e c t r u m o f t h e e l u t i n g compound f r e e f r o m t h e c o n t r i b u t i o n s o f a d j a c e n t components, column b l e e d , background, e t c .

(ref.

10).

Figure 7 i l l u s t r a t e s

how w e l l t h e inass s p e c t r u m o f n-docosane i s g e n e r a t e d f r o m t h e raw d a t a i n t h e r e y i o n where b o t h p h e n a c e t i n e (mol. w t .

179) and p a l m i t i c a c i d (mol.

wt.

256)

e l u t e ( s e e i n s e r t i n F i g . 6 s h o w i n g t h e GC-trace as w e l l as t h e mass c h r o m a t o grdms o f t h e i n o l e c u l a r i o n s o f t h e s e t h r e e compounds).

I t i s even p o s s i b l e t o

g e n e r a t e t h e i n d i v i d u a l mass s p e c t r a o f a compound and i t s d e u t e r a t e d a n a l o g w h i c h u s u a l l y e l u t e a s a s i n g l e p e a k b u t t w o o r t h r e e illass s p e c t r a l s c a n s apart.

S i m i l a r a l y o r i t h m s have been d e v e l o p e d by o t h e r s ( r e f .

o f them were a l s o i n c o r p o r a t e d i n t o c o m m e r c i a l d a t a systeias. p l e s u t i l i z e d packed c o l u m n g a s c h r o m a t o g r a p h y .

11,12)

and some

A l l t h e s e exam-

Modern c a p i l l a r y columns

w o u l d have e l i m i n a t e d t h e need f o r t h e d a t a - b a s e d improvement o f r e s o l u t i o n i n many o f t h e s e c a s e s , b u t t h e r e a l w a y s r e m a i n some u n r e s o l v e d c o m p o n e n t s t o w h i c h t h e s e t e c h n i q u e s c a n t h e n be a p p l i e d . Another piece of i n f o r m a t i o n t h a t c a n b e a u t o m a t i c a l l y e x t r a c t e d f r o m c o n t i n u o u s l y r e c o r d e d mass s p e c t r a i s t h e r e t e n t i o n i n d e x .

Algorithms f o r t h e

i d e n t i f i c a t i o n o f compounds e m e r g i n g f r o m a c h r o m a t o y r a p h b y m a t c h i n g t h e i r mass s p e c t r a a g a i n s t a c o l l e c t i o n o f t h e s p e c t r a o f o f d u t h e n t i c compounds had 13).

T h i s formed t h e b a s i s o f t h e r e c o g n i t i o n o f

i n t e r n a l reference standards (i.e.

n-hydrocarbons) o r any compound o f known

been d e v e l o p e d e a r l i e r ( r e f .

r e t e n t i o n index present i n t h e m i x t u r e being andlyzed.

These c a n t h e n be u s e d

t o c a l c u l a t e t h e r e t e n t i o n i n d i c e s c o r r e s p o n d i n g t o each o n e o f t h e c o n s e c u t i v e mass s p e c t r a r e c o r d e d d u r i n g t h e GCMS e x p e r i m e n t ( r e f .

14).

The r e t e n -

t i o n i n f o r m a t i o n c a n t h e n be u s e d e i t h e r as a window t o r e s t r i c t t h e number o f known s p e c t r a t o be compared w i t h each unknown, o r t o d i f f e r e n t i a t e b e t w e e n t w o compounds w h i c h e x h i b i t v e r y s i m i l a r mass s p e c t r a b u t e l u t e a t d i f f e r e n t r e t e n t i o n times (ref.

15).

Generally, t h i s approach r e q u i r e s t h e a v a i l a b i l i t y

o f e x p e r i m e n t a l l y d e t e r m i n e d r e t e n t i o n i n d i c e s a l o n g w i t h t h e mass s p e c t r a o f

50

a u t h e n t i c compounds, b u t i n one p a r t i c u l a r case, t h e d e r i v a t i v e s o f p e p t i d e s u s i n g o u r mass s p e c t o m e t r i c s e q u e n c i n g t e c h n i q u e ( r e f , c a n be p r e d i c t e d w i t h s u f f i c i e n t

16), r e t e n t i o n i n d i c e s

a c c u r a c y t o e l i m i n a t e t h e need f o r t h e i r e x -

p e r i m e n t a l measurement ( r e f . 1 7 ) .

c

Fig. 6. Top: Gas chromatogram ( p a c k e d c o l u m n ) i n t h e f o r m o f a t o t a l I n s e r t i s an enlargement i o n i z a t i o n p l o t o f t h e e x t r a c t o f a g a s t r i c lavage. o f a n u n r e s o l v e d segment c o n t a i n i n g a t l e a s t t h r e e components o f d i f f e r e n t peak p r o f i l e s . Bottom: Mass r e s o l v e d gas chromatogram d e r i v e d f r o m t h e mass s p e c t r a l data recorded d u r i n g t h e experiment.

51

F i g . 7 . Top: Scan 114 r e c o r d e d d u r i n g the g a s chromatogram shown i n F i g . 6 . Bottom: Reconstructed mass spectrum ( r e p r e s e n t i n g n-docosane) d e r i v e d from scan 114 ( f o r d e t a i l s see t e x t and r e f . 1 0 ) . APPLICATIONS The i n t e g r a t i o n o f gas chromatograph, mass s p e c t r o m e t e r and c o m p u t e r h a s l e d t o an i n s t r u m e n t package t h a t i s widely u s e d t o d a y i n many a r e a s o f a n a l y t i c a l , b i o l o g i c a l , environmental and c l i n i c a l r e s e a r c h .

I t s main a d v a n t a g e

i s g e n e r a l a p p l i c a b i l i t y ( l i m i t e d o n l y by t h e v o l a t i l i t y and thermal s t a b i l i t y r e q u i r e m e n t s l e v i e d by t h e g a s c h r o m a t o g r a p h ) and h i g h s e n s i t i v i t y .

These

methodologies were i n c o r p o r a t e d i n t e c h n i q u e s designed t o i d e n t i f y drugs a n d t h e i r m e t a b o l i t e s i n body f l u i d s ( r e f . 18) and t o a u t o m a t i c a l l y d i a g n o s e c e r t a i n d i s e a s e s from the abundance p a t t e r n of u r i n a r y a c i d s ( r e f . 1 9 ) .

An extreme example of the use o f a GC-MS-computer system where s e n s i t i v i t y and general a p p l i c a b i l i t y were p a r t i c u l a r l y i m p o r t a n t was t h e Molecular Ana l y s i s experiment of NASA's Viking Mission t o t h e s u r f a c e o f t h e p l a n e t Mars i n 1976. The purpose of t h i s experiment was t h e s e a r c h f o r t r a c e s o f o r g a n i c compounds i n t h e s u r f a c e m a t e r i a l and t h i s r e q u i r e d high s e n s i t i v i t y a s w e l l a s broad a p p l i c a b i l i t y because i t had t o respond t o any o r y a n i c compound t h a t

52

c o u l d be v o l a t i l i z e d o r p y r o l y z e d o u t o f t h e M a r t i a n s o i l sample.

The i n s t r u -

ment c o n s i s t e d o f a s e t of p y r o l y s i s ovens, a gas c h r o m a t o g r a p h i c c o l u m n , h y d r o g e n c a r r i e r gas, h y d r o g e n s e p a r a t o r , d o u b l e f o c u s s i n g mass s p e c t r o m e t e r and

A l l miniaturized t o weiyh l e s s than

d a t a a c q u i s i t i o n and p r o c e s s i n g system.

40 kg and f i t i n t o a cube 30 cin o n edge ( r e f .

20).

I t s automated operation,

t h a t c o u l d be c o n t r o l l e d by radio-command f r o m t h e E a r t h was h i g h l y s u c c e s s ful.

The i n s t r u m e n t d e m o n s t r a t e d t h e absence o f v o l a t i l e ,

volatilizable or

p y r o l y z a b l e o r g a n i c compounds a t t h e s u r f a c e o f two, w i d e l y s e p a r a t e l o c a t i o n s a t t h e p l a n e t w i t h a d e t e c t i o n l i m i t below t h e ppb l e v e l ( r e f .

21).

That i n -

s t r u m e n t r e p r e s e n t s t h e m o s t r e m o t e ( o v e r 325 m i l l i o n km f r o m t h e E a r t h ) y a s C h r o m a t o g r a p h ( a n d mass s p e c t r o m e t e r ) t h a t h a s e v e r b e e n o p e r a t e d . The r e s u l t s had g r e a t i m p a c t o n t h e e x p e r i m e n t s c a r r i e d o u t t o s e a r c h f o r t h e mani f e s t a t i o n o f l i v i n g systems

on Wars.

With t h e a l m o s t complete r e p l a c e m e n t o f packed columns by c a p i l l a r y columns d u r i n g t h e l a s t decade and t h e i n c o r p o r a t i o n o f more e f f i c i e n t vacuum pumps i n t o t h e mass s p e c t r o m e t e r , t h e need f o r a c a r r i e r gas s e p a r a t o r has ess e n t i a l l y disappeared.

The l o w e r f l o w t h r o u g h a c a p i l l a r y c o l u m n a l l o w s i t s

d i r e c t connection t o t h e i o n source o f t h e spectrometer, t h e i n t e r f a c i n g w i t h t h e yas chromatograph.

greatly simplifying

More r e c e n t l y , t h e t e n d e n c y h a s

become t o h i d e t h e mass s p e c t r o i n e t e r f r o m t h e c h r o m a t o g r a p h e r b y c a i n o u f l a g i n y i t as s o p h i s t i c a t e d "mass s e l e c t i v e d e t e c t o r " ( H e w l e t t - P a c k a r d 5970B) o r F i n nigan Corp.'s

"ion trap".

INTERFACING AN HPLC WITH A MASS SPECTROMETER The r e v o l u t i o n w h i c h yas c h r o m a t o g r a p h y b r o u g h t t o c h e m i s t r y , b i o c h e i n i s t r y and b i o l o g y was r e p e a t e d more r e c e n t l y by t h e i n t r o d u c t i o n o f h i g h p e r f o r -

mance l i q u i d c h r o m a t o g r a p h y (HPLC).

It d i d n o t t a k e l o n g t o e x t r a p o l a t e f r o m

t h e s u c c e s s o f GCMS t o e x p e c t a s i m i l a r p o t e n t i a t i o n b y i n t e r f a c i n g a n HPLC d i r e c t l y w i t h t h e mass s p e c t r o r n e t e r . twofold:

The a d v a n t a g e w o u l d b e a t l e a s t

f i r s t , a s p e e d i n g - u p o f d a t a p r o d u c t i o n (mass s p e c t r a ) b y t h e e l i m i -

n a t i o n o f t h e need t o c o l l e c t f r a c t i o n s , e v a p o r a t e t h e s o l v e n t and s e p a r a t e l y i n t r o d u c i n g t h e r e s i d u e i n t o t h e mass s p e c t r o m e t e r ; and, s e c o n d ,

t h e yenera-

t i o n o f t h e same t y p e o f t h r e e d i m e n s i o n a l d a t d i n h e r e n t i n c o n t i n u o u s d a t a c o l l e c t i o n w h i c h p r o v e d so u s e f u l i n t h e c a s e o f tiCMS ( m a s s c h r o m a t o g r a m s , etc.).

However, i n s p i t e o f t h e g r e a t e f f o r t s made o v e r t h e l a s t d e c a d e o n e

m u s t say t h a t p r o g r e s s i s s l o w and n o t a t a l l c o m p a r a b l e t o t h e s u c c e s s o f GCMS even i n i t s f i r s t few y e a r s o f e x i s t e n c e .

The r e a s o n f o r t h i s i s t w o f o l d .

The p r i n c i p a l c o m p a t i b i l i t y o f G C a n d

MS, b o t h o f w h i c h r e q u i r e t h e sample t o be i n t h e yas p h a s e ( w i t h t h e e x c e p t i o n o f a few, r e l a t i v e l y new s u r f a c e i o n i z a t i o n t e c h n i q u e s ) and t h e d i f f i c u l t y of c o l l e c t i n g from a yas c h r o m a t o g r a p h a v e r y s m a l l s a m p l e ( m i c r o g r a m s o r

53 ndnograms) i n a way t h a t makes i t e a s y t o i n t r o d u c e i t i n t o t h e mass s p e c t r o s eter.

On t h e o t h e r hand, t h e compounds s e p a r a t e d b y HPLC a r e n o n v o l a t i l e ,

p o l a r and o f t e n l a r g e m o l e c u l e s ( o t h e r w i s e one w o u l d u s e t h e s i m p l e r GC) w h i c h a r e t h u s n o t e a s i l y amenable t o mass s p e c t r o m e t r y ( e x c e p t u s i n g s u r f a c e i o n i z a t i o n t e c h n i q u e s ) a n d t h e m o v i n g phase i s a l i q u i d - - o f t e n

quite polar.

All

t h i s makes i t d i f f i c u l t t o i n t e r f a c e t h e l i q u i d c h r o i n a t o y r a p h v J i t h t h e mass s p e c t r o m e t e r and any such c o m b i n a t i o n must be e a s y t o o p e r a t e w i t h a h i g h succ e s s r a t e t o be s u p e r i o r t o t h e b a t c h mode, namely c o l l e c t i o n o f f r a c t i o n s and successive i n t r o d u c t i o n o f t h e sample i n t o t h e s p e c t r o m e t e r .

E a r l i e r ap-

22) i n which a small

p r o a c h e s such as t h e d i r e c t l i q u i d i n t r o d u c t i o n ( r e f .

p o r t i o n o f t h e e f f l u e n t i s vaporized i n t o t h e spectrometer o r t h e moving b e l t ( r e f . 23) w h i c h t r a n s p o r t s p a r t o f t h e sample a f t e r e v a p o r a t i o n o f t h e s o l v e n t i n t o t h e i o n s o u r c e work i n p r i n c i p l e a n d a r e now c o m m e r c i a l l y a v a i l a b l e . However, t h e y a r e u s e f u l o n l y f o r a s m a l l segment o f compounds, n a m e l y t h o s e t h a t a r e i n a r d n y e o f v o l a t i l i t y and m o l e c u l a r s i z e n o t much b e y o n d t h e a p p l i c a b i l i t y o f gas chromatography.

A t t h e same t i m e ( s i n c e 1 9 8 1 ) i t h a s b e -

come p o s s i b l e t o o b t a i n iiiass s p e c t r o m e t r i c i n f o r m a t i o n

on l a r g e , p o l a r m o l e c -

u l e s , e s p e c i a l l y by f a s t atom bombardment (FAB) mass S p e c t r o m e t r y ( r e f .

24),

w h i c h pushes t h e a p p l i c a b i l i t y o f MS f u r t h e r i n t o r e y i o n s o f i n t e r e s t w h e r e o n l y HPLC c a n be u s e d f o r s e p a r a t i o n .

T h i s may be a c h i e v a b l e by a m o v i n g b e l t

s y s t e m w h i c h t r a n s p o r t s t h e sample, d i s s o l v e d i n a s m a l l amount o f l i q u i d mat r i x ( l i k e g l y c e r o l ) i n t o t h e FAB i o n s o u r c e o f a mass s p e c t r o m e t e r . The m o s t e x t r e m e b u t p e r h a p s m o s t p r o m i s i n g d e v e l o p m e n t i s t h e " t h e r mospray" t e c h n i q u e o f V e s t a l ( r e f . 2 5 ) w h i c h combines t h e r m a l e v a p o r a t i o n w i t h s e l f i o n i z a t i o n and p r o d u c e s r e l a t i v e l y s t a b l e p r o t o n a t e d m o l e c u l a r i o n s o f l a r g e and p o l a r m o l e c u l e s ( s u c h as p e p t i d e s o f s o l e c u l a r w e i g h t b e y o n d 1 0 0 0 daltons )

.

A t t h i s writing,

HPLC-MS r e m a i n s i n t h e e x p l o r a t o r y phase, p r o m i s i n g b u t

r a r e l y a p p l i e d t o t h e s o l u t i o n o f chemical o r b i o l o g i c a l p r o b l e m s because i t i s s t i l l more p r a c t i c a l t o u s e t h e t w o systems, c h r o m a t o g r a p h a n d s p e c t r o m e t e r , s e p a r a t e l y r a t h e r t h a n d i r e c t l y coupled. COWL US I ONS M o n i t o r i n g t h e e f f l u e n t o f a gas c h r o r n a t o y r a p h w i t h a f a s t s c a n n i n g ( o r j u m p i n g ) mass S p e c t r o m e t e r c o u p l e d t o a computer-based d a t a system has widened t h e u t i l i t y o f t h i s s e p a r a t i o n method i n t w o d i r e c t i o n s :

(1) t h e i d e n t i f i c a -

t i o n o f e l u t i n y compounds h a s become e f f i c i e n t a n d h i g h l y s p e c i f i c ; a n d ( 2 ) q u a n t i t a t i v e a n a l y s i s i s much inore r e l i a b l e .

T h i s i s due t o t h e h i g h i n f o r m a -

t i o n c o n t e n t o f t h e mass s p e c t r u m and t h e p r o p e r c h o i c e o f one ( o r m o r e ) speci f i c mass a v a i l a b l e f o r q u a n t i t a t i o n o f a s p e c i f i c compound.

Mass s p e c t r o m e -

t r y a l s o a l l o w s t h e use of s t a b l e i s o t o p e l a b e l l e d i n t e r n a l s t a n d a r d s .

This

54

m e t h o d o l o g y i s now w i d e l y u s e d i n b i o m e d i c a l and e n v i r o n m e n t a l a n a l y s i s .

The

advantages o f t h i s approach m a n i f e s t t h e m s e l v e s m o s t d r a m a t i c a l l y i n c a s e s where i t i s n e c e s s a r y t o d e t e c t and q u a n t i t a t e t r a c e s o f o r g a n i c compounds i n v e r y complex m i x t u r e s where t h e s u b s t a n c e o f i n t e r e s t may be masked b y a m o r e The a n a l y s i s f o r p o l y c h l o -

a b u n d a n t component o f s i m i l a r r e t e n t i o n b e h a v i o r . r i n a t e d d i o x i n s i s a n o t o r i o u s example.

RE FER E i l C E S

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

K. Biemann and W. V e t t e r , Biochem. Biophys. Res. Cornmuns., 3 (1960) 5 78-584. A.T. James and A.J.P. M a r t i n , A n a l y s t , 77 (1952) 915; Biochem. J., 50 (1952) 679. K. Biemann. M. S p i t e l l e r - F r i e d m a n n and G. S p i t e l l e r . J. Am. Chem. SOL., 85 (1963) 631-638. J.T. Watson and K. Biemann, Anal. Chem., 36 (1964 1135-1137. J.T. Watson and K. Biemann, A n a l . Chem., 37 (1965 844-851. R. Ryhaye, Anal. Chein., 36 (1964) 759. R.A. H i t e s and K. Bieniann, Anal. Chem., 39 (1967) 965-970. R.A. H i t e s and K. Biemann, Anal. Chem., 40 (1968) 1217-1221. R.A. H i t e s and K. Biernann, Anal. Chem.. 42 (1970) 855-860. J.E. B i l l e r and K. Biemann, Anal. L e t t e r s , 7 (1973) 515-528. R.G. Dromey, M.J. S t e f i k , T.C. R i n d f l e i s c h and A.M. Duffield, Anal. Chem., 48 (1976) 1368. D. Henneberg, Adv. Mass Spectrom., 8B (1980) 1511-1531. H.S. H e r t z , R.A. H i t e s and K. Biemann, Anal. Chem., 43 (1971) 681. H. Nau and K. Biemann, Anal. L e t t e r s , 6 (1973) 1071-1081. K. Biemann and J.E. B i l l e r , i n R.F. H i r s c h (Ed.), S t a t i s t i c s , F r a n k l i n I n s t i t u t e P r e s s , P h i l a d e l p h i a , 1978,pp.259-276. J.A. K e l l e y , H. Nau, H.-J. F o r s t e r and K. Biemann, Biomed. Mass Spectrom., 2 (1975) 313-325. H. Nau and K. Biemann, Anal. Biochem., 73 (1976) 139-153. C.E. C o s t e l l o , H.S. H e r t z , T. S a k a i and K. Biemann, Clin. Chem., 20 (1974) 255-265. S.C. Gates, N. Dendrainis and C.C. Sweeley, C l i n . Chem., 24 (1978) 1674. D.R. Rushneck, A.V. D i a z , D.W. Howarth, J. Rampacek, K.W. Olson, U.D. Dencker, P. S m i t h , L. McDavid, A. Tomassian, M. H a r r i s , K. B u l o t a , K. Biemann, A.L. L a f l e u r , J.E. B i l l e r and T. Owen, Rev. S c i . I n s t r u m . , 49 (1975) 817-834. K. Biemann, J. Oro, P. T o u l i n i n , L.E. O r g e l , A.O. N i e r , D.M. Anderson, P.G. Simmonds, D. F l o r y , A.V. D i a z , D.R. Rushneck, J.E. B i l l e r and A.L. L a f l e u r , J. Geophys. Res., 82 (1977) 4641. F.W. M c L a f f e r t y and t4.A. B a l d w i n , Org. Mass Spectrom., 8 (1973) 1111. R.P.W. S c o t t , C.G. S c o t t , M. Munroe and J. Hess, J. Chromatogr., 99 (1974) 395. 14. B a r b e r , R.S. B o r d o l i , R.D. Sedywick and A.N. T y l e r , J. Chem. SOC. Chem. Commun., 7 (1981) 325-327. C.R. B l a k e l y and M.L. V e s t a l , Anal. Chem., 55 (1983) 750.

55

ROADS TO FASTER AND MORE SENSITIVE CAPILLARY GC/MS. APPLICATION COLUMNS

OF 50

pm

P.A. LECLERCQ, C.P.M. SCHUTJES AND C.A. CRAMERS Laboratory o f I n s t r u m e n t a l Analysis, Department o f Chemical Engineering, Eindhoven U n i v e r s i t y o f Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. INTRODUCTION I n isothermal gas chromatography t h e r e t e n t i o n time, tR,o f a compound can be described by :

where L i s t h e column l e n g t h ,

t h e average l i n e a r c a r r i e r gas v e l o c i t y , and

k the c a p a c i t y r a t i o o f t h e s o l u t e . While gas chromatographic r e t e n t i o n times can be reduced a t w i l l , t h e p o s s i b i l i t i e s f o r i n c r e a s i n g t h e speed o f a n a l y s i s f o r a g i v e n s e p a r a t i o n problem are 1 i m i t e d by t h e r e l a t i o n s h i p between r e t e n t i o n t i m e and column r e s o l u t i o n o r p l a t e number. The well-known r e s o l u t i o n e q u a t i o n reads : R:- 1

4

kd l+k

N3

(2)

a

where R i s t h e r e s o l u t i o n between two subsequently e l u t i n g peaks, k i s t h e c a p a c i t y r a t i o o f t h e l a s t e l u t i n g compound and a=kl/k2

i s the r e l a t i v e reten-

t i o n . The p l a t e number N=L/H, where H stands f o r the column p l a t e h e i g h t . [ R 2 / 2(u2tu1) and N= ( t R / u ) ,

and N a r e measured o p e r a t i o n a l l y as R= (tR,2-tR,1)

where u i s the standard d e v i a t i o n i n e l u t i o n time]. Combination o f eqns. ( 1 ) and ( 2 ) y i e l d s an e x p l i c i t r e l a t i o n s h i p between a n a l y s i s time and r e s o l u t i o n f o r a two-component m i x t u r e :

Eqn. ( 3 ) was d e r i v e d as such by E t t r e ( r e f . 1 ) i n 1973, and a l r e a d y conceived by P u r n e l l ( r e f . 2 ) and Giddings ( r e f . 3 ) i n t h e e a r l y s i x t i e s . H/G i s a complex pressure-dependent r e l a t i o n s h i p ( r e f . 4 ) .

I t contains the

r e s i s t a n c e t o mass t r a n s f e r o f which t h a t i n t h e s t a t i o n a r y phase, C,,

usually

can be neglected w i t h r e s p e c t t o t h a t i n the gas phase, Cm. I n the f o l l o w i n g discussion t h i s term w i 11 t h e r e f o r e be neglected. For t h i n - f i l m columns w i t h a low i n l e t - t o - o u t l e t pressure r a t i o (P=pi/po+l), the optimum chromatographic c o n d i t i o n s are g i v e n by ( r e f . 4 )

:

56

[F] opt

(4)

P1

p+1 llk2

"" -

PO

2 Cmyl

=

6k

t

24 ( k

t

+ 1

1)2

2

J -

(5)

Dm,l

i s t h e b i n a r y s o l u t e c a r r i e r gas where r i s t h e i n n e r column r a d i u s and D m,l d i f f u s i o n c o e f f i c i e n t a t u n i t p r e s s u r e pl. S u b s t i t u t i o n o f eqns. ( 4 ) and ( 5 ) i n ( 3 ) y i e l d s : 4 ( l t k ) (11k2

tR,opt,P+l

=

3

t

6k

+

1)

k2

(x22

2 2-

R r

PI Dm,l

(a-1)

Giddings ( r e f . 3 ) proved t h e o r e t i c a l l y t h a t "minimum-time o p e r a t i o n i n gas chromatography" can be achieved o n l y , and always, when t h e column i s o p e r a t e d w i t h a vacuum o u t l e t (po

-t-

0 ) . T h i s f a c t has s i n c e been overlooked, o r m i s -

i n t e r p r e t e d , b y numerous a u t h o r s . Recently, i t was r e c o n f i r m e d and experiment a l l y proven by Cramers ( r e f . 5 ) and L e c l e r c q ( r e f . 6 ) .

F o r t h i n - f i l m columns, o p e r a t e d a t vacuum o u t l e t ( P y . ) under optimum chromatographic c o n d i t i o n s , s i m i l a r r e a s o n i n g as above leads t o eqn. ( 7 ) ( r e f s . 4,5) : l + k ) 2 (11k2

tR ,opt , P a

+

6k

t

1)

c13

24 (

R3r

]

[k

3 k3

(a-1 )

PI

(7)

Dm,l

where q i s t h e dynamic v i s c o s i t y of t h e c a r r i e r gas. [Guiochon ( r e f . 7 ) d e r i v e d t h e same e q u a t i o n , w i t h a c o n s t a n t of 19.2 i n s t e a d o f 24, f o r minimum t i m e analysis]. The f o r e g o i n g e q u a t i o n s c l e a r l y show a l l f a c t o r s a f f e c t i n g t h e speed o f a n a l y s i s . I f f a s t chromatographic s e p a r a t i o n s a r e r e q u i r e d , t h e f o l l o w i n g c o n d i t i o n s s h o u l d be met. F i r s t , vacuum o u t l e t c o n d i t i o n s s h o u l d be employed ( r e f s . 3,5-7;

c f . eqn.

(6)). Second, hydrogen o r h e l i u m s h o u l d be used as c a r r i e r gas, because t h e s e gases show t h e l o w e s t q / D r a t i o ( r e f s . 3,5). Eqn. ( 7 ) i n d i c a t e s t h a t t h e m,l a n a l y s i s times i n vacuum o u t l e t chromatography, r e l a t i v e t o t h e " b e s t " gas, H2 a r e H2,2;

He,3 and N2,5 ( w h i c h i s n o t as d r a m a t i c as s t a t e d b y G i d d i n g s

(ref.3)). Eqns. ( 6 ) and ( 7 ) r e v e a l a second t o t h i r d power dependence o f tRon a / (a-1) and R. Hence t h e s t a t i o n a r y phase s h o u l d be c a r e f u l l y s e l e c t e d and

an u n n e c e s s a r i l y l a r g e r e s o l u t i o n should be avoided. The s t a t i o n a r y phase and

57

t h e s e p a r a t i o n temperature s h o u l d be tuned t o y i e l d s o l u t e c a p a c i t y r a t i o s between about one and two ( r e f s . 7,8),

f o r which t h e k - c o n t a i n i n g terms i n

eqns. (3), ( 6 ) and ( 7 ) approach minimum v a l u e s , w h i l e m a x i m i z i n g t h e r e l a t i v e r e t e n t i o n a. Once t h e c a r r i e r gas, k,

c1

and R have been s e l e c t e d , and t h e column i s

o p e r a t e d a t vacuum o u t l e t , t h e s e p a r a t i o n speed can be f u r t h e r i n c r e a s e d b y employing l i n e a r gas v e l o c i t i e s h i g h e r t h a n t h e optimum v a l u e ( r e f . 9 ) . F o r t h i n - f i l m columns w i t h vacuum o u t l e t , t h e i n l e t p r e s s u r e r e q u i r e d f o r minimum time operation i s

.

(refs. 3,7). P i ,opt,P* The l a s t p o s s i b i l i t y l e f t t o s h o r t e n a n a l y s i s t i m e s f o r a g i v e n s e p a r a t i o n

problem, i s t o reduce t h e column i n n e r d i a m e t e r 2 r (eqns. 6 , 7 ) . S i n c e H i s d i r e c t l y p r o p o r t i o n a l t o r ( f o r t h i n - f i l m columns), t h e column l e n g t h can b e reduced i n p r o p o r t i o n t o r w h i l e m a i n t a i n i n g t h e r e q u i r e d r e s o l u t i o n ( r e f s . 5 , 8 ) . F o r s h o r t , low-plate-number columns w o r k i n g a t low p r e s s u r e drops, t h e optimum c a r r i e r gas v e l o c i t y i s p r o p o r t i o n a l t o t h e column diameter, so t h a t tRi s found t o decrease as t h e square o f r. F o r columns w i t h h i g h p l a t e numbers, and hence w i t h l a r g e p r e s s u r e drops, tRi s d i r e c t l y p r o p o r t i o n a l t o r, w h i l e

u

approaches a c o n s t a n t v a l u e . I n vacuum-outlet gas chromatography, P i s always l a r g e . T h e r e f o r e , f o r any p l a t e number, t h e a n a l y s i s t i m e i s p r o p o r t i o n a l t o t h e column d i a m e t e r . I n t h i s paper t h e a p p l i c a t i o n o f narrow-bore columns i n GC/MS i s p r e s e n t e d . EXPERIMENTAL Two narrow-bore fused s i l i c a columns c o a t e d w i t h 0.07 um o f O V - 1 were used ( r e f s . 10-12).

.

The dimensions o f columns A and B were 4.1 m x 55 pm i . d . and

6.3 x 50 pm, r e s p e c t i v e l y . Both columns were o p e r a t e d w i t h h e l i u m c a r r i e r gas a t i n l e t p r e s s u r e s o f (A) 7.0 and ( 8 ) 10.0 b a r ( a b s o l u t e ) . Samples were i n j e c t e d by s p l i t t e r , w i t h a s p l i t r a t i o o f 1:4000. The c a p i l l a r y columns were d i r e c t l y i n s e r t e d i n t o t h e i o n source o f a F i n n i g a n (Sunnyvale, C a l i f . ,

USA) model 4000 quadrupole GC/MS i n s t r u m e n t .

Between t h e GC oven and t h e i o n source, t h e column was enclosed i n a h e a t e d i n t e r f a c e oven. To e l i m i n a t e p o s s i b l e " c o l d s p o t s " , t h e temperature o f t h i s i n t e r f a c e oven s h o u l d n o r m a l l y be m a i n t a i n e d above t h e temperature o f t h e GC oven. I n o u r s i t u a t i o n , 60 cm, i . e .

10-15% o f t h e column l e n g t h was embedded i n

t h i s i n t e r f a c e oven, w h i c h was k e p t a t c o n s t a n t temperatures o f 100, 200 o r 25OoC, depending on t h e samples analyzed. A s u b s t a n t i a l number o f p l a t e s i s t h e r e f o r e l o s t u n l e s s t h e temperatures o f t h e GC and i n t e r f a c e ovens a r e maintained approximately equal.

58

The mass spectrometer was o p e r a t e d i n t h e e l e c t r o n i m p a c t ( E I ) and n e g a t i v e i o n chemical i o n i z a t i o n ( N I C I ) mode under t h e f o l l o w i n g c o n d i t i o n s : e l e c t r o n energy 70 eV, e l e c t r o n c u r r e n t 0.30 mA, i o n s o u r c e temperatures ( E I ) 250°C and 2OO0C ( N I C I ) . I n t h e l a t t e r mode a m i x t u r e o f CH4/N20 (1:l) was employed f o r t h e p r o d u c t i o n o f OH- r e a c t a n t i o n s . The GC/MS c o m b i n a t i o n was c o u p l e d t o a Nova 4/S (Data General, Westboro, Mass.

, USA)

mini computer sys tem.

The i n s t r u m e n t c o n t r o l and d a t a a c q u i s i t i o n s o f t w a r e were developed i n o u r l a b o r a t o r y , e n a b l i n g f a s t scanning r a t e s o f o v e r 3000 amu/s. RESULTS AND DISCUSSION To keep mass d i s c r i m i n a t i o n e f f e c t s w i t h i n a c c e p t a b l e l i m i t s , t h e scanning speed o f t h e mass s p e c t r o m e t e r has t o be i n c r e a s e d p r o p o r t i o n a l l y t o t h e i n creased speed o f a n a l y s i s . P r e f e r a b l y f i v e t o t e n mass s p e c t r a a r e a c q u i r e d d u r i n g t h e e l u t i o n o f s i n g l e compound. T h e r e f o r e , a speed o f 10 scans/s i s a p p r o p r i a t e f o r 50 prn columns.

1004

w

0

200

400

bOO

5CRN5 C

B00 10

1000120014001b001800200002~00?400~h00

X.01

SECONDS/5CANI

-->

F i g . 1. TIC chromatogram o f a t e s t m i x t u r e a t 100°C on column A. DMP = 2,6-dimethylphenol; DMA = 2 , 6 - d i m e t h y l a n i l i n e .

59

Fig. 1 i l l u s t r a t e s a reconstructed " t o t a l ion current" chromatogram of a t e s t mixture, separated on column A and 100°C. About 65,000 t h e o r e t i c a l p l a t e s a r e observed f o r n-dodecane, a t k = 7 , which i s about 10,000 p l a t e s l e s s than found with a F I D . The DMP and DMA peaks represent about 40 pg per compound. Yet high-quality EI mass s p e c t r a were obtained a t a r a t e of ten per second over a mass range of 35-200 amu ( r e f s . 8,12).

R

I I n

d

K u k. Y

W

I

w 4 +I4 w

n

t-u

U

z

U

0

U J

W

a a

a-12 a-u w w

=-

tTTrrrrrrrrrrri 0 200 400 b O O 800

5CRN5

C

10

I

I I

-

W

z w w

-Iz - I w

W

E Z 0 4

-I 0 m

>I-

> w

a a u

w

YJLP catl

z w

-299

Y

I I I I I I I I I I I I I

1000120014001b001800Z0002~00240DZb002E003000

36- 0 4

5ECOND5/5CRN 1

-- >

Fiq. 2. TIC chromatogram of Bergamot o i l (column A). Fig. 2 shows a TIC chromatogram of Bergamot o i l separated on column A with a temperature program (20s a t 8OoC, 15'/min t o 140OC). A scan r a t e o f ten spectra per second over a mass range of 35-250 amu was employed. The separat i o n time i s only f i v e minutes. This c l e a r l y demonstrates t h a t 50 i.d. c a p i l l a r y columns i n p r a c t i c e do allow a f i v e times f a s t e r speed of a n a l y s i s compared t o conventional 0.25 mm i . d . columns with equal p l a t e numbers (eqn. ( 7 ) ) . This gain i n speed i s mainly r e a l i z e d by the reduction of the p l a t e height, since t h e c a r r i e r gas v e l o c i t y remains almost constant due t o an increased compression of the gas ( r e f s . 8,lO).

60

F i g . 2 a l s o shows t h a t narrow-bore columns a r e r e a d i l y overloaded. (The peaks o f limonene and l i n a l y l a c e t a t e correspond w i t h f o u r t o f i v e nanogram). The sample c a p a c i t y o f a column i s p r o p o r t i o n a l t o t h e t h i r d power of t h e d i a m e t e r . Overloaded peaks a r e g e n e r a l l y observed when more t h a n 1-2 ng o f a compound a r e a p p l i e d t o a 50 pm i . d . column ( r e f s . 8,12). T y p i c a l E I s p e c t r a o f a m a j o r and a m i n o r compound, o b t a i n e d under t h e f a s t - s c a n n i n g c o n d i t i o n s employed, a r e p r e s e n t e d i n F i g . 3.

F i g . 3. Background-corrected E I mass s p e c t r a o b t a i n e d d u r i n g f a s t - s c a n n i n g conditions. F o r i d e n t i f i c a t i o n o f t h e s p e c t r a o b t a i n e d , use was made o f t h e "mass s p e c t r a l r e o r o d u c i b i lity based r e t r i e v a l system" (MSRR), developed b y Van It K l o o s t e r e t a l . ( r e f . 13), a t t h e S t a t e U n i v e r s i t y o f U t r e c h t , The Nether-

l a n d s . With t h i s computer program, t h e s p e c t r a c a n be compared t o t h e WileyM c L a f f e r t y d a t a base, c o n t a i n i n g 39,000 r e f e r e n c e s p e c t r a . S y s t e m a t i c e f f e c t s such as mass d i s c r i m i n a t i o n a r e accounted f o r by t h e MSRR program.

A r e p r o d u c i b i l i t y f u n c t i o n i s used t o c a l c u l a t e a g o o d n e s s - o f - f i t c r i t e r i o n c a l l e d t h e " s i m i l a r i t y i n d e x " , S I ( v a l u e d between 0 - l o o ) , f o r a l l p o s s i b l e compound c a n d i d a t e s s e l e c t e d by t h e computer f r o m t h e r e f e r e n c e f i l e . T h i s c r i t e r i o n i n d i c a t e s t h e degree of correspondence between t h e measured spectrum and a s e l e c t e d r e f e r e n c e spectrum. F o r s p e c t r a o b t a i n e d w i t h c o n v e n t i o n a l c a p i l l a r y columns, t h e MSRR program i s known t o p e r f o r m v e r y w e l l . The E I s p e c t r a o f most compounds i n Bergamot o i l were e a s i l y i d e n t i f i e d by t h e MSRR system. Even m i n o r compounds such as a-bergamotene, w i t h a peak i n F i g . 2 c o r r e s p o n d i n g t o a p p r o x i m a t e l y 60 pg, were c o r r e c t l y i d e n t i f i e d ( F i g . 4 a1 though t h e S I - v a l u e was g e n e r a l l y l o w e r t h a n observed w i t h c o n v e n t i o n a l columns.

,

61

SCAN 2516

51

MEU

1

80.0

40

3

33.6

40

COMPOUNU NAMEr SERIAL NR. MOL. WEIGHT,

BRUT0 FORMULA

ALPHA-BERGAMOTENE 13550, MW= 2049 C15 H24 (I,E)-ALPHA-FARNESENE

38051~MW= 204.

CIS

H24

3

30.9

ALPHA-TRANS-BETA-BERGAMOTENE MW= 204.

C15 H24

4

20.1

ALPHA-CEDRENE 13588, MW= 204.

C l 5 H24

13551,

5

16.8

3187105003 ALPHA CEURENE 3 8 0 9 5 , MW= 2 0 4 , Cl5 H24

F i g . 4. P a r t i a l l i b r a r y search r e s u l t s f o r t h e spectrum o f F i g . 3b n r . 2516 f r o m F i g . 2), showing t h e f i v e b e s t matches.

(scan

S i n c e no s p e c i a l p r e c a u t i o n s , as c l e a n i n g t h e i o n source and t h e l i k e , were taken, t h e s e n s i t i v i t y o f t h e narrow-bore GC/MS method i s s t r i k i n g and needs e l a b o r a t i o n . I n c o n v e n t i o n a l c a p i l l a r y column GC/MS " i d e n t i f i c a t i o n l i m i t s " a r e i n t h e o r d e r o f 1 ng p e r compound. I d e n t i f i c a t i o n l i m i t s a r e d e f i n e d h e r e as t h e minimal amount needed t o produce i n t e r p r e t a b l e mass s p e c t r a . The small e s t s i g n a l t h a t can be d e t e c t e d i s a s i n g l e i o n , b u t s t a t i s t i c a l l y such an e v e n t i s v e r y u n r e l i a b l e . F o r t h i s reason, i o n s t a t i s t i c s d i c t a t e t h a t f i v e i o n s p e r mass s p e c t r a l peak a r e c o n s i d e r e d a minimum f o r r e l i a b l e d e t e c t i o n d u r i n g scanning ( r e f . 14). Hence, r e q u i r i n g a s i g n a l - t o - n o i s e r a t i o o f t w e n t y , t h e base peak i n a scanned mass spectrum s h o u l d r e p r e s e n t a t l e a s t 100 i o n s . An average E I mass spectrum would, t h e r e f o r e , r e q u i r e 103-10 4 i o n s t o be c o l l e c t e d d u r i n g scanning, which e q u a l s a b o u t

Coulomb o r 10-10-10-9 g

o f compound p e r scan, depending on t h e mass s p e c t r a l r e s o l u t i o n and assuming

6.

an i o n i z a t i o n e f f i c i e n c y o f 1:lO

When t h e scanning r a t e i s i n c r e a s e d i n p r o p o r t i o n t o t h e s e p a r a t i o n speed, no e f f e c t on t h e s e n s i t i v i t y i s expected. When t h e amount o f m a t e r i a l i s k e p t c o n s t a n t , t h e number o f i o n s c o l l e c t e d d u r i n g a scan i s c o n s t a n t as l o n g as t h e number o f scans p e r GC peak w i d t h i s t h e same. N e v e r t h e l e s s , t h e observed i d e n t i f i c a t i o n l i m i t s were f o u n d t o be p r o p o r t i o n a l t o t h e a n a l y s i s t i m e and hence t o t h e column d i a m e t e r (eqn. ( 7 ) ) .

A t e n t a t i v e e x p l a n a t i o n f o r t h e g a i n i n s e n s i t i v i t y by d e c r e a s i n g t h e column diameter, i s t h e reduced n o i s e l e v e l measured by f a s t e r scanning. Back2 3 ground i o n c u r r e n t s , w h i l s t v a r y i n g w i t h mass, a r e o f t h e o r d e r o f 10 -10 i o n s p e r second. T h e r e f o r e , i n f a s t e r scanning experiments l e s s background ions a r e detected.

62

Moreover, the gas f l o w from 50 pm columns i s about 25 times l e s s than from 0.25 mm columns, and so i s the bleeding mass flow. A t t h i s p o i n t i t should be mentioned t h a t a d d i t i o n o f make-up gas (He : 1-5 ml/min) d i d n o t i n f l u e n c e the s e n s i t i v i t y . Careful1 p o s i t i o n i n g o f the column o u t l e t i n the i o n source block, however, appeared t o be o f utmost importance.

"3

M

Fig. 5. TIC chromatogram, reconstructed from N I C I spectra, o f C3-C11 (Column A ) . The peaks represent about 10 pg per compound.

ketones

Fig. 5 shows the t o t a l i o n i z a t i o n chromatogram o f a mixture o f C3-C11 ketones analyzed i n the N I C I (OH-) mode. Again, ten spectra per second were scanned over a mass range o f 35-200. I n t h i s s o f t i o n i z a t i o n mode, fragmentat i o n i s l a r g e l y suppressed : I n a l l mass spectra the ( M - l ) -

peak accounted

f o r over 60% o f the t o t a l i o n c u r r e n t . This explains the odd peak shapes i n F i g . 5: the data system was overloaded by GC-peaks corresponding w i t h only 10 pg per compound. Although n o t y e t v e r i f i e d experimental l y , these r e s u l t s show t h a t sub-picogram q u a n t i t i e s can be detected i n the scanning mode, i n d i c a t i n g femtogram s e n s i t i v i t y i n the

F i g . 6. TIC chromatogram o f a condensate o f Dutch natural gas on column 6 , programmed from 30-2OOoC a t 20°C/min (Off-scale peaks represent n-Cl0 through n-CI6).

63

I I

A .

n

64 s e l e c t e d i o n m o n i t o r i n g mode, where l o n g e r d w e l l t i m e s a r e used. F i g s . 1,2 and 5 i l l u s t r a t e t h a t t e n t o t w e n t y s p e c t r a p e r e l u t i n g GC peak can be a c q u i r e d when scanning a t a r a t e o f t e n s p e c t r a p e r second. I t i s known t h a t a b o u t t h r e e s p e c t r a p e r GC peak 0 a r e needed i n o r d e r t o be a b l e t o r e l i a b l y r e c o n s t r u c t t h e chromatographic peak shapes ( r e f . 1 5 ) . The i n f l u e n c e o f t h e scanning r a t e on t h e appearance o f t h e chromatogram was i n v e s t i g a t e d w i t h column B. F i g . 6 shows a TIC chromatogram o f crude o i l c o n t a i n e d i n n a t u r a l gas, r e c o n s t r u c t e d f r o m t h e E I mass s p e c t r a , a c q u i r e d f i v e t i m e s p e r second o v e r a mass range o f 35-350. The a n a l y s i s t i m e i s a b o u t 1 2 minutes. A p p a r e n t l y , t h e s e p a r a t i o n e f f i c i e n c y i s n o t d e t e r i o r a t e d by t h e lowered scanning r a t e , a l t h o u g h o n l y t h r e e t o s i x mass s p e c t r a p e r GC peak were taken.

I I

I

I

980

I 800

SCRNS C

I

I

I

1200 5

I 1b00

I

I 2880

I

I 2480

M - 0 1 5ECOND5/SCRY3

I

r 2800

r

r 3200

r

l

%BE

--->

F i g . 7. TIC chromatogram o f a commercial t h i n n e r (column programmed f r o m 35 t o 80OC a t 15oC/min.

B). Temperature

F i g . 7 shows t h e TIC chromatogram o f an i n d u s t r i a l " t h i n n e r " ,

reconstructed

from 3600 E I mass s p e c t r a . The s p e c t r a were scanned o v e r a mass range o f 35200 amu a t a r a t e o f 20 s p e c t r a / s . The t o t a l s e p a r a t i o n t i m e i s 3 m i n u t e s . F a s t e r scanning i s c u r r e n t l y l i m i t e d b y t h e d a t a system and s o f t w a r e used. The m a j o r peaks a r e overloaded, b u t i t can be seen t h a t about 50 s p e c t r a p e r GC peak a r e a c q u i r e d . A t t h i s r a t e i n s t r u m e n t a l t i m e c o n s t a n t s become apparent.

65

CONCLUSIONS Summarizing, i t can be concluded t h a t t h e c o u p l i n g o f 50 p m c a p i l l a r y columns t o a mass spectrometer i s b e n e f i c i a l , b o t h i n terms o f speed o f a n a l y s i s and s e n s i t i v i t y . The a n a l y s i s time i s reduced i n p r o p o r t i o n t o t h e column i . d .

and, t h e r e f o r e , t h e sample throughput r a t e can be increased sub-

s t a n t i a l l y , thus making use o f t h i s expensive instrument more a t t r a c t i v e .

E I s p e c t r a obtained from 50 pg amounts can u s u a l l y be c o r r e c t l y i d e n t i f i e d . Accurate chromatographic and mass s p e c t r a l data f o r narrow-bore c a p i l l a r y GC peaks can o n l y be obtained a t f a s t GC/MS scanning r a t e s .

REFERENCES

1. L.S. E t t r e , Open Tubular Columns; An I n t r o d u c t i o n , Perkin-Elmer, Norwalk, Conn., 1973, p.13. 2. J.H. P u r n e l l and C.P. Quinn, i n R.P.W. S c o t t (Ed.), Gas Chromatography 1960, Bu t t e r w o r t h s , 1960, p. 184. 3. J.C. Giddings, Anal. Chem., 34 (1962) 314. 4. C.A. Cramers, F.A. Wijnheymer and J.A. R i j k s , J. High Res. Chromatogr., Chromatogr. Commun. , 2 (1979) 329. 5. C.A. Cramers, G.J. Scherpenzeel and P.A. Leclercq, J. Chromatogr., 203 (1981) 207. 6. P.A. Leclercq, G.J. Scherpenzeel, E.A.A. Vermeer and C.A. Cramers, J. Chromatogr., 241 (1982) 61. 7. G. Guiochon, Anal. Chem., 50 (1978) 1812. 8. C.P.M. Schutjes, D i s s e r t a t i o n , Eindhoven U n i v e r s i t y o f Technology, Eindhoven, 1983. 9. C.P.M. Schutjes, P.A. Leclercq, J.A. R i j k s , C.A. Cramers, C. Vidal-Madjar and G. Guiochon, J . Chromatogr., 289 (1984) 163. 10.C.P.M. Schutjes, E.A.A. Vermeer, J.A. R i j k s and C.A. Cramers, J. Chromatogr. 253 (1982) 1. ll.C.P.M. Schutjes, E.A.A. Vermeer and C.A. Cramers, J. Chromatogr., 279 (1983) 49. 12.C.P.M. Schutjes, E.A.A. Vermeer, G.J. Scherpenzeel, R.W. B a l l y and C.A. Cramers, J. Chromatogr., 289 (1984) 157. 13.P. C l e i j , H.A. van ' t K l o o s t e r and J.C. van Houwelingen, Anal. Chim. Acta, 150 (1983) 23. 14.J.R. Chapman, Computers i n Mass Spectrometry, Academic Press, London, 1978. 15.P.A. Leclercq, i n J.A. R i j k s (Ed.), C a p i l l a r y Chromatography, E l s e v i e r , Amsterdam, 1983, p. 763.

This Page Intentionally Left Blank

61

HISTORY AND SPECIAL AUSTRIAN CONTRIBUTIONS

TO

CHROMATOGRAPHY

Erika CREMER Institute of Physical Chemistry, University Innsbruck, Austria INTRODUCTION The history of chromatography demonstrates a fascinating scientific and technical achievement. Its knowledge gives the possibility to understand and estimate the great inventions in this field. In Table I early applications of chromatographic methods in the period between 1512 and 1903 are given and their scientific publications documented. In addition to this, there may exist a relatively large number of practical uses o f chromatography where the inventors are unknown. There is an old Swedish method o f purifying spirits by distillation through a bread filter. This is similar to a method still used in the second world war by certain prisoner of war guards. They percolated gasoline fractions through bread to obtain potable alcohol.

Table I (1512 - 1903) Year

Name

Living in

1512

BRUNSWICK pharmacist

Strassburg

1822

RUNGE chemist

Berlin

1859 1861

RUNGE Oranienburg SCHOENBEIN Base1 and GOPPELSROEDER

1898 1903

Carrier

Preparative

Detection

no

no

/1/

mostly no

yes

/2/

yes yes

/3/

yes no

/5/ /6/

yes

/7/

f ronta1 ana-

GOPPELSROEDER D av id T .DAY La kewood/Oh i o TSWETT Russian botanist

Method

Italy Switzerland Russia

lysis Displacement chr.of dyes on paper -' I _

"Capi1 laranalysis" Separation of dyes and org. compounds

-1'-

Yes

Yes Prepa rat i ve no Displacement chromatogr"Tswettography" Yes Separation of Components o f Ch lorophy11 _'I-

/4/

68

BRUNSWICK /1/ was able to separate alcohol from water by frontal analysis but used no carrier gas and no detection unit. RUNGE /2,3/ dropped a solution containing coloured ions onto paper and from there the so called "Grown Pictures" arose. He was delighted with the reproducibility o f his coloured pictures. He searched for practical applications but the time was not yet ripe to think of the great possibilty of its use as an analytical method. In the picture beneath an example of Rungz's extraordinary achievements is given in a black and white copy, in its original colours it is however much m r e attractive. SCHOENBEIN /4/ and GOPPELSROEDER /5/ improved Runge I s method considerably and called their approach to paper chromatography "capillary analysis". By the use of an elution medium they obtained clear separations o f the zones. Goppelsroeder collected and published these interesting results in numerous publications /5/ and could prove that his detection limits were for certain substances in the ppb range.

Black and white copy o f a "grown picture" from Runge.

69

D.T.DAY / 6 / used preparative displacement chromatography, an early variant of LS Chromatography, but had no carrier fluid and no detection system. TSWETT /7/ was the first scientist who developed the earlier attempts to a complete chromatographic sytem. He really already fulfilled the four conditions which characterise modern chromatography at his time: 1. the utilization o f a stationary phase, 2. the application of the sample in the form of a small pulse, 3. the use of a solvent as elution medium, and 4. a detection system which was in this case coloured rings travelling through the column and ready to be collected individually at the column exit. This complete analytical system is so characteristic that I proposed t o use the name TSWETTOGRAPHY in modern chromatography. It was not until 20 years after the great achievements of Tswett that BERL /8/ used a preparative gaschromatographic method for the separation of several compounds, which were analyzed by a refractometer. For the transport in the column he used a displacement medium (steam) and thus obtained overlapping zones and therefore not complete separations. In 1931 KUHN,WINTERSTEIN and E.LEDERER / 9 / achieved the separation of the colour compounds o f egg-yolk and a short time later KUHN and LEDERER /lo/ obtained the separated caratinoides by applying a preparative Tswett chromatography . In the next 10 years many experiments were started to use adsorption for the separation of gases, beginning with the separation of rare gases through fractional adsorption by PETERS /ll/. We may cite here TISELIUS /12/ and CLAESSEN /13/, TURNER /14/, EUCKEN and KNICK /15/, HENJES /16/, EDSE, SUHR and HARTECK /17,18/, KUECHLER and WELLER /19/, who all tried to elute the substances without the use o f a carrier gas. It was feared at this time that longitudinal diffusion would make the separation impossible. WICKE /20/ originating from the school of Eucken was especially concerned with these problems. He compared eight different "sorptographic methods" and showed in 1940 that one may expect to achieve the best chromatographic separations in the liquid phase with a carrier fluid. The results compared with distillation were however unsatisfactory due to the relatively long separation time and the small amounts obtained. DAMKOEHLER and THEILE /21/ made experiments on Adsorbtion-Tswettography in the gas phase on a preparative scale, very secretly in an institute for motor research in Braunschweig. They separated methanol and ethanol as well as cyclohexane and benzene using earthenware as adsorbent. The break through of the compounds was signalized by the use of a detector. The concentration profiles were broad and irregular, caused by diffusion, condensation and

overloading of the column, so that an analytical evaluation, which the authors had not intended /22/, would also not have been possible. To quote from a book by NEUFELD /23/: "At these prapartive dimensions the efficiency of the method in the milligram region remained hidden". HESSE and coworkers /24/ succeeded in 1941 in separating ternary mixtures by combining distillation and adsorption. They even used a carrier gas which they led through the distillation apparatus and the adsorption column. In this year also appeared the important invention of liquid-liquid chromatography by MARTIN and SYNGE /25/. Martin and Synge opened up the way to microanalysis. The target to obtain high sensitivity was in reach. How fast it was achieved Martin says in "75 Years of Chromatography" /26/:"the silica partition column requires a few milligrams, but within a 34 years period the quantity of sample needed has been reduced by a factor of 10 12.1' Of course an imnense work of development was necessary with changes of the stationary phase as well as of the mobile phase, and last but not least of the detectors. In order not to exceed the volume of this contribution I now have to concentrate on the description of the chromatographic development in Austria.

GAS-ADSORPTION CHROMATOGRAPHY During the second world war Austria did not exist but was included in the German Reich. Science was guided from above and the state was only interested in research relevant to the war or to a lesser extent to economy. Only German orginal literature was obtainable and we had only little knowledge of foreign scientific work. 1941 I was coworker of a team /27/ in Innsbruck which was occupied with the catalytic hydration of acetylene to ethylene. A fast analytical method for the determination of both these gases was urgently desired for a precise analysis o f the kinetic data. I thought of the possibility of separating both these compounds bacause of their difference in adsorption strength. In 1935 I got in contact with this method during an investigation of the catalytic transformation of ortho to para hydrogen on solid oxygen /28/. From this kinetic experiment could be concluded that ortho hydrogen was more strongly adsorbed than para hydrogen. As expected it was found that a step-wise desorption using a vacuum pump made it possible to obtain enriched ortho hydrogen in higher concentrations then it occurs in normal hydrogen. Before the application of such an adsorption separation process it was necessary to measure the adsorption isotherms o f acetylene and ethylene. These measurements were carried out by A.KUNTE /29/ during his master thesis which

71

was derminated in 1943. The difference in the adsorption energy, the sorbence was carbon, was so low that a fast separation in a single run was not to be expected. In this situation I had the idea that a carrier gas could solve the problem. Using a simplified model where boats (molecules) are floating down a river (mobile phase) and from time to time anchor on the shore (stationary phase) led to the equation (I) where represents the energy value necessary for the desorption of the molecules from the stationary phase. The energy adsorption difference of two substances is given by

wherebyht is the retention time t R minus to /30/. The use of a carrier gas should therefore produce large differences in the retention times even in the cases were only small differences in the lambda values occur. The first values for their application on LSC were obtained experimentally by R.KNOEPFLER / 3 U . After the war at the end of the year 1945 Fritz PRIOR was interested in a ph.D.work and asked me if I could offer him a scientific theme. I was convinced that he was very well able to carry out the above mentioned experiments where a carrier gas (hydrogen) should be used as mobile phase for the development of a gas chromatogram. The situation for experimental work was still extremely difficult. Practically no equipment and spare parts could be bought. Only the instrumentation and material in the institute which were not lost by war occurrences such as bombings and removals were available. We were glad that Kunte's equipment was still under this category. It contained a self-made heat conduction cell and was to be used for the new experiments. I still had a small piece of a 7 micrometer Wollaston-wire to repair the cell. The hydrogen was produced by a Kipp-apparatus belonging to a high school laboratory. Fig.1 and 2 show chromatograms which were obtained with the described equipment as early as in the year 1946. Carbondioxide and air could be clearly separated. Also the separation of acetylene and ethylene could be achieved in 12 minutes with an overlap of approximately 10 percent. A more complete separation is shown in Fig.3 with a separation time of 8 minutes (CREMER and MUELLER) /33/. Due to difficulties in publishing the results in Austria and in other countries, this research work was first presented only in ph.D.theses and lectures /31,32/. Slowly international scientific relations were restored. In May 1950 it was possible for me to present the chromatogram of Fig.3 on an internationally attented meeting of the Bunsen society in Marburg /33/. Another important contact occured in the summer of the same year: Sir Robert ROBINSON

72 G

24

I Scale deflection

Signal

1

n \ tms CO,

I

I

cog

1

y t m s air

time 8

16

24

32

Fig.1 First analytical gas chromatogram, detector HCD, sample amount 0.5 to 10 mg (Cremer and Prior, Innsbruck 1946) /31/

lime

40 (minute)

Fig .2 Same substances as in 1. Baseline-drift deducted. Perfect repeatability of rel. time and area data. First precise analytical chromatograms /31,32/.

(GB) visited our institute in Innsbruck during his holidays in the Tyrolean mountains. I showed him our apparatus and some chromatograms, in which he obviously was very interested. He advised us to include propylene in our investigations. Within 3 minutes we could separate this compound from ethylene. The measured retention value (Alambda) is shown in Fig.4. Sir Robert's interest and acknowledgement were for me a real satisfaction,because my closer colleagues showed more scepticism than approval for this new method. My ealier connections with research on adsorption /34/ made it understandable that in addition to the application of chromatography for analytical problems, a certain interest in theoretical and practical adsorption processes would always remain. HAUPT /31/ and ROSELIUS /35/ investigated the influence of poisoning the surface o f the stationary phase on the adsorption of the carrier gas. Fig.5 shows the proportionality of the retention times (tR) with the free surface. In Fig.6 the retention times are related to different water surface layers. This picture already shows the change from a pure gas adsorption chromatography to a gas distribution chromatography. The difference of the retention times with and without an adsorption layer, as well as its influence on the peakform, is given in Fig.7.

73

sc

Ideflection

t

Fig.3 Chromatogram by Cremer and Muller (1950): Separation of moistened silicagel (LAC) 0-28

0,39

9,C2H4

and C2%

on

0-65

b

L

CH2CHCl

Fig.4

AX in

kcal, (measurement Prior /32/ and Mueller /33/), precurser of the retention index. Retention index rel-to C2H4, C02 and C2H2.

Fig.5 Dependency of the retention times versus the quantity of H S measured by HAUPT 2 /31/. Fig.6 C02 on silicagel with different amounts of water present /31 ,35/.

a

Ilr

-1 0 f 9 - 6 8 10 12 I(, 16 18 20 mln

b

4060 -c

Fig.7a Separation of noble gases at 2OoC, column: 80x0.4 cm, carrier gas: hydrogen, velocity of flow: 30 ml/min, sample: about 1 mg, column material: carbon (CREMER and ROSELIUS /35/). Fig. 7b The same with CO as carrier gas 2 The ph.D.thesis of J.F.K.HUBER /3l/,now full Professor in Vienna, was mainly concerned with the relation of the adsorption isotherms with the retention volumes. He also was engaged in the problem of separating optical isomers but the time was too early to obtain convincing results. He is the author of over 60 papers of chromatography (see /26/, p 159); My strong involvement in the o ,p-hydrogen-transformation were the reason for tests to separate both these spin modifications through chromatography. MOORE and WARD /36/ were however earlier then we, and obtained separation on a aluminium oxid column. With my co-workers BACHMANN and BECHTOLD /37/ we finally achieved the separation on a molecular sieve column (Fig.8). Pure molecular sieves allow a complete separation of these hydrogen modifications. On molecular sieves with iron contamination one observes a simultaneous o,p-transformation which leads to the formation of a bridge between both these peaks. We called this phenomenon "Simultaneous-reaction chromatography". This effect also occured sometimes in experiments where Hans HUBER /38/ measured adsorbtion isotherms using the chromatographic method, when the temperature was too high (Fig.9, see also KRAMER 47).

75

a

2 30

20

10

0

10

50

10

50

mm

Fig.8 Chromatograms of 0- and p-hydrogen and 7'K on a molecular sieve, a) pure, b) after contact with C02 (BACHMANN, BECHTOLD and CREMER /37/)

J

,

0

mail

,

10

,

20

b

30 mm

-PlTORRI

Fig.9 Adsorption isotherms for hexane, measured gaschromatographically /38/.

HEAD-SPACE ANALYSIS We began investigations on the flavor of honey in 1960 on 'account of a contact with Dr.DUISBERG, the head of the Honey Research Institute in Bremen. He brought us into contact with Professor MECKE (Physical Chemistry Institute, Freiburg, Br.) who intented to analyse these flavors spectroscopically. He needed a chromatographic preseparation. At the same time we also tested enrichment methods but encountered contamination through solvents. In this connection it appeared desirable to extract the flavor directly from the gas space above the honey. It was clear that only a very sensitive detector, in combination with the chromatographic equipment, could produce the necessary signals. K.FRIEDRICH, of the Freiburger institute, was able to produce with their FID detector interesting chromatograms which we could not yet make in Innsbruck with our heat conductivity cells. The work of DOERRSCHEIDT (Innsbruck) and FRIEDRICH (Freiburg) /39/ was submitted in May 1961 and was certainly an early example of the head-space-method.

76

My co-worker Manfred RIEDMANN continued this research in his ph.D.thesis (1961-1964). He also applied the head-space-method for the investigation of a criminal case. I repeat this story as it was told in a laudation /40/: Once a farmer came to the court with the suspicion that he might be poisoned by the bottle of wine presented to him. The contents of the bottle were chemically analyzed and found to be in good condition. However, in the Institute of Physical Chemistry in Innsbruck it was proved by gas chromatography that the gas between stopper and wine contained traces of terpertine substitute. The normal chemical analysis did not find what the sensitive nose of the farmer suspected. Through chromatography the problem was solved. The farmer was not to be poisoned, but the children-in-law had used the bottle before to store terpentine substitute. We asked the court to refund our expenses with approximately US$ 100. As answer we were told that the value of this case was too low for such a sum and therefore we obtained no compensation. RIEDMANN /41/ made progress in the research of honey flavor by showing that the peak tailing, which is connected with the adsorption energy, can be used for the identification of alcohols. The chromatogram in Fig.10 shows a special plot which can be used when many substances with large concentration differences are separated. We called it a "line diagram", where the logarithm of the amount is plotted against the logarithm of the relative retention time. 4

. c

a

70f

4

a

5-

I 2 .

Fig.10 Line-chromatogramm of honey flavours /41/. Log of the peak area against A A/2,3RT.

CHROMATOGRAPHIC DETECTORS In the years 1945 t o 1951 Klaus BIEMANN /42,43,44/ studied in Innsbruck and I was his teacher in the field of physical chemistry b u t his thesis was in the f i e l d of organic chemistry. During his stay in Innsbruck he aquired an early knowledge in gas chromatography, t o which he l a t e r made a very important with mass contribution by solving structural problems in combination spectroscopy detection system /32-35/. He certainly will t e l l you more a b o u t i t in his own lecture here i n Urbino. We also were interested in using mass spectrometers as detector units. Concerning the already mentioned honey flavor, Dr. DUISBERG arranged contacts f o r us t o Dr. JENCKEL (MAT Bremen), who supplied us with a suitable mass spectrometer. RIEDMANN was able t o identify by t h i s means several honey flavor compounds b u t o n l y those which occurred in a relatively high concentration. I n Fig.11 one examples o f these experiments i s given.

Fig.11 a ) Mass spectrum o f diethylether with continuous input of a c a p i l l a r chromatogram into the ion source. b ) Mass spectrum of diethylether following the API-catalogue (M.RIEDMANN, Thesis 1964)

At approximately the same time I began in cooperation with KRAUS and BECHTOLD /45/ to develop a very sensitive detection system according to the principle of a halogen leak detector. K.HABLIK was afterwards engaged with the testing and improvement of selective detectors during his dissertation /31/. He also could show, that under special operation conditions, the detector showed a good selectivity for nitrogen (Fig.12) /45/. Manfred RIEDMANN, who in the meantime had changed t o an industry position (Hewlett Packard, Boeblingen, FRG) was very successful in the further development of a extremely sensitive and reliable nitrogen detector. This detector was for instance also used at the Olympic games in Munich (1972) to carry out the requested doping tests.

Fig. 12 Line-chromatogram showing the selectivity for a) Parathion versus DDT (DDD) and hexan, b) nitromethane versus hydrocarbons H-GRUBER (now full Professor of Physical Chemistry in Innsbruck) who was also concerned in his ph.D.work in our institute with the comparison o f calorimetrically measured adsorption energies with thermodynamic parameters o f gas chromatography. He was engaged in the development of a detector on the principle of gas diffusion fuel cell and constructed together with H.HUCK a detector for the determination of ethanol in respiratory air ("alcohol spy") /46/.

THEORETICAL INVESTIGATIONS As early as 1944 the connection between adsorption energy and the retention time had been theoretically formulated by equation (I). This equation had already been used in the thesis of KNOEPFLER /31/. J.F.K.HUBER helped in his Innsbrucker times to find more precise

I9

definitions for several chromatographic terms. His further engagement in the application and development of the van Deempter equation and the theoretical evaluation of the column switching technique are certainly known to the audience. You will hear further details in Prof.HUBER's lecture. Of interest was also the theoretical treatment of the ortho-para hydrogen transformation by R.KRAMER, who showed that the reaction constants can be obtained by evaluating the form of the chromatographic peaks (Fig.13) /47/. New theoretical approaches are coming from the technical university of Graz where WEGSCHEIDER /48/ shortens the optimization process for chromatographic separations successfully by iteration methods.

Fig.13 Chromatogram of o-and p-hydrogen with simultaneous conversion -experimental curve,-oocalculated /47/ THIN-LAYER CHROMATOGRAPHY The development of thin-layer chromatography gave new impulses to adsorption chromatography. In this connection interest in very thin and ultra-thin layers arose. We already had contact with the Balzers AG, Liechtenstein, and I discussed this problem Dr.Th. KRAUS /49/ and asked him if he could supply us with some vapor deposited ul.tra-thin layer material. He advised us to try indiumoxide layers and meant that "if one layer works, then this one", and he was right. The liquid phase spreads out on the thin-layer support in a very thin film, which is 2-4 pn thick. This research work was carried out by NAU in his ph.D.thesis /31/. DEUTSCHER and FILL /50/ tried also thin layer materials (e.g.Mg0) for their separations. In some cases they also got a very quick separation. By this method some test substances of dyes could be separated within 10 seconds.

80

$[

RX

Fig. 14 Chromatogram from the thesis of E.SEIDL showing the separation o f aminoaclds (50 pg) on a thin layer o f indium oxide. Reference sample: 40 pg prolin. Autoradiograms and the evaluation by a scanning photometer /51/

....-

0 2 4 6 T mm

I

START

1

FRONT

I

START

t

FRONT

Fig.15 Electron microscopic picture of an indium oxide layer /52/.

T o increase the sensitivity for small amounts E.SEIDL /31,51/ used in her ph.D.work radioactive nuclides or radioactive labelled substances. By these means the "thin-film- thin-layer chromatography" was further improved which could easily be documented by autoradiography. Without difficulty the detection

of picogram amounts i s possible (Fig.14). The determination limit of radioactive labelled compounds lay in the region of g. In Fig.15 the electron microscopic picture of an indiumoxide layer i s shown. Dr.PULKER /31,52/ (Balzers AG,Liechtenstein), an alumnus of our institute, kindly reproduced it for us.

GAS CHROMATOGRAPHY OF HIGH POLYMERS H.NEWESELY,Tyrolyse by birth, who has studied in Innsbruck i s now Professor at the Free University o f Berlin and head of the Institute of Dentale Technology and Material Science. He uses gas chromatography very successfully for the identification of high polymers in this field /53,54/.

LIQUID CHROMATOGRAPHY (HPLC AND GPC) W.LINDNER of the Pharmaceutical Chemistry Institute of the University in Graz, works on the trace anylysis of complex multi-component matrixes. He was especially successful in the chromatographic separation of enantiomers /55/. Another interesting subject, on which intensive work is being done at present in Austria, is biomass analysis. 0.BOBLETER made his first experiments for the degradation o f biomass in the early fifties in the Institute of Physical Chemistry in Innsbruck, over which I presided at this time. The chances that a respectable fraction of the 120 billion tons of yearly plant growth on the continents may be used by the new developed hydrolysis processes (hydrothermolysis and enzymatic hydrolysis) give a optimistic outlook for the future. The earlier difficulties in analyzing the great number of carbohydrates, organic acids, ketones, phenolic and furfural compounds could be overcome by developments in the institute of 0.BOBLETER. For the determination of the molecular weight of celluloses a new GPC method was developed where the sample is directly dissolved in cadoxen and the complete molecular size distribution can be obtained in a short time /56/ (0.BOBLETER and W.SCHWALD). As the last picture I can present you one of the latest chromatograms obtained by G-BONN, the leader of the analytical group in the aforementioned institute. In Fig.16 the separation of oligosaccharides, monosaccharides and sugar degradation products was determined within 25 minutes by the application of a coupled column device /57/.

82

1

13

J

10

20

i 30

Fig. 16 Optimized separation of a standard mixture l . . . glucose, 2-7...DP ( = degree of polymerisation, glucooligomeres), 8...xylose, 9... fructose, 1 1 ... dihydroxyacetone, 12... 1,6-anhydro-D-glucose, 13... hydroxymethylfurfural, 14... furfural, 15... ethanol. Chromatographic conditions: coupled column system - Ca loaded ion exchange stationary phase and Ag loaded ion exchange stationary phase, column temperature: 95Oc, mobile phase: water, flow-rate: 1,2 ml/min, refractive index detection (G.EONN /57/)

Because I was asked to describe the contribution of Austrians to the development of chromatography I would like to repeat the names o f the scientists who were mentioned in this lecture and born in Austria (including the German-speaking part of Old-Austria): Kuhn, Lederer, Harteck, Kuechler, Kunte, Knoepfler, Prior, Muller, Eiemann, Haupt, Gruber, Eechtold, Kraus, Eachmann, Kramer, J.F.K.Huber,Wegscheider, Hans Huber, Riedmann, Hablik, Nau,

83

Deutscher, Fill, E.Seid1, Lindner, Newesely, Pulker, Bobleter, Schwald, Bonn. Those who still are engaged in chromatography are now distributed over a large part of the world: Cambridge (Mass.,USA), Wien (A), Graz (A), Waldbrunn (FRG),Balzers (Liechtenstein), Berlin (FRG), Innsbruck (A).

References /1/ BRUNSWICK, Liber de arte distillandi, see A.Bitte1, Thesis, Tubingen 1957 /2/ F.F.RUNGE, Thesis, Berlin 1822 /3/ F.F.RUNGE, Musterbilder fur Freunde des Schoenen usw., Heft 1 , Munchen 1850, Der Bildungstrieb der Stoffe, veranschaulicht in selbstaendig gewachsenen Bildern, Oranienburg 1859 /4/ SCHOENBEIN, Verh.d.Naturf.Ges. zu Basel, 1861, 111, 249 /5/ GOPPELSROEDER, see I.Houben, Die Methoden der org.Chemie, Band 1 , Allg. Teil, 1925, p 203 /6/ D.T.DAY, Proc.Am.Phil.Soc. 36, 112, 1897 (by courtesy of Prof.Jerzy

S. Kowalczyk, University of Gdansk) /7/ M.S.TSWETT, Proc. Warsaw Soc.Nat.Sci.Biol.Sekt. 14.6, 1903 /8/ E.BERL and O.SCHMIDT, Angew.Chem., 36, 247, 1925 /9/ R.KUHN, WINTERSTEIN and E.LEDERER, Hoppe Seilers Physiol .Chem.197, 141, 1931 / l o / R.KUHN and E.LEDERER, ibid. 200, 108, 1931 /11/ PETERS, Z.Phys.Chem. Abt.A, 148, 1 , 1930 PETERS and O.LOHMAR, ibid. 180, 44, 1939 /12/ A.TISELIUS, Ark.Chem.Mineralog.Geol., Ser.B, 14,1940 /13/ CLAESSEN, ibid., Ser.A, 25, 1 , 1946 /14/ TURNER, Nat.Petr.News 35, R234, 241, 1943 /15/ A.EUCKEN and KNICK, Brennstoffchemie 17, 241, 1936 /16/ HENJES, Oel und Kohle, Ver.Erdoe1 und Teer, 14, 1079, 1938 /17/ EDSE, P.HAR1ECK and SUHR, Angew-Chemie 52, 32, 1939 /18/ P.HARTECK and SUHR, ibid. 56, 120, 1943 /19/ L.KUCHLER and WELLER, Microchim-Acta 14, 1939 /20/ E-WICKE, Kolloid Z. 86, 167, 295, 1939, 93, 129, 1940 E.WICKE and E.WEYDE, ibid. 90, 156, 1940, Angew.Chem.B 19,15, 1947 /21/ G.DAMKOEHLER and H.THEILE, Chemie 56, 1943, Beihefte zur Zt.d.VDCH Nr.49 1944 /22/ By error caused by print or translation this "not" is missing in:E.CREMER, J.of High Resolution Chromatography and Chromatography Communications,

1979, p 8

84

The same mistake happened also p 9 in the fourth row from beneath. Read: "not be solved" /23/ S.NEUFELD, Chronologie Chemie, Verlag Chemie Weinheim 1977 /24/ G.HESSE and EILBRACHT, Liebigs Ann.Chern., 405, 546, 1941 /25/ A.I.P.MARTIN and R.L.M.SYNGE, 6iochem.J. 35, 1941 /26/ L.S.ETTRE and A.ZLATKIS, 75 Years Of Chromatography, J.of Chromatog. Library, Elsevier, Amsterdam, 1979,p296 /27/ E-CREMER, C.A.KNORR and H.PLIENINGER, Z.Elektrochem.47, 41, 737, 1941 /28/ E.CREMER, Z.Phys.Chem.B 28, 383, 1935, and Handb.d.Katalyse, Bd.1, 338, 1941 /29/ A.KUNTE, Master Thesis, Innsbruck 1943 /30/ E.CREMER, paper send to Naturwissenschaften, Nov.29th, 1944, see /26/ p 25, and Chromatographia 9, 364, 1976 /31/ list of theses performed in the Institute of Physical Chemistry of the University of Innsbruck, concerning chromatography: R-KNOEPFLER (1946), F.PRIOR (1947), R.MUELLER (1950), H.GRUBER (1956), R.ROSELIUS (1957), J.F.K.HUBER (1960), E.BECHTOLD (1962),M.RIEDMANN (1964), K.HABLIK (1967),H.NAU (1969), R.KRAMER (1971), E.SEIDL (1971). /32/ E.CREMER and F.PRIOR, Z.Elektrochem.55, 66, 1951, for early lectures see footnote 20, p 69 /33/ E.CREMER, Discussion remark, Z.Elektrochem.55, 85, 1951 R.MUELLER, Thesis, 1950 E.CREMER and R.MUELLER, Z.Elektrochem.55,217, 1951 /34/ E.CREMER, Monatshefte fur Chemie, 77, 126, 1946 /35/ E.CREMER and L.ROSELIUS, Advances in Catalysis I X , 659, 1957, Angew. Chemie 70, 47, 1958 /36/ W.R.MOORE and H.R.WARD, J.Am.Chem.Soc., 80(1958)2909 /37/ L.BACHMANN, E.BECHTOLD and E.CREMER, J.of Catalysis 1 , 113, 1962 /38/ E.CREMER and H.F.HUBER, Gaschromatographie, Academic Press, New York 1962

/39/ W.DOERRSCHEIDT and K.FRIEDRICH, J.Chromatogr. 7, 13, 31, 1961 /40/ O.BOBLETER, Beitraege zur Technikgeschichte Tirols, Heft 8, 1977 /41/ M.RIEDMANN, Berichte d.Bunsenges. Bd.69, 840, 1965 E.CREMER and M.RIEDMANN, Z.Anal.Chem. Bd.212, 1965 /42/ J.T.WATSON and K.BIEMANN, Anal.Chem.37, 844, 1965 K.BIEMANN and J.T.WATSON, Monatsh.Chem.96, 305, 1965 /43/ R.A.HITES and K.BIEMANN, Anal .Chem.40, 1217, 1968 /44/ J.A.KELLEY, H.NAU, H.J.FORSTER and K.BIEMANN, Biomed.Mass.Spectrom.2, 313, 1975 /45/ E.CREMER, Th. KRAUS, E.BECHTOLD, Chemie- Ingenieur-Technik, 9(1961)632

85

E.CREMER, J.of Gaschromatography 331, 1967 /46/ 0.BOBLETER and H-GRUBER, Kolloid Zt.Bd.151, 110, 1957 E.CREMER, H.GRUBER and H.HUCK, Chromatographia 2, 197, 1969 H.L.GRUBER and H.HUCK, 3rd 1nt.Symposium on Fuel Cells, Bruessel 1969 E.CREMER and H.L.GRUBER, J.of Gas Chromatography, 8, 1965 /47/ R.KRAMER, J.Chromatogr. ,107,241, 1976 E.CREMER and R.KRAMER, J.Chromatogr., 107(1975)253 /48/ 0-MATTHIAS and W.WEGSCHEIDER, J.Chromatogr., 258( 198311 1 /49/ E.CREMER, Th.KRAUSS and H.NAU, ZAC 245, 37 (1969) /50/ E.CREMER, F.DEUTSCHER, P.FILL and H.NAU, J.Chromatog.48, 132, 1970 /51/ E.CREMER and E.SEIDL, Chr.3, 17, 1970 E.CREMER and E.SEIDL, Monatshefte 101, 1614, 1970 E.CREMER, R.KRAMER and E.SEIDL, J-Chromatogr., 69, 135, 1972 /52/ H.K.PULKER, Coatings on glass, Thin Film Science and Technol.6, Elsevier 1984, 499 /53/ H.NEWESELY, Krauland Festschrift, Schriftenreihe der Freien Universitat Berlin 1977 p 439 /54/ H.NEWESELY, Eichner's Werkstoffe und ihre Verarbeitung 4.Aufl.,Bd.l,p 95, Huthig Verlag, Heidelberg,l981 /55/ W.LINDNER,J.Chrornatogr.,l51(1978)406, W.LINDNER, Abstract 15th 1nt.Symposium on Chromatography, Nurnberg, FRG, 1984 /56/ O.BOBLETER, W.SCHWALD, Austrian patent applied /57/ G.BONN, J-Chromatogr., in press

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87

THE FULL UTILIZATION OF THE VARIABLES OF OPEN - TUBULAR COLUMNS L. S. ETTRE Chromatography D i v i s i o n The Perkin-Elmer C o r p o r a t i o n , Norwalk CT 06856

(U.S.A.)

INTRODUCTION I n gas chromatography, t h r e e s p e c i a l r e q u i r e m e n t s a r e u s u a l l y emphasized: h i g h e f f i c i e n c y , r e a s o n a b l e a n a l y s i s t i m e and s u f f i c i e n t sample c a p a c i t y . Since t h e i r i n t r o d u c t i o n almost 30 y e a r s ago, o p e n - t u b u l a r ( c a p i l l a r y ) columns have been primarilJlconsidered as a t o o l p r o v i d i n g e f f i c i e n c i e s h i t h e r t o i m p o s s i b l e . W h i l e t h e e f f i c i e n c i e s o f packed columns u s u a l l y r e p r e s e n t a few thousand t h e o r e t i c a l p l a t e s , i t i s n o t r a r e t o have c a p i l l a r y columns p r o v i d i n g hundreds o f thousands o f t h e o r e t i c a l p l a t e s . N a t u r a l l y , emphasis on e f f i c i e n c y a u t o m a t i c a l l y r e s t r i c t s t h e o t h e r r e q u i r e m e n t s : i f very h i g h absolute e f f i c i e n c y i s required, it w i l l take longer t o get i t and t h e amount o f sample which can be i n t r o d u c e d i s l i m i t e d . E f f i c i e n c y , sample s i z e and r e t e n t i o n t i m e a r e m a i n l y c o n t r o l l e d by t h e s e l e c t i o n o f t h e column v a r i a b l e s : l e n g h t , d i a m e t e r , t h e l i q u i d phase f i l m t h i c k n e s s and, n a t u r a l l y , t h e c h o i c e o f t h e l i q u i d phase. It had been recognized s i n c e p r a c t i c a l l y t h e b e g i n n i n g o f t h e use o f o p e n - t u b u l a r columns t h a t , by t h e c a r e f u l s e l e c t i o n o f t h e s e v a r i a b l e s (and a l s o o f t h e o p e r a t i n g conditions

such as temperature, t h e c a r r i e r gas and i t s v e l o c i t y ) , good

r e s o l u t i o n can be o b t a i n e d even on columns w i t h a r e l a t i v e l y poor a b s o l u t e efficiency.

(1)

.

I n f a c t , o p e n - t u b u l a r columns w i t h a wide v a r i e t y o f

d i a m e t e r s and l e n g t h s have been u t i l i z e d . The v a r i a t i o n o f t h e f i l m t h i c k n e s s had been more r e s t r i c t e d f o r reasons t o be mentioned l a t e r , b u t , w i t h i n t h i s r e s t r i c t i o n , i t has a l s o been v a r i e d . I n f a c t , e a r l i e r columns u s u a l l y had

a relatively thick f i l m .

I n a d d i t i o n t o t h e l e n g t h , diameter, f i l m t h i c k n e s s and t h e l i q u i d phase, t h e r e i s one more column v a r i a b l e : t h e t u b i n g m a t e r i a l . F o r o v e r a decade, t h i s v a r i a b l e had been w i d e l y i n v e s t i g a t e d . A c t i v i t i e s i n t h i s f i e l d f i n a l l y l e d around 1970-1972 t o t h e u n i v e r s a l acceptance o f g l a s s and. more r e c e n t l y

88

(1979-1980), f u s e d - s i l i c a as t h e t u b e m a t e r i a l . 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 t h e u n i v e r s a l acceptance o f g l a s s somewhat c o i n c i d e d w i t h an overemphasis o f a b s o l u t e e f f i c i e n c y . Probably f o r t h i s reason, e a r l y e x t e n s i v e work on wider-bode and s h o r t e r columns was p r a c t i c a l l y f o r g o t t e n ; i n f a c t , a l i t t l e o v e r a y e a r ago, t h e i n t r o d u c t i o n o f 0.5

-

0.75 mm i . d .

columns was c o n s i d e r e d by many as an e n t i r e l y new i n v e n t i o n . The s i t u a t i o n w i t h t h e v a r i a t i o n o f t h e l i q u i d phase f i l m t h i c k n e s s i s somewhat d i f f e r e n t . E a r l y columns were a c t u a l l y c o a t e d w i t h a somewhat t h i c k e r f i l m . However, t h e f i l m t h i c k n e s s as a m a j o r v a r i a b l e had n o t been emphasized m a i n l y due t o t e c h n i c a l reasons, a l t h o u g h some p i o n e e r i n g work had been done i n t h i s f i e l d . I n t h e p a s t y e a r , o p e n - t u b u l a r columns w i t h a wide d i a m e t e r and a t h i c k

f i l m s t i r r e d a c o n s i d e r a b l e i n t e r e s t which i s s t i l l growing. Today, such columns a r e a v a i l a b l e f r o m a number o f s u p p l i e r s . The purpose o f t h i s paper i s t w o f o l d . F i r s t , i t w i l l b r i e f l y summarize t h e e v o l u t i o n o f t h e p a s t 25 y e a r s which l a i d t h e f o u n d a t i o n on which t h e most r e c e n t developments a r e based.

I n a d d i t i o n , i t w i l l discuss t h e

t h e o r e t i c a l r e l a t i o n s h i p s d e s c r i b i n g t h e i n f l u e n c e o f t h e t h r e e most i m p o r t a n t column v a r i a b l e s ( l e n g t h , d i a m e t e r and f i l m t h i c k n e s s ) on column efficiency,

r e s o l u t i o n , sample c a p a c i t y and t h e r e t e n t i o n t i m e .

EVOLUTION OF OPEN-TUBULAR COLUMNS WITH DIFFERENT PARAMETERS Length and Diameter E a r l y columns made by Golay d u r i n g t h e development o f o p e n - t u b u l a r columns had an i n t e r n a l d i a m e t e r ( i . d . )

o f 0.8-1.4

mm (2.3)

. The

first

columns were s h o r t , o n l y a few meters long. Then, m a i n l y f o r convenience ( t o have a c o n t r o l l a b l e p r e s s u r e d r o p ) , l o n g e r (100-300 f t ) t u b e s have been used i n column p r e p a r a t i o n . A t t h a t t i m e e x t e n s i v e i n v e s t i g a t i o n s were c a r r i e d o u t on t h e p o s s i b i l i t i e s o f s h o r t columns p r o v i d i n g v e r y f a s t a n a l y s i s . F o r example, a l l t h r e e chromatograms used as i l l u s t r a t i o n i n t h e fundamental p a t e n t on o p e n - t u b u l a r columns (4,5) were o b t a i n e d on 5-10 m l o n g columns w i t h 0.25

mm i . d . ,

under v e r y h i g h f l o w , r e p r e s e n t i n g a n a l y s i s t i m e s o f

o n l y a few seconds. A few y e a r s l a t e r such s h o r t (1-10 m) columns w i t h small (0.035-0.127

mm) i . d . have been a l s o e x p l o r e d by Desty e t a l . (6-8).

89

The first chromatograms on open-tubular columns illustrating real applications shown by Golay at the 1958 Amsterdam Symposium (9) were obtained on 45 m long, 0.25 mm i.d. columns and, for practical reasons, this remained the most popular internal diameter. However, 0.50 mm i.d. columns have also been very popular practically since the beginning; for example,Scott has used both 0.25 and 0.50 nun i.d. tubing in his initial investigations (10) and then settled with 0.50 mm i.d. for his detailed studies (11).

In our

laboratoires, we have also used both internal diameters from the beginning, and, in 1960-1962, published a number of data and chromatograms obtained on such columns (12-18); in fact, 50% of the chromatograms shown in a major lecture presented at the 13th Pittsburgh Conference (19) on the occasion of the fifth anniversary of open-tubular columns were obtained on 0.50 mm i.d. columns. In their book published in 1962, Dal Nogare and Juvet (20) also mention that these two (0.25 and 0.50 mm) are the most frequently used internal diameters. Columns with both diameters have been commercially available since almost th,e beginning o f open-tubular column gas chromatography.. The selection of 0.25 and 0.50 mm (0.010 and 0.020 in.) as the internal diameter was mainly due to the fact that such stainless steel tubes were comnercially available. The third diameter in which metal tubes were marketed was 0.76 mm (0.030 in.) and the use of such columns has also been reported in the early period of open-tubular column development. In 1963, Zlatkis and Walker (21) investigated the applicability of such copper tubing, and this was soon followed by Teranishi and Mon (22) who introduced 0.76 mm i.d. stainless steel columns. In their fundamental studies on foods and food flavours carried out for over a decade, these columns played an important role. Columns with 0.86 mm (0.034 in.) i.d. have also been investigated in 1959-1963 by a number of researchers, more notably by Zlatkis and Kaufman (23) and by Schwartz et al. (24). In addition Zlatkis and Kaufman also investigated the performance of a one-mile long open-tubular column with 1.68 mm i.d., which probably was the longest column ever made. However, this investigation was more out of curiosity than due to any practical need. Further investigations on larger-diameter columns were initiated by the report of Kovats, in 1961, showing a chromatogram obtained on a 2 mm i .d.

90

column u s i n g a 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 ( 2 5 ) . We, i n o u r l a b o r a t o i r e s , have a l s o s t u d i e d i n d e t a i l such w i d e r - d i a m e t e r columns. J e n t z s c h and Hovermann, i n Germany, i n v e s t i g a t e d columns w i t h 1 mm i . d .

(26-28) w h i l e we

r e p o r t e d on t h e performance o f 1.55 mm i . d . columns ( 2 9 ) . W i t h t h e s e columns, thermal c o n d u c t i v i t y d e t e c t o r s c o u l d a l s o be employed. A t t h e same t i m e , Quiram o f Esso ( 3 0 ) a l s o r e p o r t e d on t h e a p p l i c a t i o n o f long, 1.55 mm i . d . o p e n - t u b u l a r columns w i t h f l o w r a t e s as h i g h as 800 mL/min. As a r e s u l t of t h i s , he c o u l d analyze n - c e t y l a l c o h o l (1-hexadecanol a t 175"C, i . e . ,

, c16

H33OH; BP=344"C)

169°C below i t s b o i l i n g p o i r l t , i n e i g h t minutes.

I t s h o u l d be n o t e d h e r e t h a t , as a c o n c l u s i o n o f t h e work o f J e n t z s c h and Hovermann, o p e n - t u b u l a r columns w i t h 1 mm i . d . ( t h e s o - c a l l e d Columns')

"

Macro Golay

have been marketed f r o m 1962 on f o r a number o f y e a r s by Bodenseewerk

Perkin-Elmer & Co. I t i s somewhat amusing t o see t h a t now, 20 y e a r s l a t e r , 0.53 mm i.d. o p e n - t u b u l a r columns a r e c a l l e d "megabore" columns by some. E v i d e n t l y , t h e i n f l a t i o n o f t h e l a s t 20 y e a r s a l s o extended t o t h e f i e l d of nomenclature...

I d i s c u s s e d t h e e a r l y e v o l u t i o n o f o p e n - t u b u l a r columns w i t h d i f f e r e n t diameter i n a c o n s i d e r a b l e d e t a i l m a i n l y t o show t h a t l a r g e r - d i a m e t e r columns have been i n v e s t i g a t e d and used s i n c e t h e b e g i n n i n g o f o p c n - t u b u l a r column gas chromatography. I t i s , t h e r e f o r e ,

i m p o r t a n t t h a t present-day s t u d i e s f u l l y

u t i l i z e t h e accumulated knowledge and e x p e r i e n c e o f t h e past. I n t h e secon p a r t o f t h e 1 9 6 0 ~g l~a s s s t a r t e d t o g r a d u a l l y r e p l a c e metal as t h e column m a t e r i a l and, by about 1970-72,

t h e change became complete.

Glass t u b i n g c o u l d be prepared i n everybody's l a b o r a t o r y , u s i n g r e l a t i v e l y l o w c o s t i n s t r u m e n t a t i o n . F o r p r a c t i c a l reasons t h e g l a s s drawing machines were m a i n l y s e t t o produce t u b i n g w i t h an i . d .

o f 0.20-0.27

mm. T h i s p r o b a b l y

c o n t r i b u t e d t o t h e f a c t t h a t t h e advantages o f l a r g e r diameter

columns have

been m o s t l y f o r g o t t e n . The i n t r o d u c t i o n o f f u s e d s i l i c a i n 1979 d i d n o t change t h e s i t u a t i o n e x c e p t t h a t about t h e same time, 0.32 mm i . d . columns have a l s o been i n t r o d u c e d , m a i n l y t o p r o v i d e t h e p r o p e r columns f o r c o l d , on-column sample i n t r o d u c t i o n . F i n a l l y , i n t h e f a l l o f 1983, f u s e d - s i l i c a columns o f 0.53 inn i . d .

(30-31) and g l a s s c a p i l l a r y columns o f 0.75 mm i . d .

(32) have

been i n t r o d u c e d , combining t h e advantages o f w i d e r d i a m e t e r and a t h i c k e r f i l m . U n t i l r e c e n t l y , most of t h e o p e n - t u b u l a r columns used i n p r a c t i c e were f a i r l y long. The reason f o r t h i s was p r o b a b l y more p s y c h o l o g i c a l t h a n r e a l need.

91

For t h i s reason, except t h e e a r l y work o f Golay ( 4 ) and Desty e t a l . (6-8) very l i t t l e data can be found on s h o r t columns and f a s t a n a l y s i s . A n o t a b l e exception i s Marco who, i n 1964, demonstrated t h e a n a l y s i s o f C1-C12 a l c o h o l s on a 9 m x 0.50 mm i.d.

column i n l e s s than f o u r minutes ( 3 3 ) . I n 1977,

Johansen (34) published a d e t a i l e d study demonstrating t h e

advantages of

u s i n g s h o r t (10-15 m) glass open-tubular columns, g r e a t l y r e d u c i n g a n a l y s i s t i m e w h i l e s t i l l p r o v i d i n g adequate e f f i c i e n c y . The d i s c u s s i o n u n t i l now o n l y d e a l t w i t h t h e i n c r e a s e i n diameter. Unt r e c e n t l y , except Desty's p i o n e e r i n g work i n 1960-61, done i n t h e f i e l d o f small i . d .

( c0.2

(7,8)

very l i t t l e was

mm) open-tubular columns. T h i s f i e d

i s now under i n t e n s i v e i n v e s t i g a t i o n (35-37).

L i q u i d Phase F i l m Thickness While we have r e l a t i v e l y e x t e n s i v e l i t e r a t u r e s i n c e t h e beginning o f open-tubular column gas chromatography on t h e v a r i a t i o n o f t h e t u b e diameter, very l i t t l e i n f o r m a t i o n i s a v a l a i b l e on t h e v a r i a t i o n o f t h e f i l m t h i c k n e s s . There a r e m a i n l y t h r e e reasons f o r t h i s : ( a ) Metal columns were g e n e r a l l y prepared w i t h a t i c k e r f i l m , about 0.5-0.6p,

m a i n l y t o reduce t h e a c t i v i t y o f t h e i n s i d e tube surface.

(b) For a l o n g time,the

s o - c a l l e d dynamic c o a t i n g method was w i d e l y used.

Using t h i s method i t i s d i f f i c u l t t o know t h e a c t u a l f i l m t h i c k n e s s . I n o t h e r words, one d i d n o t know t h e a c t u a l f i l m t h i c k n e s s o f t h e column prepared. ( c ) U n t i l r e c e n t l y , colurrns w i t h a f i l m t h i c k n e s s above about 1 p m were n o t s t a b l e : most o f t h e excess phase was l o s t through bleeding. Looking a t t h e e a r l i e r l i t e r a t u r e we can f i n d o n l y a l i m i t e d number o f p u b l i c a t i o n s s p e c i f i c a l l y d e a l i n g w i t h t h e d e l i b e r a t e use o f t h i c k e r f i l m s . Franken and Rutten (38), i n 1972, showed t h e advantages o f columns having a f i l m thickness of about 1 u m i n reducing t h e t u b e ' s a c t i v i t y a g a i n s t h i g h - b o i l i n g p o l a r compounds and a l s o

i n c r e a s i n g sample c a p a c i t y which

improved t h e p o s s i b i l i t y o f t r a c e a n a l y s i s . Grob and Grob, i n 1977, explored columns w i t h f i l m s up t o 1.85

m t h i c k and recommended t h e use o f d i f f e r e n t

f i l m thicknesses, depending on t h e a c t u a l s e p a r a t i o n problem ( 3 9 ) . I n our l a b o r a t o i r e s , we have recognized s i n c e t h e beginning t h e need f o r

92

t h e v a r i a t i o n o f t h e f i l m t h i c k n e s s e s o r , more c o r r e c t l y , t h e phase r a t i o . F o r example, Jentzsch and Hovermann, i n t h e i r s t u d i e s (26,28) p o i n t e d o u t as e a r l y as 1961-1963 t h e need t o p r o p e r l y a d j u s t t h e r a t i o o f t u b e r a d i u s o v e r f i l m t h i c k n e s s . More r e c e n t l y , A v e r i l l and March , i n 1976, ( 4 0 ) i l l u s t r a t e d , by p r a c t i c a l examples, t h e d i f f e r e n c e s i n t h e s e p a r a t i o n o f t h e e a r l y peaks i n complex n a t u r a l samples when changing t h e f i l m t h i c k n e s s f r o m 0.25 t o 0.50,km.

A t t h a t t i m e , columns w i t h two t h i c k n e s s e s a l s o became c o m m e r c i a l l y

a v a l a i b l e . I n 1979 Johansen ( 4 1 ) p u b l i s h e d h i s p i o n e e r i n g s t u d y on t h e char a c t e r i s t i c s of 0.27 mm i . d . colmumns coated w i t h a 1-Am f i l m . We have widel y used these columns f o r t h e a n a l y s i s o f m i x t u r e s w i t h a w i d e b o i l i n g range

such as gasol i n e s (see e.g. r e f . 4 2 ) . I n 1978-1982 Mackay and Hussein p u b l i s h e d a s e r i e s o f v e r y i n t e r e s t i n g papers (43-48) on t h e p r e p a r a t i o n o f l a r g e - b o r e c o a t e d (LBC) columns c o n s i s t i n g o f 7.6-15.3

m l o n g Tygon and m e t a l tubes w i t h 2-4.7 mm i . d . and c o a t e d w i t h

SE-30 methyl s i l i c o n e , h a v i n g a f i l m t h i c k n e s s up t o 19,u.m. These columns were used t o t r a p f l a v o r compounds f r o m gases o r s o l u t i o n s , by c o n d u c t i n g t h e gas, t h e headspace o f t h e l i q u i d , or even t h e l i q u i d i t s e l f , t h r o u g h t h e column a t room temperature. D e s o r p t i o n o f t h e t r a p p e d substances t o o k p l a c e by h e a t i n g t h e LBC column a t 250°C f o r 12 minutes i n

a high nitrogen flow.

I n t h e i r papers, Mackay and Hussein a l s o i n d i c a t e t h a t t h e y checked t h e c h r o matographic p r o p e r t i e s o f t h e LBC columns: a 50 f t (15.3 m) column had about 4000

-

5000 t h e o r e t i c a l p l a t e s .

As mentioned e a r l i e r t h e main o b s t a c l e f o r u s i n g o p e n - t u b u l a r columns prepared w i t h a t h i c k e r f i l m had been t h e i r i n s t a b i l i t y : p a r t o f t h e l i q u i d phase was soon l o s t t h r o u g h b l e e d i n g . Recent advantages i n t h e p r e p a r a t i o n of i m m o b i l i z e d ("bonded") phases by i n s i t u p o l y m e r i z a t i o n f i n a l l y changed t h i s s i t u a t i o n and made t h i c k - f i l m columns a p r a c t i c a l r e a l i t y . The f i r s t d e t a i l e d r e p o r t s on such columns w i t h f i l m t h i c k n e s s e s up t o 8 p m were p u b l i shed i n 1983 (49-52) and were f o l l o w e d by more papers i n 1984 (53-58). Today, such t h i c k - f i l m columns w i t h d i f f e r e n t diameters a r e a v a i l a b l e f r o m a number o f suppl i e r s , Up t o now I o n l y d e a l t w i t h t h e e v o l u t i o n o f c a p i l l a r y columns prepared w i t h a t h i c k e r f i l m . However, t h e p o s s i b i l i t y and need o f t h i n n e r - f i l m columtis

93

have a l s o been e x p l o r e d i n t h e p a s t . I n t h e i r e a r l y work, Desty e t . a l . ( 7 ) i n v e s t i g a t e d t h e performance o f columns prepared w i t h t h i n (0.08-0.18

urn) f i l m s

and Grob and Grob, when s t u d y i n g t h e p o s s i b i l i t y o f v a r y i n g t h e f i l m t h i c k n e s s (39), a l s o prepared columns h a v i n g f i l m t h i c k n e s s e s as low as 0.018,um.

In

t h e same year, Adam e t a l . ( 5 9 ) demonstrated t h e advantages o f u s i n g a g l a s s o p e n - t u b u l a r column prepared w i t h a v e r y t h i n , l e s s t h a n O . l p m ,

(actually

around 0 . 0 5 p m ) f i l m f o r t h e a n a l y s i s o f v e r y - h i g h - b o i l i n g substances (amino a c i d d e r i v a t i v e s ) i n a r e l a t i v e l y s h o r t t i m e ( about 20 m i n u t e s ) . THE INFLUENCE OF COLUMN VARIABLES ON CHROMATOGRAPHIC PERFORMANCE I n t h e second p a r t of t h i s paper, we want t o summarize t h e i n f l u e n c e o f t h e column v a r i a b l e s on chromatographic performance..

I n t h i s discussion, tube

r a d i u s and f i l m t h i c k n e s s w i l l be d i s c u s s e d t o g e t h e r because t h e y a r e i n t e r r e l a t e d , a f f e c t i n g t h e phase r a t i o o f t h e column. Phase R a t i o and I t s I n f l u e n c e The phase r a t i o

(p) o f

a column r e p r e s e n t s t h e r a t i o o f t h e volumes o f

t h e gas (VG) and l i q u i d (VL) phases i n t h e column:

p

(1 j

vG/vL

=

The phase r a t i o i s a fundamental c h a r a c t e r i s t i c o f a chromatographic column and i s r e l a t e d 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 ( K ) and t h e c a p a c i t y f a c t o r ( k ) : K

Using

(2)

= pk

t h e same l i q u i d phase and keeping t h e temperature c o n s t a n t , K w i l l a l s o

remain c o n s t a n t . Thus, on a column w i t h a l a r g e phase r a t i o t h e c a p a c i t y f a c t o r w i l l be small and v i c e versa. Comparing two columns prepared w i t h t h e same l i q u i d phase and o p e r a t e d a t t h e same temperature, we can w r i t e t h a t k2

(3)

=

T h i s means t h a t , by t h e p r o p e r s e l e c t i o n o f t h e phase r a t i o , t h e c a p a c i t y f a c t o r o f a g i v e n s o l u t e can be a d j u s t e d . F o r an o p e n - t u b u l a r column t h e phase r a t i o i s r e l a t e d t o t h e i n s i d e t u b e r a d i u s ( r c ) and t h e t h i c k n e s s o f t h e l i q u i d phase f i l m ( d f ) :

P=

rr

(

-

df)2

(4)

2 rc df Assuming t h a t rc))df,

eq. 4 can be s i m p l i f i e d i n t h e f o l l o w i n g form:

94

13

*

rc/2df

F o r t h i c k e r - f i l m columns t h e use o f eq. 5 p r e s e n t s some inaccuracy; e.g.,

if

t h e column r a d i u s i s 0.16 mn and t h e f i l m t h i c k n e s s 5 p m , t h e e r r o r i n t h e phase r a t i o w i l l be 6.6%. However, f o r most c a l c u ; a t i o n s ,

t h i s e r r o r can be

neglected.

Eq. 5 shows t h a t t h e phase r a t i o can be a d j u s t e d by t h e p r o p e r s e l e c t i o n o f t h e t u b e d i a m e t e r o r t h e f i l m t h i c k n e s s , o r b o t h . Thus, t h e same phase r a t i o can be o b t a i n e d on a v a r i e t y o f columns. F o r example, Table I l i s t s t h r e e s e t s o f columns w i t h t h e same phase r a t i o . L e t us e.g., have k = 0.5 on t h e columns w i t h

p= 250;

assume t h a t we

then, t h e c a p a c i t y f a c t o r o f t h e

same s o l u t e ( assuming, n a t u r a l l y , t h e same l i q u i d phase and t h e same column temperature) w i l l be 2.5 on t h e columns w i t h with

?/J

p=

50, and 5.0 on t h e columns

= 25 ( c f . eq. 3). The p o s s i b i l i t y o f d e c r e a s i n g t h e phase r a t i o i s

p a r t i c u l a r l y i m p o r t a n t t o improve r e s o l u t i o n o f e a r l y emerging peaks. T h i s

w i l l be discussed below when i n v e s t i g a t i n g t h e number o f t h e o r e t i c a l p l a t e s required t o achieve a given r e s o l u t i o n . Table I Parameters o f o p e n - t u b u l a r columns h a v i n g v a r i o u s phase r a t i o ( ) v a l u e s ( c a l c u l a t e d u s i n g eq. 5). Tube

F i l m thickness f o r

i.d.

radius

=250

=50

flm

P

ym

100 250 3 20 530 750

50 12F 1 co 265 375

0.10 0.25 0.32 0.53 0.75

m 0.50 1.25 1.60 2.65 3.75

=25 m 1.0 2.5 3.2 5.3 7.5

Sometimes t h e phase r a t i o i s t o be increased. T h i s i s t h e case i n t h e a n a l y s i s o f h i g h - b o i l i n g compounds where we want t o reduce t h e c a p a c i t y f a c t o r s i n o r d e r t o speed up a n a l y s i s , o r t o reduce t h e t i m e t h e s o l u t e m o l e c u l e s a r e exposed t o a h i g h temperature i n t h e column. Here, a g a i n , we can change b o t h t h e t u b e d i a m e t e r and t h e f i l m t h i c k n e s s . F o r example, Adams e t a l . ( 5 9 ) analyzed amino a c i d d e r i v a t i v e s on a column w i t h a phase r a t i o o f a b o u t 1350

(0.27 mn i.d.,

0.05 ),Lm f i l m ) . The same e f f e c t c o u l d be achieved u s i n g a

95

1.55 nun i . d . column w i t h a 0.29 p.m f i l m o r a 0.75 mm i . d .

column w i t h a

0 . 1 4 p film. S i n c e t h e same phase r a t i o can be o b t a i n e d on columns w i t h d i f f e r e n t parameters, t h e a c t u a l s e l e c t i o n o f t h e parameters w i l l depend on o t h e r f a c t o r s such as e.g., capacity

i n s t r u m e n t a t i o n ( c h o i c e o f d e t e c t o r , i n l e t system) o r sample

.

A b s o l u t e Column E f f i c i e n c y The a b s o l u t e column e f f i c i e n c y expressed as t h e HETP (H) i s d e s c r i b e d b y t h e Golay e q u a t i o n :

CG

=

1 t 6k t l l k 2 3 (1 t k)2

CL where

=

k 3(1 t k ) *

(rc)2 8DG 2(df)2 DL

.

.

(9)

B r e p r e s e n t s t h e l o n g i t u d i n a l gaseous d i f f u s i o n w h i l e CG and CL a r e

t h e r e s i s t a n c e t o mass t r a n s f e r terms i n t h e gas and l i q u i d phases r e s p e c t i v e l y , 0 i s t h e average l i n e a r gas v e l o c i t y , k i s t h e c a p a c i t y f a c t o r o f t h e solu-

t e , DG and DL a r e t h e r e s p e c t i v e c o e f f i c i e n t s d e s c r i b i n g t h e s o l u t e ' s d i f f u s i o n i n t h e gas and l i q u i d phase, rc i s t h e i n s i d e t u b e r a d i u s and df i s t h e f i l m t h i c k n e s s o f t h e l i q u i d phase. L o o k i n g a t eqs. 8 and 9 i t seems as an i n c r e a s e i n t h e r a d i u s and t h e f i l m t h i c k n e s s w i l l s i g n i f i c a n t l y i n c r e a s e t h e r e s i s t a n c e t o mass t r a n s f e r terms and t h u s , t h e HETP w i l l be i n c r e a s e d and a b s o l u t e column e f f i c i e n c y decreased. T h i s i s t r u e ; however, t h e r e l a t i o n s h i p i s v e r y complex because change i n t h e r a d i u s and/or t h e f i l m t h i c k n e s s a l s o changes t h e v a l u e o f t h e c a p a c i t y f a c t o r ; t h u s , i n eqs. 8-9 n o t o n l y t h e second f r a c t i o n changes b u t a l s o t h e f i r s t . I n a r e c e n t paper ( 5 0 ) we have i n v e s t i g a t e d t h i s q u e s t i o n i n d e t a i l . These s t u d i e s showed t h a t t h e r e d u c t i o n i n t h e a b s o l u t e column

e f f i c i e n c y due t o an i n c r e a s e o f t h e t u b e r a d i u s and/or t h e f i l m t h i c k n e s s i s g e n e r a l l y l e s s t h a n t h e r e d u c t i o n i n t h e column e f f i c i e n c y r e q u i r e d t o a c h i e v e a g i v e n r e s o l u t i o n f o r a g i v e n s o l u t e p a i r . I n o t h e r words, w h i l e a b s o l u t e column e f f i c i e n c y i s decreased, r e s o l u t i o n may a c t u a l l y be improved.

96 Required Column E f f i c i e n c y Chromatography i s e s s e n t i a l l y a s e p a r a t i o n t e c h n i q u e : o u r goal i s t o sep a r a t e a l l t h e components p r e s e n t i n t h e sample. T e s t i n g t h e column w i t h a s i n g l e s o l u t e and e x p r e s s i n g t h e a b s o l u t e e f f i c i e n c y as t h e number o f t h e c r e t i c a l p l a t e s o r t h e HETP c o r r e s p o n d i n g t o t h i s s o l u t e does n o t n e c e s s a r i l y t e l l us whether we can a c t u a l l y r e s o l v e two peaks, however h i g h t h e e f f i c i e n c y may be. The reason f o r t h i s i s t w o f o l d : ( a ) Both t h e t h e o r e t i c a l p l a t e number and t h e HETP depend on t h e c a p a c i t y f a c t o r o f t h e p a r t i c u l a r s o l u t e . ( I t s h o u l d be n o t e d t h a t t h e same i s a l s o t r u e o f t h e number o f e f f e c t i v e p l a t e s and t h e c o r r e s p o n d i n g h e i g h t e q u i v a l e n t f o r one e f f e c t i v e p l a t e values which a l s o depend on t h e capac i t y f a c t o r . ) T h i s means t h a t i f t h e e f f i c i e n c y

i s established f o r a

peak a t k l , t h i s v a l u e w i l l n o t g i v e t h e e f f i c i e n c y f o r a n o t h e r peak a t k2. ( b ) The p l a t e number measured f o r peak A w i l l n o t p r e d i c t whether t h i s peak can a c t u a l l y be separated f r o m peak €3. The reason f o r t h i s i s t h a t t h e a b s o l u t e e f f i c i e n c y c a l c u l a t e d f o r a peak and t h e e f f i c i e n c y r e q u i r e d t o s e p a r a t e i t f r o m a n o t h e r peak a r e two d i f f e r e n t terms. Only t h e comparison o f t h e s e two values can g i v e us i n f o r m a t i o n on t h e a c t u a l s e p a r a t i o n . Since t h e concept o f t h e p l a t e number r e q u i r e d t o s e p a r a t e two peaks i s o f t e n n o t f u l l y understood, i t i s w o r t h w h i l e t o d i s c u s s i t i n more d e t a i l . L e t us assume t h a t we have two c l o s e l y spaced peaks s p e c i f i e d by t h e i r relative retention

d

( a ) and

the respective capacity factors

(10)

= k2/kl

and we want t o s e l a r a t e them by a r e s o l u t i o n o f o f t h e o r e t i c a l p l a t e s r e q u i r e d (nreq)

R. I n t h i s case, t h e number

t o a c h i e v e t h i s r e s o l u t i o n can be ex-

Dressed as

where n r e q and k r e f e r t o t h e second peak o f t h e p a i r . Table I 1 l i s t s a c t u a l values o f t h e r e q u i r e d t h e o r e t i c a l p l a t e number f o r two peaks h a v i n g a r e l a t i v e r e t e n t i o n o f o( = 1.05 which a r e t o be separated by b a s e l i n e r e s o l u t i o n

( R = 1.5). L e t us, e.g.

, compare

two columns on which t h e r e s p e c t i v e capa-

97

Table I1 Number o f t h e o r e t i c a l p l a t e s r e q u i r e d (nreq) t o a c h i e v e b a s e l i n e r e s o l u t i o n o f two peaks h a v i n g a r e l a t i v e r e t e n t i o n o f a = 1.05, as a f u n c t i o n o f t h e c a p a c i t y f a c t o r ( k 2 ) o f t h e second peak ( c a l c u l a t e d a c c o r d i n g t o eq. 1 1 ) .

k2 0.1 0.2 0.5 1 .o 2.0 5.0 10.0 20.0

“eq 1,921,000 571 ,540 142,880 63,500 35,720 22,21u 19,210 17,500

% 100.00 29.75 7.44 3.31 1.86 1.16 1 .oo 0.91

c i t y f a c t o r o f t h e second peak o f t h i s p a i r i s 0.2 and 2.0 As we have seen e a r l i e r ( c f . eq. 3 ) such a change i n t h e c a p a c i t y f a c t o r can be a c h i e v e d by r e d u c i n g t h e phase r a t i o by a f a c t o r o f t e n , e.g.,

from

p = 250 t o p= 25

(see Table I ) . While on t h e f i r s t column we need 571,540 t h e o r e t i c a l p l a t e s t o a c h i e v e t h e d e s i r e d r e s o l u t i o n , we o n l y need 35,720 p l a t e s on t h e second column, 6.25% o f t h e f o r m e r v a l u e . Table I 1 and t h i s example

i l l u s t r a t e t h e p r a c t i c a l advantages o f chan-

g i n g t h e phase r a t i o : o n l y a f r a c t i o n o f t h e column e f f i c i e n c y i s r e q u i r e d f o r e a r l y emerging peaks, and t h u s , t h e l o s s i n a b s o l u t e column e f f i c i e n c y due t o t h e i n c r e a s e i n t u b e r a d i u s and/or t h e f i l m t h i c k n e s s i s amply compensated.

As shown i n Table 11, t h e same e f f e c t i s l e s s i m p o r t a n t f o r peaks w h i c h a l r e a d y have a h i g h e r c a p a c i t y f a c t o r on t h e f i r s t column. F o r example, chang i n g f r o m k = 2 t o k = 20 o n l y reduces t h e r e q u i r e d p l a t e number t o h a l f o f t h e o r i g i n a l v a l u e (35,720 v s . 17,500).

T h i s r o u g h l y corresponds t o t h e chan-

ge i n t h e a b s o l u t e column e f f i c i e n c y i f changing t h e phase r a t i o by a f a c t o r o f t e n . I n o t h e r words, f o r l a t e r peaks, r e s o l u t i o n i s a f f e c t e d o n l y t o a m i n o r degree. The examples above were based on 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 . However, t h e advantage o f d e c r e a s i n g t h e phase r a t i o i n i m p r o v i n g r e s o l u t i o n can a l s o be demonstrated by p r a c t i c a l examples. Johansen, i n 1979 ( 4 1 ) , compared two

98 0.27 mm i . d . g l a s s columns h a v i n g t h e r e s p e c t i v e l e n g t h s o f 100 and 55, m and f i l m t h i c k n e s s e s

o f 0.2 and 0.92hm. The c o r r e s p o n d i n g phase r a t i o va-

l u e s were 337.5 and 73.4 The column e f f i c i e n c i e s expressed as t h e HETP f o r a peak h a v i n g t h e same c a p a c i t y f a c t o r ( k = 2.5) were 0.28 and 0.41 mm r e s p e c t i v e l y . I n o t h e r words, t h e a b s o l u t e e f f i c i e n c y o f t h e column prepared w i t h a 4.6 t i m e s t h i c k e r f i l m was reduced by 31.7

%.However,

the resolution

o f l o w - b o i l i n g compounds was much improved: f o r t h e n-butane/isobutane p a i r i t was 1.66 vs. 4.50.

I f we e x t r a p o l a t e t o a 100-m l o n g t h i c k - f i l m column

(see below), t h e a c t u a l r e s o l u t i o n would be as h i g h as 6.1,

r e p r e s e n t i n g an

improvement by a f a c t o r o f 3.7. T h i s means t h a t w h i l e on t h e t h i n - f i l m column

(R= 1.54) was achieved f o r t h e isobutane/n-butane peak

baseline r e s o l u t i o n

p a i r , when a n a l y z i n g t h e same compounds on t h e t h i c k - f i l m column h a v i n g t h e same l e n g t h one c o u l d a c t u a l l y p l a c e f i v e more peaks between t h e s e two and s t i l l f u l l y s e p a r a t e them. Table I 1 1 S e p a r a t i o n number v a l u e s measured on d i f f e r e n t g l a s s c a p i l l a r y columns ( i . d . = 0.27 rim) c o a t e d w i t h m e t h y l s i l i c o n e phase. Column temperature: 69°C. n - P a r a f f in pair

100 m df=O. 2 pm

55 m d= ,O .92 p m

100 m i df=O. 92

c3-c4

2.4

6.3

8.9

c4-c5

5.4

13.3

18.3

'5-'6

12.5

23.2

31.6

'6-'7

24.0

35.5

48.2

C7-Cg

42.0

43.5

59.0

C8-h

60.3

61 .2

82.9

*

pm

extrapolated

Johansen a l s o compared t h e s e p a r a t i o n number ( " T r e n n z a h l " ) v a l u e s o b t a i ned on t h e two columns. These g i v e t h e number o f peaks one can s e l a r a t e between two normal p a r a f f i n peaks. Table I11 g i v e s t h e v a l u e s o b t a i n e d . As seen, t h e column having a 4.6 t i m e s t h i c k e r f i l m b u t r e p r e s e n t i n g o n l y h a l f t h e l e n g t h o f t h e t h i n - f i l m column a c t u a l l y gave much h i g h e r s e p a r a t i o n number v a l u e s . The comparison i s more c o r r e c t i f columns o f equal l e n g t h a r e

99

compared. The values l i s t e d by Johansen f o r t h e 55 m column can e a s i l y be e x t r a p o l a t e d t o a 100-m column c o n s i d e r i n g t h e r e l a t i o n s h i 1 between t h e sep a r a t i o n number (SN) and r e s o l u t i o n ( R ) SN

R - 1

=

(12)

and t h e f a c t t h a t r e s o l u t i o n i s r e l a t e d t o t h e square r o o t o f column l e n g t h (see below). Hence :

SN2

-+

1

The l a s t e x t r a p o l a t e d t o a 100-m l o n g column having a 0 . 9 2 - p f i l m . The improvement i s t h e b e s t f o r e a r l y peaks and l e s s f o r t h e l a t e r p a r t o f t h e chromatogram: i n the C3-C4 r e g i o n t h e number o f peaks which can be separated i s increased by a f a c t o r o f 3.7 ( 2.4 vs. 8.9), w h i l e i n t h e C4-C5 r e g i o n i t i s increased by a f a c t o r o f 3.4 (5.4 vs. 18.3), and i n t h e C 8 - k ~r e g i o n i t i s increased by 37.5 % (60.3 vs. 82.9). Column Length As mentioned i n t h e i n t r o d u c t i o n , open-tubular columns have been i d e n t i f i e d f o r a l o n g time w i t h l o n g tubes. One o f t h e reasons f o r t h i s was undoubtedly t h e d e s i r e f o r a h i g h a b s o l u t e column e f f i c i e n c y : e v e r y t h i n g being equal, column l e n g t h and p l a t e number a r e l i n e a r l y r e l a t e d . I t is,however, important t o r e a l i z e t h a t w h i l e column l e n g t h i s l i n e a r l y r e l a t e d t o t h e p l a -

(4

t e number, i t i s q u a d r a t i c a l l y r e l a t e d t o r e s o l u t i o n : L

=

16 H R2

(k;

1)

This means t h a t i f we compa e two columns having d i f f e r e n t l e n g t h s (L1 and L2) b u t o t h e r w i s e i d e n t i c a l c h a r a c t e r i s t i c s , then t h e r e s o l u t i o n o f t h e same s o l u t e p a i r i s r e l a t e d t o t h e square r o o t o f t h e column l e n g t h r a t i o :

f i r s t , one has t o increase column l e n g t h by a f a c t o r o f f o u r i n o r d e r t o doub l e r e s o l u t i o n . Obviously, t h i s is a h i g h p r i c e t o pay, because t h e r e t e n t i o n time i s d i r e c t l y r e l a t e d t o column l e n g t h (see below). On t h e o t h e r hand, i t a l s o means t h a t i f we reduce column length, r e s o l u t i o n i s l e s s a f f e c t e d than

100

t h e r e d u c t i o n i n column l e n g t h . F o r example, i f a 50-m l o n g open..tubular column p r o v i d e s a r e s o l u t i o n o f R1 then, r e d u c i n g t h e column l e n g t h t o 10 m ( t h a t means by 80 % ) w i l l s t i l l p r o v i d e a r e s o l u t i o n o f R2

=

( 10/50)’/2

R1

=

0.45 R1

i n o t h e r words, 20 % o f t h e o r i g i n a l column l e n g t h w i l l s t i l l g i v e a l m o s t h a l f t h e r e s o l u t i o n o f t h e l o n g column. Table I V C h a r a c t e r i s t i c s d a t a f o r t h e s e p a r a t i o n o f methyl s t e a r a t e ana Ult!dLe on two columns prepared w i t h d i e t h y l e n e g l y c o l r l i c c i n a t e l i q u i d phase; column temperature: 180°C. A f t e r E t t r e and March 160).

Length I n t e r n a l diameter L i q u i d phase l o a d i n g L i q u i d phase f i l m t h i c k n e s s Phase r a t i o Av. l i n e a r gas v e l o c i t y ( h e l i u m ) R e s o l u t i o n o f methyl s t e a r a t e / o l e a t e R e t e n t i o n t i m e o f methyl o l e a t e C a p a c i t y f a c t o r o f methyl o l e a t e

* support:

m mm % P n cm/sec

min

Packed*

Capi 1 l a r y

2.44 2.16 10.0 10.3 6.8 1.54 35.62 58.6

45.72 0.25 0.52 120 9.0 7.06 51.65 5.1

Chromosorb W 80/100 mesh.

A good p r a c t i c a l example has been shown i n 1974 by E t t r e and March ( 6 0 ) u s i n g t h e methyl s t e a r a t e

-

o l e a t e s e p a r a t i o n f o r t h e comparison o f v a r i o u s

columns. Table I V l i s t s t h e c h a r a c t e r i s t i c d a t a o f a packed and an open-tubul a r column, b o t h o p e r a t e d a t optimum v e l o c i t y . I f we accept a r e s o l u t i o n o f R=1.54 ( w h i c h c o u l d be o b t a i n e d on t h e packed column, r e p r e s e n t i n g b a s e l i n e s e p a r a t i o n ) , t h e n we would o n l y need a 2.2-m l o n g o p e n - t u b u l a r column (4.76 % of t h e o r i g i n a l

l e n g t h ) and t h e r e t e n t i o n t i m e o f methyl o l e a t e on t h i s s h o r t

column would o n l y be 2.46 m i n u t e s . Such a v e r y s h o r t o p e n - t u b u l a r column m i g h t be i m p r a c t i c a l b u t e.g.,

a r e d u c t i o n o f t h e column l e n g t h t o 10 m

(21.9 % o f t h e o r i g i n a l l e n g t h ) would s t i l l p r o v i d e a r e s o l u t i o n o f R=3.3, almost 50 % o f t h e o r i g i n a l r e s o l u t i o n . The r e t e n t i o n t i m e o f methyl o l e a t e on t h i s 10-m l o n g column would be 11.3 minutes.

101

Sample C a p a c i t y I n c r e a s i n g t h e sample c a p a c i t y o f a column i s i m p o r t a n t f o r t h r e e reasons: ( a ) It s i m p l i f i e s t h e t e c h n i q u e o f sample i n t r o d u c t i o n ; ( b ) i t improves t h e minimum d e t e c t a b i l i t y o f t h e chromatographic system; and ( c ) i t i n c r e a s e s t h e a b s o l u t e sample amount ( and t h u s , t h e c o n c e n t r a t i o n o f t h e s o l u t e i n t h e c a r r i e r gas) which may be i m p o r t a n t i n c e r t a i n a p p l i c a tions. T y p i c a l cases f o r ( c ) a r e t h e s o - c a l l e d hyphenated t e c h n i q u e s (combining GC w i t h a s p e c t o s c o p i c method) o r u s i n g a 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 i n t h e GC system. I t i s d i f f i c u l t t o c h a r a c t e r i z e t h e sample c a p a c i t y o f an o p e n - t u b u l a r

column because t h e r e i s no accepted d e f i n i t i o n f o r i t . The usual d e f i n i t i o n ( " n o t more t h a n 10% r e d u c t i o n i n column e f f i c i e n c y " ) i s o f l i t t l e use. T h i s

&,"'

f o l l o w s f r o m eq. 14 f r o m w h i c h one can d e r i v e t h a t R2

=

R1

Thus, a 10% r e d u c t i o n i n e f f i c i e n c y ( 1 0 % i n c r e a s e i n HETP) o n l y r e p r e s e n t s a 5 % r e d u c t i o n i n r e s o l u t i o n which i s n e g l i g i b l e .

As d i s c u s s e d r e c e n t l y b y E t t r e ( 6 1 ) a b e t t e r way seems t o be t o compare t h e r e l a t i v e e f f i c i e n c i e s o f o p e n - t u b u l a r columns u t i l i z i n g t h e r e l a t i o n s h i p f i r s t d e s c r i b e d i n 1957 by van Deemter e t a l . ( 6 2 ) and e l a b o r a t e d i n d e t a i l by Keulemans ( 6 3 ) . A c c o r d i n g t o t h i s , t h e maximum sample c a p a c i t y o f a column (Vmax) can be expressed as

By v a r i o u s s u b s t i t u t i o n s f o r VL and K, eq. 17a can a l s o be w r i t t e n i n t h e f o l -

l o w i n g forms: "max

aK VG - n1/2

K

I n eqs. 17a-c VG and VL a r e t h e volumes o f t h e gas and l i q u i d phases i n t h e column, K and k a r e t h e p a r t i t i o n c o e f f i c i e n t and t h e c a p a c i t y f a c t o r of t h e s o l u t e , n i s t h e t h e o r e t i c a l p l a t e number f o r t h e s o l u t e peak and aK i s a

102

factor. These e q u a t i o n s p e r m i t us t o e v a l u a t e t h e i n f l u e n c e o f t h e column v a r i a b l e s on sample c a p a c i t y i n general terms and t o compare t h e sample c a p a c i t y o f v a r i o u s columns f o r t h e same s o l u t e . o f eq. 17a-c shows t h a t t h e i n c r e a s e i n t h e

The e x p r e s s i o n on t h e R.H.S.

sample c a p a c i t y by i n c r e a s i n g t h e t h e volumes o f t h e gas and l i q u i d phases i n t h e column s i g n i f i c a n t l y depends on t h e p a r t i t i o n c o e f f i c i e n t ( K ) and hence, on t h e c a p a c i t y f a c t o r ( k ) o f t h e s o l u t e . I n o t h e r words, one must c l e a r l y use t h e same s o l u t e when comparing two co!umns;

expressing t h e increase o f

t h e sample c a p a c i t y f o r one s o l u t e does n o t mean t h e same i n c r e a s e f o r anot h e r s o l u t e . AS shown below, t h e i n c r e a s e i n t h e sample c a p a c i t y i s much small e r f o r a peak emerging e a r l y on a t h i n - f i l m column t h a n f o r a l a t e r - e m e r g i n g peak. Eq. 17c a l s o shows t h a t i f i n c r e a s i n g t h e column l e n g t h w h i l e keeping t h e o t h e r parameters c o n s t a n t , sample c a p a c i t y w i l l n o t i n c r e a s e w i t h t h e same f a c t o r . F o r example, i f we double column l e n g t h , t h e n t h e v a l u e o f VG w i l l double; however, t h e denominator o f t h e R.H.S.

o f eq. 17c w i l l i n c r e a s e o n l y

by t h e square r o o t o f two. As t h e o v e r a l l r e s u l t , t h e sample c a p a c i t y w i l l i n c r e a s e by t h e square r o o t o f two:

Next, l e t us i n v e s t i g a t e t h e change i n t h e sample c a p a c i t y o f two columns w i t h t h e same l e n g t h b u t d i f f e r e n t t u b e d i a m e t e r and f i l m t h i c k n e s s . I n t h i s way t h e v a l u e o f VG w i l l change; n a t u r a l l y , t h e p l a t e number w i l l a l s o change. I n t h i s d e d u c t i o n we assume t h a t t h e v a l u e o f aK i s c o n s t a n t f o r t h e two columns (see below). L e t us express t h e changes i n t h e t h r e e terms i n eq. 17c by t h e f a c t o r a, b and c : k2 By

=

bkl

nz

=

cn 1

t h e p r o p e r s u b s t i t u t i o n i n t o eq. 17c, we can express t h e r a t i o o f t h e

sample c a p a c i t y o f t h e two columns i n t h e f o l l o w i n g way:

L e t us compare two c o l u n m h a v i n g t h e same l e n g t h and i n t e r n a l diameter; i n t h i s way, VG may be considered a s c o n s t a n t * ( a = l ) . L e t us f u r t h e r assume t h a t

103 t h e r e s p e c t i v e f i l m t h i c k n e s s e s a r e 0.25 and 2.51111 and t h a t t h e e f f i c i e n c y o f column i s 50 % o f t h e e f f i c i e n c y o f t h e t h i n - f i l m column; i n

the t h i c k - f i l m

o t h e r words, b = l U and c=0.5. T h e r e f o r e :

L e t us now c o n s i d e r two s o l u t e s w i t h t h e r e s p e c t i v e c a p a c i t y f a c t o r s o f 0 . 2 and 2.0 on t h e t h i n - f i l m column. By s u b s t i t u t i n g t h e s e v a l u e s f o r k l i n t o eq. 19, we o b t a i n t h a t t h e sample c a p a c i t y o f t h e f i r s t s o l u t e w i l l be i n c r e a sed by a f a c t o r o f 3.5, w h i l e t h e sample c a p a c i t y o f t h e second s o l u t e w i l l be i n c r e a s e d by a f a c t o r o f 9.9. Table V Comparison o f v a r i o u s columns f r o m t h e p o i n t o f sample c a p a c i t y ?

L

i.d.

m

m

*

w

vG

mL

C+

RSC

RSC

(C)

(PI 2.2 6.2 11 33 58

ML L

0.32

250 160 80 26.7 16

2.0 3.125 6.25 18.73 31.25

166,500 105,000 102,000 81,000 66,000

1.467 2.398 3.383 2.323 2.264

10.78 30.52 54.09 161.05 284.24

100 285 502 1494 2636

0.53

0.5 1.0 5.0

265 132.5 26.5

1.89 3.77 18.87

72,000 69,000 45,000

6.594 6.569 6.371

71.02 119.28 596.77

659 1106 5199

14 24 114

0.53

5.0

26.5

18.87

15,000

2.124

323.65

3001

66

9.61

4,000

2.939

493.00

4572

100

30

2

n

k

0.25 0.5 1.0 3.0 5.0

0.25

10

df

Packed""

26

*Symbols: L = l e n g t h , i . d . = i n t e r n a l d i a m e t e r , df = l i q u i d phase f i l m thickness, phase r a t i o , k = c a p a c i t y f a c t o r , n = number o f t h e o r e t i c a l p l a t e s , VG = volume o f gas phase i n t h e column, C * = see eq. 20, RSC = r e l a t i v e sample c a p a c i t y ( C = r e l a t i v e t o t h e o p e n - t u b u l a r column w i t h 0.25 mm i . d . and 0 . 2 5 ) ~ m f i l m ; P = r e l a t i v e t o t h e packed column).

p=

* Y 2.16

mm i . d . , 5 % l i q u i d phase; d a t a f o r t h e phase r a t i o and VG a r e t a k e n from r e f . (61).

Table V compares t h e sample c a p a c i t y o f o p e n - t u b u l a r columns h a v i n g d i f f e r e n t diameter, l e n q t h and f i l m t h i c k n e s s . F o r t h i s comparison a c a p a c i t y f a c t o r o f k=2.0 i s assumed on t h e 0.25 mm i . d . column h a v i n g a f i l m t h i c k n e s s of 0.25

)Am.

The c a p a c i t y f a c t o r s o f t h e same s o l u t e on t h e o t h e r columns were

c a l c u l a t e d a c c o r d i n g t o eq. 3.

The number o f t h e o r e t i c a l p l a t e s l i s t e d f o r t h e

104 d i f f e r e n t columns a r e based on d a t a g i v e n i n t h e l i t e r a t u r e and i n s u p p l i e r s ' brochures. The values o f t h e v e r t i c a l column marked as C *correspond

t o the

v a r i a b l e i n eq. 17c:

I n t h e v e r t i c a l column marked as RSC(C) t h e r e l a t i v e sample c a p a c i t y values a r e g i v e n , by c o n s i d e r i n g t h e v a l u e o f C * f o r t h e f i r s t column ( 3 0 m, 0.25 mm i.d.,

0.25pm f i l m ) as 100. I n Table V, an a t t e m p t

i s a l s o made i n comparing t h e sample c a p a c i t y o f t h e

o p e n - t u b u l a r columns w i t h t h e sample c a p a c i t y a 2 m, 1 / 8 i n . 0.d.

o f a packed column. Date f o r

(2.16 m i . d . ) packed column a r e g i v e n i n t h e l a s t h o r i z o n -

t a l l i n e o f t h e t a b l e and t h e l a s t v e r t i c a l column marked as RSC(P) i n d i c a t e s t h e sample c a p a c i t i e s o f t h e o p e n - t u b u l a r column r e l a t i v e t o t h i s packed column. I t should be emphasized t h a t any comparison o f sample c a p a c i t i e s c a l c u l a t e d from eqs. 17a-c and eq. 18 assumes t h a t t h e v a l u e o f f a c t o r aK i s i d e n t i c a l f o r a l l t h e columns compared. K l i n k e n b e r g , i n 1960 (64), d i s c u s s e d t h e problem o f aK, i n d i c a t i n g t h a t i t i s n o t a c o n s t a n t , b u t depends on t h e s o l u t e and some u n s p e c i f i e d parameters. We a r e comparing h e r e d a t a c a l c u l a t e d f o r t h e same s o l u t e and t h u s , t h e f i r s t p a r t o f t h i s u n c e r t a i n t y i s e l i m i n a t e d . We bel i e v e t h a t f o r s i m i l a r columns, aK would n o t change t o o much and t h e r e f o r e , t h e r e l a t i v e sample c a p a c i t y values l i s t e d under RSC(C) may be c o n s i d e r e d as approximate values, n o t f a r o f f t h e r e a l v a l u e s . One s h o u l d be more c a u t i o u s w i t h t h e values l i s t e d under RSC(P) because here, we assume t h e e q u i v a l e n c y o f t h e a K values o f o p e n - t u b u l a r columns and o f a packed column. S t i l l , we b e l i e v e

t h a t t h e s e values r e p r e s e n t a good i n d i c a t i o n o f t h e t r e n d , even i f t h e numer i c a l v a l u e s may be o f f . R e t e n t i o n Time The r e l a t i o n s h i p between r e t e n t i o n t i m e ( t R ) , column l e n g t h ( L ) , t h e capac i t y f a c t o r ( k ) , and t h e average l i n e a r gas v e l o c i t y

(u)

i s expressed by t h e

following relationship: tR

=

(L/u) (1 + k)

(21)

According t o t h i s e q u a t i o n , r e t e n t i o n t i m e i n c r e a s e s w i t h an i n c r e a s e i n t h e c a p a c i t y f a c t o r . I n o t h e r words, i f we decrease t h e phase r a t i o (e.g.,by i n c r e a s i n g t h e f i l m t h i c k n e s s ) , r e t e n t i o n t i m e w i l l i n e v i t a b l y i n c r e a s e because

105

the capacity f a c t o r i s increased. However, eq. 16 does n o t n e c e s s a r i l y mean a s i g n i f i c a n t i n c r e a s e i n t h e t o t a l a n a l y s i s t i m e . L e t us n o t f o r g e t t h a t o u r p r i m a r y goal i s t o i n c r e a s e t h e c a p a c i t y f a c t o r s o f e a r l y peaks which a r e d i f f i c u l t t o s e p a r a t e on a t h i n - f i l m column. Peaks which would emerge l a t e r , u s u a l l y do n o t have s e p a r a t i o n problems. Hence, here, a s i g n i f i c a n t i n c r e a s e i n t h e i r c a p a c i t y f a c t o r can u s u a l l y be avoided by t h e proper s e l e c t i o n o f column temperature. L e t us n o t f o r g e t t h a t an i n c r e a s e o f 25-30°C w i l l about h a l v e t h e c a p a c i t y f a c t o r . I n o t h e r words, we can keep t h e e l u t i o n t i m e o f l a t e r peaks w i t h i n t h e r e q u i r e d t i m e range by p r o g r a m i n g t h e column temperature t o a h i g h e r v a l u e and/or u s i n g a h i g h e r p r o gram r a t e . Our most r e c e n t p u b l i c a t i o n ( 6 1 ) d i s c u s s e d i n d e t a i l t h e i n f l u e n c e o f temperature and how r e t e n t i o n t i m e s can be reduced on t h i c k - f i l m columns by t h e p r o p e r c h o i c e o f t h e temperature p r o f i l e . There a r e two more ways t o improve a n a l y s i s t i m e on t h i c k - f i l m columns. The f i r s t i s o b v i o u s l y t o use a s h o r t e r column. We have d i s c u s s e d above i n d e t a i l t h e r e l a t i o n s h i p between f i l m t h i c k n e s s (phase r a t i o ) and t h e r e q u i r e d number o f t h e o r i c a l p l a t e s . O b v i o u s l y , i f much s m a l l e r column e f f i c i e n c y i s r e q u i r e d , one can use a s h o r t t h i c k - f i l m column and s t i l l o b t a i n adequate r e s o l u t i o n i n a much s h o r t e r t i m e . The advantages o f s h o r t columns have a l r e a d y been d i s c u s s e d e a r l i e r and here, we o n l y want t o r e f e r t o t h e most r e c e n t i n f o r m a t i o n on t h i s p o s s i b i l i t y (65, 6 6 ) . The o t h e r way t o improve a n a l y s i s

t i m e i s t o use a h i g h e r gas v e l o c i t y .

T h i s p o s s i b i l i t y , r e d u c i n g a n a l y s i s t i m e on l o n g e r columns by i n c r e a s i n g t h e c a r r i e r gas v e l o c i t y , has beerl discussed i n d e t a i l 15 y e a r s ago ( 6 7 ) ; i t f o l l o w s from t h e optimum p r a c t i c a l gas v e l o c i t y concept o f S c o t t and Hazeldean ( 1 1 ) d e s c r i b e d i n 1960. T h i s i s p a r t i c u l a r l y easy t o do w i t h w i d e r - b o r e columns because t h e y have a l o w f l o w r e s i s t a n c e and column e f f i c i e n c y does n o t s i g n i f i c a n t l y change w i t h i n c r e a s i n g v e l o c i t y , p a r t i c u l a r l y i f hydrogen i s used as t h e c a r r i e r gas ( 6 8 ) . Another advantage o f u s i n g a l o n g e r column a t a h i g h e r v e l o c i t y f o l l o w s from eq. 17c: t h e sample c a p a c i t y i s a c t u a l l y improved because V G remains t h e same w h i l e t h e p l a t e number w i l l be somewhat s m a l l e r .

T h i s b r i e f summary shows t h a t t h e r e a r e a number o f ways t o keep t h e anal y s i s t i m e w i t h i n a reasonable range w h i l e s t i l l h a v i n g adequate r e s o l u t i o n and sampl e c a p a c i t y

.

106

CONCLUSIONS We a r e now w i t n e s s i n g t h e r e n a i s s a n c e o f o p e n - t u b u l a r columns and t h e i n c r e a s i n g u n d e r s t a n d i n g o f t h e i n f l u e n c e o f t h e column v a r i a b l e s on column p e r formance. T h i s paper r e p r e s e n t s an a t t e m p t t o o u t l i n e t h e e v o l u t i o n o f o u r u n d e r s t a n d i n g and t o p r e s e n t t h e t h e o r e t i c a l background f o r t h e p r o p e r s e l e c t i o n o f these v a r i a b l e s . I t can be expected t h a t t h e more we a r e a b l e t o s e l e c t t h e column parame-

t e r s a c c o r d i n g t o o u r need, t h e more w i d e l y w i l l o p e n - t u b u l a r columns be u t i l i z e d i n t h e general p r a c t i c e . Whether t h e y w i l l c o m p l e t e l y r e p l a c e packed columns depends on a number o f f a c t o r s . S t i l l , one can s a f e l y p r e d i c t t h a t t h e developments o f t h e l a s t c o u p l e o f y e a r s

-

which a r e based on o u r accumulated

knowledge gained i n t h e p a s t 20 y e a r s - w i l l c e r t a i n l y l e a d us t o a much more general u t i l i z a t i o n o f t h e s e columns, n o t o n l y f o r t h e s e p a r a t i o n o f v e r y complex m i x t u r e s b u t a l s o i n t h e day-today a n a l y s i s o f r e l a t i v e l y s i m p l e samples.

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See t h e general t e x t b o o k s p u b l i s h e d i n 1961-1965 on o p e n - t u b u l a r columns: ( a ) R. Kaiser, Chromatographie i n d e r Gasphase, 11. K a p i l l a r Chromatographie, B i b l i o g r a p h i s c h e s I n s t i t u t , Mannheim; 1 s t edn.: 1961, 2nd edn.: 1966. ( b ) R. K a i s e r , Chromatography i n Gas Phase, 11. C a p i l l a r y Gas Chromatography, B u t t e r w o r t h s , London, 1963 ( E n g l i s h t r a n s l a t i o n o f t h e 1 s t edn. o f r e f . l a ) . ( c ) L.S. E t t r e , Open T u b u l a r Columns i n Gas Chromatography, Plenum Press, New York NY, 1965. Golay, Report on November 15, 1956. Quoted on p.3 o f r e f . l c .

2

M.J.E.

3

M.J.E. Golay, i n V.J. Coates, H.J. Noebels and I . S . Fagerson (Eds.), Gas Chromatography (1957 L a n s i n g Symposium) , Academic Press, New York NY, 1958 pp. 1-13.

4

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107 7

D.H. Desty and A. Goldup, i n R.P.W. S c o t t ( E d . ) , Gas Chromatography 1960 (Edinburgh Symposium), B u t t e r w o r t h s , London, 1960, pp. 162-183.

8

D.H. Desty, A. Goldup and T.W. Swanton, i n N. Brenner, J.E. C a l l e n and M.D. Weiss (Eds.), Gas Chromatography (1961 L a n s i n g Symposium) , Academic Press, New York NY, 1962, pp. 105-135.

9

M.J.E. Golay, i n D.H. Desty (Ed.), Gas Chromatography 1958 (Amsterdam Symposium), B u t t e r w o r t h s , London, 1958, pp.36-55.

S c o t t , N a t u r e (London) 183 (1959) 1753.

10

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R.P.W. S c o t t and G.S.F. Hazeldean, i n R.P.W. S c o t t (Ed.), Gas Chromatography 1960 (Edinburgh Symposium), B u t t e r w o r t h s , London, 1960, pp. 144-161.

12 W . A v e r i l l , I n s t r u m e n t News 12 ( 1 ) (1960/1961) 6. 13

W. A v e r i l l , i n N. Brenner, J.E. C a l l e n and M.D. Weiss (Eds.), Gas Chromatography (1961 L a n s i n g Symposium), Academic Press, New York NY, 1962, pp. 1-6.

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L.S. E t t r e and W. A v e r i l l , Anal. Chem. 33 (1961) 680.

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L.S. E t t r e and W. A v e r i l l , i n N.D. Cheronis (Ed.), Proc. 1 s t I n t . Symp. Microchemical Techniques, I n t e r s c i e n c e , New York NY, 1962, pp. 715-732.

16

L.S. E t t r e , W. A v e r i l l and F.J. Kabot, Gas Chromatographic A n a l y s i s of F a t t y Acids, No. GC-AP-001 , The Perkin-Elmer Corp., Norwal k CT, 1962.

17

L.S. E t t r e , Instument News 13 ( l a ) (1961/62) 1 .

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L.S. E t t r e , Research/Development f o r I n d u s t r y 1962 ( 1 5 ) 42.

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S. Dal Nogare and R.S. J u v e t Jr., Gas-Liquid Chromatography, I n t e r s c i e n c e , New York NY, 1962, pp, 268-269.

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25

E. Kovats, Meeting o f t h e ( B r i t i s h ) Chromatography D i s c u s s i o n Group, B i r mingham, A p r i l 21, 1961; see H. S t r i c k l e r and E. Kovats, J. Chromatog. 8 (1962) 289.

26

D. J e n t z s c h and W. Hovermann, i n M. van Swaay ( E d . ) , Gas Chromatography 1962 (Hamburg Symposium), B u t t e r w o r t h s , London , 1962, pp. 204-21 5 .

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D. Jentzsch and W. Hovermann, J. Chromatog. 11 (1963) 440.

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B.L. Ryder, J. P h i l l i p s , L.L. P l o t c z y k and M. Redstone, 3 5 t h P i t t s b u r g h Conf. A n a l . Chem. Appl. Spectroscopy, A t l a n t i c City NJ, March 5-9, 1984; A b s t r a c t s , Paper No. 497.

32

H. S i l v i s , L.M. S i d i n s k y , W.F. F a t u l a and N.H. Mosesman, 2nd I n t . Symp. C a p i l l a r y Chromatography, Terrytown NY, October 10-12, 1983.

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J.R.P. Marco, GC N e w s l e t t e r (The Perkin-Elmer Corp.) 1 ( 3 ) (1964); see pp. 127-128 o f r e f . l c .

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N.G.

35

C.P.M. S c h u t j e s , E.A. Vermeer, J.A. R i j k s and C.A. Cramers, i n R.E. K a i s e r (Ed.), C a p i l l a r y Chromatography (1981 H i n d e l a n g Symposium), I n s t . o f Chromatography, Bad Durkheim, 1981, pp. 687-702.

36

C.P.M. S c h u t j e s , E.A. 253 (1982) 1 .

37

J.V. Hinshaw J r . , J.L. DiCesare and J.E. P u r c e l l , 3 5 t h P i t t s b u r g h Conf. A n a l . Chem. Appl. Spectroscopy, A t l a n t i c C i t y NJ, March 5-9, 1984; A b s t r a c t s , Paper No. 21.

38

J . J . Franken and G.A.T.M. R u t t e n , i n S.G. P e r r y and E.R. A d l a r d (Eds.), Gas Cgromatography 1972 (Montreux Symposium), A p p l i e d Science P u b l i s h e r s , Barking, 1973, pp. 75-88.

39

K. Grob J r . and K. Grob, Chromatographia 10 (1977) 250.

40

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D.A.M. Mackay and M.M. Hussein, i n G. Charalambous (Ed.), A n a l y s i s o f Foods and Beverages - Headsoace Technique?, Academic Press, New York NY, 1978, pp. 283-357.

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D.A.M.

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111

SELECTORS FOR CHIRAL RECOGNITION I N CHROMATOGRAPHY*

E . GILAV Department o f Organic Chemistry, The Weizmann I n s t i t u t e o f Science Rehovot 76100, I s r a e l

INTRODUCTION T h i s symposium o f f e r s an e x c e l l e n t o p p o r t u n i t y t o r e v i e w t h e c o n t r i b u t i o n s o f v a r i o u s groups o f r e s e a r c h e r s t o t h e p r o g r e s s o f chromatography. I n o u r l a b o r a t o r y , we have c o n c e n t r a t e d m a i n l y on t h e r e s o l u t i o n o f o p t i c a l isomers by p a r t i t i o n i n g processes. I w i s h t o d e s c r i b e here t h e essence of o u r c o n t r i b u t i o n , namely t h e i n t r o d u c t i o n o f v a r i o u s c l a s s e s o f s o l v e n t s which have unique c a p a c i t y f o r c h i r a l r e c o g n i t i o n . There a r e two p o s s i b l e approaches f o r s e p a r a t i n g enantiomers by chromatoOne i s based on t h e f o r m a t i o n of d i a s t e r e o m e r i c d e r i v a t i v e s . I n t h i s

graphy.

case, t h e handedness r e q u i r e d f o r r e s o l u t i o n i s i n t r o d u c e d c o v a l e n t l y i n t o t h e o p t i c a l l y a c t i v e molecules.

a-Amino a c i d s , f o r i n s t a n c e , a r e e s t e r i f i e d w i t h

an asymmetric a l c o h o l , o r an asymmetric a c y l group i s a t t a c h e d t o t h e n i t r o g e n . I t i s a method which was expected t o work w e l l , as i t i s n o t h i n g b u t a v a r i a n t

o f P a s t e u r ' s c l a s s i c a l approach t o t h e r e s o l u t i o n v i a c r y s t a l l i z a t i o n o f d i a stereomeric s a l t s .

Corresponding a n a l y t i c a l procedures have been w i d e l y and

s u c c e s s f u l l y used, though t h e y have c e r t a i n i n h e r e n t disadvantages ( r e f . 1 ) . Though we d i d some r e s e a r c h on d i a s t e r e o m e r s , we focused o u r e f f o r t s essent i a l l y on t h e second a l t e r n a t i v e , where t h e handedness needed f o r r e s o l u t i o n i s p r o v i d e d by a n o p t i c a l l y a c t i v e environment i n t h e phase, and n o t i n t r o d u c e d c o v a l e n t l y i n t o t h e molecules t o be separated.

When we s t a r t e d t h i s work i n

1965, nobody b e l i e v e d t h a t i t c o u l d be done. I n f a c t , most s c i e n t i s t s i n t h e f i e l d were convinced t h a t t h e r e c o u l d n o t p o s s i b l y o c c u r a s u f f i c i e n t l y l a r g e energy d i f f e r e n c e i n t h e i n t e r a c t i o n between t h e

D- and L - s o l u t e w i t h an asym-

metric solvent.

T h i s was t h e f e e l i n g o f even those known as unorthodox t h i n k e r s T h i s view had some e x p e r i m e n t a l b a s i s , because a number o f communications had been p u b l i s h e d , i n which i t was c l a i m e d t h a t such r e s o l u t i o n s c o u l d be e f f e c t e d , b u t nobody was a b l e t o reproduce these r e s u l t s , and some o f them were shown t o

be d e f i n i t e l y wrong.

* T h i s r e v i e w i s d e d i c a t e d t o P r o f e s s o r A.J.P. M a r t i n w i t h t h e e x p r e s s i o n o f greatest admiration f o r h i s c o n t r i b u t i o n s o f genius t o science.

112 Ifhen we s t a r t e d o u r work w i t h B. Feibush ( r e f . 2 ) and R. C h a r l e s ( r e f . 3 ) we based o u r s e l v e s on t h e f o l l o w i n g two i d e a s .

F i r s t o f a l l , n a t u r e can do i t ,

enzymes d i f f e r e n t i a t e between enantiomers.

T h e r e f o r e , we t h o u g h t o f examining

systems which had some o f t h e p r o p e r t i e s o f an enzyme.

I n o t h e r words, we

decided t o t r y phases w i t h -CO- and -NH- f u n c t i o n s grouped around an asymmetric c e n t e r , capable o f f o r m i n g hydrogen bonds w i t h s u i t a b l e s o l u t e s , i .e. d e r i v a t i v e s of a-amino a c i d s .

Secondly, we reasoned t h a t we had t o a m p l i f y t h e

e f f e c t , because we expected i t t o be v e r y s m a l l .

T h i s meant t h e use of l o n g

capillaries. T h i s approach l e d t o immediate success. was N - t r i f l u o r o a c e t y l (TFA)

-

Our f i r s t s t a t i o n a r y c h i r a l phase

L - i s o l e u c i n e l a u r y l e s t e r on which t h e N-TFA-a-

amino a c i d i s o p r o p y l e s t e r s were separated ( r e f s . 3, 4 ) . Since t h e r e s o l u t i o n o f o p t i c a l isomers by g.1.c.

on asymmetric phases was a t

t h e t i m e a c o n t r o v e r s i a l m a t t e r , we had t o produce evidence t h a t what we had separated were indeed enantiomers, and n o t , f o r i n s t a n c e , some k i n d o f a r t i f a c t s . As we worked w i t h c a p i l l a r i e s , we c o u l d n o t do t h e obvious t h i n g , i . e . s e p a r a t e t h e compounds c o r r e s p o n d i n g t o t h e peaks and measure t h e i r o p t i c a l r o t a t i o n .

So

we prepared m i x t u r e s o f D- and L - a - a c i d s i n v a r y i n g r a t i o s and measured t h e peak areas.

They were found t o be p r o p o r t i o n a l t o t h e amounts o f t h e r e s p e c t i v e i s o -

mers ( s e e F i g . 1 , upper c u r v e ) .

Then we r e v e r s e d t h e c o n f i g u r a t i o n o f o u r phase

by u s i n g t h e corresponding d e r i v a t i v e o f

D - i s o l e u c i n e , and i n j e c t e d t h e same

sample of e n r i c h e d m i x t u r e s as above ( F i g . 1, l o w e r c u r v e ) .

We expected a r e -

v e r s a l of t h e o r d e r o f t h e l a r g e and small peaks, as compared w i t h t h e upper curve, and t h i s indeed t o o k p l a c e .

The two curves a r e n o t , i n c i d e n t a l l y , m i r r o r

images of each o t h e r , as t h e y should be, because o u r column c o a t i n g t e c h n i q u e was n o t y e t good enough and each c a p i l l a r y had a somewhat d i f f e r e n t b e h a v i o u r .

A t a l a t e r stage, when we had developed phases e f f i c i e n t enough t o produce good r e s o l u t i o n on packed columns, we a l s o c a r r i e d o u t p r e p a r a t i v e s e p a r a t i o n s . ORD measurements proved d e f i n i t e l y t h e e n a n t i o m e r i c n a t u r e o f t h e f r a c t i o n s

collected (ref. 5).

A s mentioned above, i t had been assumed a t one t i m e t h a t e n a n t i o m e r i c d i f f e r e n t a t i o n by c h i r a l s o l v e n t s , i f a t a l l t a k i n g p l a c e , would be v e r y s m a l l .

It i s

perhaps t h e most i m p o r t a n t r e s u l t o f o u r experiments t o have demonstrated t h a t , q u i t e on t h e c o n t r a r y , t h e r e a r e c e r t a i n s e l e c t o r / s e l e c t a n d p a i r s which have relatively large resolution coefficients . amide N-TFA-isopropyl

Thus, on N-1 a u r o y l -L-Val i n e - t - b u t y l -

e s t e r s o f l e u c i n e have a"z1.3

a t 13OoC ( r e f . 6 ) , which on

e x t r a p o l a t i o n t o O°C l e a d s t o a v a l u e o f a=3.5-4.0,

corresponding t o A A G = ~ . ~

Cal/mole.

C o n s i d e r a b l y h i g h e r c o e f f i c i e n t s were o b t a i n e d i n systems i n v o l v i n g

*a=resol u t i o n c o e f f i c i e n t s = r I I / I = c o r r e c t e d r e t e n t i on t i q e o f t h e enantiomer emerging l a s t o v e r t h a t emerging f i r s t , which a c c o r d i n g t o t h e case may be rL/d O r ~ D / L .

113 L-Phase

F i g . 1 . Chromatogram o f enantiomers o f N-TFA- cr-aniino a c i d i s o p r o p y l e s t e r s w i t h N-TFA-L-isoleucine l a u r y l e s t e r (L-phase) and N-TFA-D-isoleucine l a u r y l e s t e r (D-Phase), r e s p e c t i v e l y , as t h e s t a t i o n a r y 1 i q u i d . L-Phase: column loom, 98,000 p l a t e s w i t h r e s p e c t t o N-TFA-alanine i s o p r o p y l e s t e r . D-Phase: column 72m, 57,000 p l a t e s w i t h r e s p e c t t o N-TFA-D-alanine i s o p r o p y l e s t e r . Temperature 9OOC, c a r r i e r gas, n i t r o g e n p r e s s u r e , 2Op.s.i. The e s t e r s were d e r i v e d from amino a c i d s c o n s i s t i n g o f unequal amounts o f enantiomers. c o o r d i n a t i o n w i t h m e t a l s (see below).

I t s h o u l d be p o i n t e d o u t t h a t t h e s e l e c -

t i v i t y i n c h i r a l chromatographic p a r t i t i o n i n g systems i s approaching t h a t f o u n d

i n h o s t - g u e s t i n t e r a c t i o n s i n v o l v i n g c h i r a l crown e t h e r s prepared from t h e rigid 2,2-dihydroxy-1 , l ' - b i n a p h t h y l

by Cram e t a l . ( r e f . 7 ) .

Another s t r i k i n g f e a t u r e o f r e c e n t r e s e a r c h concerns t h e scope o f t h e phenomenon.

Indeed a v a r i e t y o f c l a s s e s o f compounds have been found t o show s t e r e o -

s e l e c t i v e i n t e r a c t i o n s and new examples o f c h i r a l d i f f e r e n t i a t i o n a r e c o n s t a n t l y b e i n g found i n v o l v i n g b o t h s e l e c t o r / s e l e c t a n d p a i r s o f known and y e t unknown types.

Here I s h a l l d i s c u s s t h o s e types o f c h i r a l s e l e c t o r molecules which have

been p i o n e e r e d i n o u r l a b o r a t o r y . SELECTOR TYPES R e s o l u t i o n o f o p t i c a l isomers by chromatography i s a s c r i b e d t o t h e r a p i d and r e v e r s i b l e f o r m a t i o n i n t h e column 3 f d i a s t e r e o m e r i c complexes between t h e c h i r a l component o f t h e phase ( s e l e c t o r ) and t h e c h i r a l s o l u t e s ( s e l e c t a n d s ) .

I f these s e l e c t o r - s e l e c t a n d a s s o c i a t e s d i f f e r s u f f i c i e n t l y i n t h e i r f r e e energy o f formation, the r e s u l t i n g d i f f e r e n c e s i n p a r t i t i o n i n g c o e f f i c i e n t s w i l l l e a d t o t h e s e p a r a t i o n o f t h e enantiomers.

I t has been shown t h a t such s t e r e o s e l e c t i v e complexes can be based on d i f f e r e n t types o f intermol e c u l a r f o r c e s , e . g . hydrogen bonding , charge t r a n s f e r (CT)

114

complexation, c o o r d i n a t i o n t o m e t a l s , i o n i c and d i p o l e - d i p o l e a t t r a c t i o n s , o r combinations o f two o r more o f such i n t e r a c t i o n s .

I n our l a b o r a t o r y , we have

d e a l t w i t h systems based e s s e n t i a l l y on t h e f i r s t t h r e e types o f i n t e r a c t i o n s . S e l e c t o r s o f t h e hydroqen-bondi ng t y p e

As d i s c u s s e d i n t h e i n t r o d u c t i o n , t h e f i r s t phase on which enantiomers were r e s o l v e d , gas c h r o m a t o g r a p h i c a l l y c o n s i s t e d o f d e r i v a t i v e s o f a-amino a c i d s capa b l e o f f o r m i n g hydrogen bonds w i t h s u i t a b l e s o l u t e s .

They were N-acyl e s t e r s

of n a t u r a l l y o c c u r r i n g a-amino a c i d s h a v i n g t h e g e n e r a l f o r m u l a :

a where R ' = a1 k y l o r c y c l o a l k y l . Though these compounds had p e r m i t t e d t o demonstrate t h e v e r y f e a s i b i l i t y o f c h i r a l d i f f e r e n t a t i o n by asymmetric s o l v e n t s , t h e y were n o t v e r y e f f i c i e n t , * and were soon superceded by o t h e r d e r i v a t i v e s . I n p u r s u i t o f o u r s t u d i e s , we i n v e s t i g a t e d t h e e f f e c t o f t h e v a r i a t i o n o f t h e s t r u c t u r e on t h e s t e r e o s e l e c t i v i t y o f phases o f t y p e

a.

We r e c o g n i s e d i n

p a r t i c u l a r , t h e i n f l u e n c e o f t h e b u l k o f t h e groups R and R ' , t h e i n c r e a s e of which has an enhancing e f f e c t ( e . g . i P r , i B u , p h e n y l ) , p r o v i d e d a c e r t a i n s i z e i s n o t exceeded ( r e f . 4 ) .

L a t e r , we t h o u g h t o f a n o t h e r way o f r a i s i n g t h e r e s -

o l u t i o n f a c t o r s , namely, t h e i n t r o d u c t i o n o f a second -NH- group i n t o t h e molec u l e t o i n c r e a s e t h e number o f hydrogen bonds w i t h t h e s o l u t e .

Dipeptide deriv-

a t i v e s have t h i s f e a t u r e and gave f a r b e t t e r r e s o l u t i o n than p r e v i o u s l y achieved.

A t y p i c a l r e p r e s e n t a t i v e o f t h i s c l a s s o f s o l v e n t s , i n c o r p o r a t i n g t h e experi e n c e c o n c e r n i n g t h e e f f e c t o f t h e b u l k o f t h e s u b s t i t u e n t s , i s t h e N-TFA-L-valy l - L - v a l i n e cyclohexyl e s t e r ( r e f . 8).

The

CL

values observed on t h i s phase were

o f t e n o f t h e o r d e r o f 1.1-1.2 o r even h i g h e r , a t l l O ° C

( r e f . 9, F i g . 2 ) .

The

general f o r m u l a o f these s o l v e n t s i s :

where i n general R ' = c y c l o h e x y l

*

cg

R e s o l u t i o n c o e f f i c i e n t s were o f t h e o r d e r o f 1.02-1.05 f o r N-TFA-a-amino a c i d e s t e r s a t 9OOC ( r e f . 4 ) **llc511 and llC711 stand f o r c y c l e - 5 and -7, r e s p e c t i v e l y , and i n d i c a t e t h e number of atoms i n t h e r e s p e c t i v e m o i e t i e s capable o f f o r m i n g hydrogen bonded r i n g s w i t h s u i t a b l e solutes.

115

150

I20

90

60

30

0

TIME (MINUTES) F i g . 2. Chromatogram o f t h e N-TFA i s o p r o p y l e s t e r s o f t h e r e l a t i v e l y v o l a t i l e amino a c i d s on a 500 f t column a t 1lOOC. I t was shown by C o r b i n e t a l . ( r e f . 1 0 ) and S. W e i n s t e i n e t a1 ( r e f . 1 1 ) t h a t

t h e N - t e r m i n a l amino a c i d s i n f l u e n c e t h e s t e r e o s e l e c t i v i t y f a r more s t r o n g l y than t h e C-terminal ones (see, a l s o below, s e c t i o n on d i a m i d e s ) .

Some a t t e m p t s

were made t o t e s t t h e b e h a v i o u r o f c o r r e s p o n d i n g t r i p e p t i d e s ( d e r i v e d from ( v a l ) 3 ( r e f . 8), and from (leu),

( r e f . 10).

However, i t t u r n e d o u t t h a t t h e

e x t e n s i o n o f t h e p e p t i d e c h a i n somewhat decreases t h e s e l e c t i v i t y . Q u i t e a number o f i n v e s t i g a t o r s c o n t r i b u t e d subsequently t o t h e development Of

t h i s t y p e o f s o l v e n t s , s u b s t i t u t i n g d i f f e r e n t amino a c i d s f o r ( v a 1 ) p ( r e f s .

12-14).

The purpose o f these a u t h o r s was t o i n c r e a s e t h e thermal s t a b i l i t y of

t h e phases, w h i l e c o n s e r v i n g t h e i r s e l e c t i v i t y . C e r t a i n progress was indeed made and column temperatures o f e.g. asp phase, r e f . 1 4 ) , were r e p o r t e d .

Smith and !lonnacott ( i 5 )

165OC (phe-

r e p l a c e d N-TFA i n

t h e ( v a l ) 2 d e r i v a t i v e by N-octadecanoyl and mixed i t w i t h a diamide; t h e y c a r r i e d o u t s e p a r a t i o n s a t 170OC. phases, t y p e

W i t h t h e advent o f t h e n e x t g e n e r a t i o n o f

s o l v e n t s were, however l a r g e l y abandoned.

I t i s i n t e r e s t i n g t o m e n t i o n t h a t Koenig e t a l . ( r e f . 1 6 ) demonstrated t h a t

N-TFA4L-pro)p-0- c y c l o h e x y l p e r m i t s t o r e s o l v e t h e N-TFA i s o p r o p y l e s t e r o f p r o line.

T h i s o b s e r v a t i o n means t h a t s t e r e o s e l e c t i v i t y i s achieved s o l e l y by

d i p o l e - d i p o l e i n t e r a c t i o n s , as no hydrogen bond can form i n t h i s case between s o l v e n t and s o l u t e . Ten t o f i f t e e n y e a r s ago, phases o f t y p e

were i n use, m a i n l y f o r t h e

e n a n t i o m e r i c a n a l y s i s o f amino a c i d s , e.g. i n m e t e o r i t e a n a l y s i s o r i n t h e hydrolysates o f s y n t h e t i c peptides.

A l s o , s o l u t e s o t h e r than a-amino a c i d s

116

a r e r e s o l v e d on t y p e

b solvents

and, e s p e c i a l l y , p r i m a r y a-amino a l o c h o l s i n

t h e form o f t h e i r N-TFA 0 - a c y l e s t e r s .

A d i f f e r e n t l i n e o f development was pursued by O i e t a l . ( r e f . 16) who l i n k ed two d i - o r t r i p e p t i d e r e s i d u e s t o a c e n t r a l m u l t i f u n c t i o n a l nucleus (1,3,5t r i a z i n e ) and t h e r e b y i n c r e a s e d t h e m o l e c u l a r w e i g h t and thermal s t a b i l i t y t o form molecules c l o s e l y r e l a t e d t o t y p e

b.

F o r i n s t a n c e , N,Ni-[2,4-(6-ethoxy-1-

3 - 5 - t r i a z i n e ) i d y l I b i s ( L - v a l y l -L-Val ine i s o p r o p y l e s t e r ) (OA-.200) and N , N ' - [ 2 , 4

( 6-ethoxy-l,3,5-triazine)diyl]bis ( L - v a l y l - L - v a l y l -L-Val i n e i s o p r o p y l e s t e r (On 300) were i n t r o d u c e d ( o p e r a t i n g temperatures mentioned reached 160OC f o r OA300).

I t i s noteworthy t h a t O i e t a l . r e p o r t e d u n i q u e r e s o l u t i o n s on these

phases, e.g. t h a t o f f r e e a l c o h o l s and o f a c i d amides w i t h an u n d e r i v z t i z e d ahydroxy group ( r e f s . 16, 1 7 ) . Another c l a s s o f phases h a v i n g t h e s t r u c t u r e

c turned

o u t t o be s t i l l more

C -

e f f i c i e n t t h a n t h e d i p e p t i d e d e r i v a t i v e s and a p p l i c a t i v e t o a wide v a r i e t y o f solutes.

The h i g h e s t a values a r e , i n g e n e r a l , f o u n d f o r a-amino a c i d and 2-

amino a l c o h o l d e r i v a t i v e s .

Diamides were f i r s t mentioned i n a p a t e n t ( r e f . 1 8 )

and e x p e r i m e n t a l d a t a p r e s e n t e d i n t h e s c i e n t i f i c l i t e r a t u r e i n 1971 by Feibush ( r e f . 19). Considerable r e s e a r c h was c a r r i e d o u t i n o u r l a b o r a t o r y on t h e s t r u c t u r a l e f f e c t s o f R1, R 2 and R3, and t h e r e s u l t s were r e c e n t l y summarized i n a r e v i e w article (ref. 6).

An i m p o r t a n t f i n d i n g was t h a t a t - b u t y l m o i e t y a t t a c h e d t o

t h e n i t r o g e n i n R3 [-N-C(CH3)2CH2-] selectivity.

i s p a r t i c u l a r l y e f f e c t i v e i n promoting

Another i m p o r t a n t f e a t u r e i s t h a t l e n g t h e n i n g o f t h e a l k y l c h a i n s

i n e i t h e r R 1 o r R 3 reduces t h e e f f i c i e n c y o f t h e phases, c o r r e s p o n d i n g t o t h e decrease o f t h e r e l a t i v e importance o f t h e " a c t i v e s i t e " i n t h e m o l e c u l e .

As t o

R2, i t was found t h a t i s o p r o p y l ( V a l ) , i s o b u t y l ( l e u ) and b e n z y l (phe) gave t h e best solvents.

The l e u c i n e d e r i v a t i v e s o f t e n exceed t h e performance o f t h e v a l -

i n e phases, which f o r a l o n g t i m e had been c o n s i d e r e d t o g i v e t h e h i g h e s t stereoselectivi ty.

These diamides may have such h i g h s e l e c t i v i t y f o r a-amino

a c i d d e r i v a t i v e s t h a t r e s o l u t i o n can be r e a d i l y achieved on packed columns ( r e f . 20).

T h i s can be more c o n v e n i e n t i n some cases t h a n t h e use o f c a p i l l a r y

117 columns, as g e n e r a l l y p r a c t i c e d i n g.c.1.

enantiomer a n a l y s i s ( F i g . 3 ) .

columns a l s o p e r m i t t h e c a r r y i n g o u t o f p r e p a r a t i v e s e p a r a t i o n s , e.g.

Packed

f o r peak

id e n t i f ic a t i o n purposes.

L Y

0

5

10

I5

20

25

30

35

TIME Im~nuleil

F i g . 3 . Chromatogram o f t h e N-TFA i s o p r o p y l e s t e r s o f a l a , Val and l e u ( e n r i c h ed i n t h e L - i s o m e r ) . Column: 2.1 m x 4 mm. S t a t i o n a r y phase: 10% - N - l a u r o y l L-Val ine t - b u t y l a m i d e , c o a t e d on 60-80 mesh Chromosorb W-AW, 130OC. With l o n g c h a i n R1 and R3 groups as, f o r i n s t a n c e , i n N-docosanoyl-L-valine-

2-(2-methyl)-heptadecylamide, columns have been r u n a t temperatures as h i g h as 2OO0C ( r e f . 21) ( F i g . 4 ) . As f o r many o t h e r t y p e s o f s o l v e n t s , constancy o f t h e o r d e r o f emergence f o r a g i v e n c l a s s o f compounds has been found t h r o u g h o u t (e.g. f o r N-acyl a-amino e s t e r s on L-phases).

L - a f t e r t h e D-isomer

Thus, t h e o r d e r o f e l u t i o n can s e r v e

f o r t h e d e t e r m i n a t i o n o f t h e c o n f i g u r a t i o n o f unknown compounds. Type 4, h a v i n g been r e c o g n i z e d as p a r t i c u l a r l y e f f i c i e n t , has been mod f i e d i n a v a r i e t y o f ways by a s e r i e s o f a u t h o r s .

The diamide m o i e t y ( d e r i v e d from

V a l ) has been i n t r o d u c e d i n p o l y m e r i c backbones by Frank e t a l . ( r e f . 22) e t a l . ( r e f . 23) and Koenig e t a1 . ( r e f . 2 4 ) .

O i e t a1

.

Saad

( r e f . 17) have used

t h e p r i n c i p l e mentioned above, namely t h e l i n k a g e t o a c e n t r a l s - t r i z z i n e r i n g , t o synthesize t h e solvent

N ,N ' ,N"-[2,4,6-(

113,5-triazine) t r i y l] t r i s (N-lauroyl-

L - l y s i n e t - b u t y l a m i d e ) (OA-400). These m o d i f i c a t i o n s have l e d t o v e r y good phases, though on t h e whole, l e s s s e l e c t i v e than t h e o r i g i n a l t y p e

c compounds.

The thermal s t a b i l i t y o f some of

them i s e s p e c i a l l y h i g h , and t h e y have found wide a p p l i c a t i o n .

Except f o r t h e

c l a s s i c a l r e s o l u t i o n s o f t h e d e r i v a t i v e s o f a-amino a c i d s and 2-amino a l c o h o l s , many a d d i t i o n a l c l a s s e s o f compounds, e.g. a-hydroxy c a r b o x y l i c a c i d s , TFA

118

TEMPERATURE 195°C I

LYS

F i g . 4. Chromatogram o f N-TFA i s o p r o p y l e s t e r s o f o r n i t h i n e and l y s i n e . Column, s t a i n l e s s s t e e l c a p i l l a r y , 50 m x 0.5 mm, coated w i t h N-docosanoyl-L-valine-2- (2-methyl )-n-heptadecyl amide. d e r i v a t i v e s o f sugars, e s t e r s o f a r o m a t i c d i o l s , a r o m a t i c d i k e t o n e s , and a

methylphosphonofluoridate [MeFP0.0CH(CH3)C(CH3)3, Soman] have been separated on them.

R e s o l u t i o n s o f v a r i o u s c l a s s e s o f s o l u t e s on t y p e

c phases

have r e c e n t l y

been reviewed ( r e f s . 24, 2 5 ) . There have a l s o been r e l e v a n t developments i n HPLC.

By r e a c t i n g N-acyl-L-

v a l i n e w i t h aminated s i l i c a g e l , c h i r a l s u p p o r t s have been prepared by Dobashi e t a l . ( r e f . 27) on which a s e r i e s o f N-acyl e s t e r s o f a-amino a c i d s were e f f i c i e n t l y resolved. I n t h e same l a b o r a t o r y , Oobashi and Hara ( r e f . 28) have a l s o shown t h a t N-

acetyl-L-valine-t-butylamide can s e r v e as a c h i r a l a d d i t i v e t o t h e i n o b i l e phase (n-hexane-CHC13) f o r r e s o l v i n g N - a c e t y l - t - b u t y l

on s i l i c a g e l .

e s t e r s o f v a r i o u s a-amino a c i d s

Thus y e t a n o t h e r mode o f a p p l i c a t i o n o f t y p e

4s o l v e n t s

for

c h i r a l r e c o g n i t i o n has been demonstrated. The mechanism o f c h i r a l r e c o g n i t i o n f o r a-amino a c i d d e r i v a t i v e s i s g e n e r a l l y assumed t o i n v o l v e e s s e n t i a l l y t h e hydrogen bonded s o l v e n t - s o l u t e p a i r s shown i n F i g . 5.

Such a s s o c i a t i o n s have been proposed i n analogy t o t h e p l e a t e d s h e e t

B - s t r u c t u r e found i n p r o t e i n s .

Arguments i n s u p p o r t o f t h i s mechanism have

119

F i g . 5 . Scheme o f t h e hydrogen bonded a s s o c i a t i o n o f a N - a c y l - L - v a l i n e t - b u t y l amide phase w i t h a N-TFA i s o p r o p y l e s t e r o f an L-a-amino a c i d . The c o r r e s ponding L - s o l v e n t D - s o l u t e a s s o c i a t i o n i s l e s s s t a b l e and has l o w e r r e t e n t i o n time. been discussed i n r e f . 6.

However, as mentioned above, v a r i o u s c l a s s e s o f com-

pounds which cannot f o r m t h e proposed hydrogen bonded r i n g s , a r e a l s o r e s o l v e d

on diamides.

Thus, o t h e r s o l v e n t - s o l u t e a s s o c i a t e s , l e a d i n g t o c h i r a l d i f f e r -

e n t i a t i o n , must e q u a l l y occur.

I n t e r c a l a t i o n models o f t h e s o l v e n t - s o l u t e -

s o l v e n t t y p e a r e one o f t h e forms which such i n t e r a c t i o n s The diamides have a s i m p l e r c o n s t i t u t i o n t h a n t y p e

could take.

b solvents.

They c o n t a i n

o n l y one asymmetric c e n t e r , and f u r t h e r m o r e , have one C 7 and one C 5 m o i e t y , whereas t h e d i p e p t i d e d e r i v a t i v e s possess one C7 and

two C 5

sides.

I t should

be n o t e d t h a t i n o t h e r cases a l s o , e.g. t h a t o f diamides d e r i v e d f r o m t r i f u n c t i o n a l amino a c i d s ( s e r , asp) ( r e f . 2 9 ) , t h e i n t r o d u c t i o n o f t h e t h i r d p o l a r group was found t o be unfavourable.

Probably, competitive formation o f l e s s

s t e r e o s e l e c t i v e a s s o c i a t e s w i t h t h e s o l v e n t reduces t h e o v e r a l l e f f i c i e n c y . On t h e b a s i s o f t h e above r e s u l t s , we made a search f o r t h e s i m p l e s t s t r u c -

120

t u r e s capable o f c h i r a l r e c o g n i t i o n . An amide group l i n k e d t o an asymnetric c e n t e r t h r o u g h carbon seemed an essent i a l feature.

Then, a l o n g a c y l group was a t t a c h e d t o t h e c a r b o n y l ( N - l a u r o y l ) t o make t h e phases s u f f i c i e n t l y n o n - v o l a t i l e f o r gas chromatography. F u r t h e r , we chose t o a t t a c h a hydrogen and a methyl t o t h e asymmetric carbon, and examined a s e r i e s o f s i m p l e groups as t h e f o u r t h s u b s t i t u e n t o f t h a t atom. I t t u r n e d o u t t h a t an a r o m a t i c group o f s u f f i c i e n t s i z e , such as naphthalene, gave t h e desired e f f e c t ( r e f . 30).

Thus, t y p e

s o l v e n t was developed:

T h i s phase was p a r t i c u l a r l y e f f i c i e n t f o r t h e r e s o l u t i o n o f N-acyl amines and o f a - s u b s t i t u t e d c a r b o x y l i c a c i d s , XCH(R)C02H, i n t h e form o f t h e i r N - t - b u t y l d e r i v a t i v e s (e.q. X = a - h a l o , a - a l k y l , a-phenyl) ( r e f s . 30, 3 1 ) . X-ray spectroscopy showed t h a t t h e s e l e c t o r arranges i t s e l f i n t h e c r y s t a l l i n e s t a t e , as w e l l as i n t h e m e l t , i n a 5-8 t r a n s l a t i o n a l a r r a y , as shown i n F i g . 6, w i t h t h e hydrogen bonds i n t h e p l a n e o f t h e paper and t h e n a p h t h y l r i n g s oriented perpendicularly t o i t ( r e f . 32).

I n t e r c a l a t i o n i n t o t h e hydrogen bond-

ed a r r a y o f t h e s e l e c t o r m a t r i x i s assumed t o e x p l a i n t h e mechanism of r e s o l u t i o n , i l l u s t r a t e d i n F i g . 6 f o r N-TFA-a-phenylethylamine h o s t and g u e s t have t h e same c o n f i g u r a t i o n ,

as t h e s o l u t e .

When

t h e o r i g i n a l m o t i f o f hydrogen bond-

i n g i s preserved, t h e a r o m a t i c groups make p l a n e - t o - p l a n e c o n t a c t s and t h e hydrogen atom l i n k e d t o t h e asymmetric carbon i s wedged between two a r o m a t i c r i n g s as i n t h e o r i g i n a l s t a c k .

The methyl group, on t h e o t h e r hand, p o i n t s

away f r o m t h e s t a c k ( F i g . 6 a ) . F o r unequal c o n f i g u r a t i o n of h o s t and g u e s t ( F i g . 6b), i n t e r c a l a t i o n , w h i l e m a i n t a i n i n g t h e h o s t s t r u c t u r e , r e q u i r e s t h a t t h e methyl group be wedged between t h e two n a p h t h y l groups, l e a d i n g t o severe overcrowding, t h a t i s , a worse f i t between h o s t and guest t h a n f o r t h e RR combination. These p r e d i c t i o n s o f t h e model a r e i n agreement w i t h t h e observed o r d e r o f emergence (R a f t e r t h e S-solUte on t h e R phase). O t h e r arguments and evidence s u p p o r t i n g t h e i n t e r c a l a t i o n model a r e g i v e n i n r e f . 31. A f t e r t h e s t e r e o s e l e c t i v i ty o f N - l a u r o y l - R ( o r S ) - a - ( 1 - n a p h t h y l ) e t h y l a m i n e was demonstrated, v a r i o u s m o d i f i c a t i o n s o f t h i s t y p e o f s o l v e n t appeared i n t h e literature.

Thus, O i e t a l . have d e r i v a t i z e d t h e amine f u n c t i o n w i t h an asym-

121

a

b

F i g . 6. A c h i r a l g u e s t m o l e c u l e , N-TFA-a-phenylethylamine, i n s e r t e d i n t o t h e Hbonded s t a c k of (R)-N-lauroyl-a-(1-naphthy1)ethylamine: ( a ) g u e s t o f R c o n f i g u r a t i o n ; ( b ) g u e s t of S c o n f i g u r a t i o n . When e s t i m a t i n g t h e i n t e r m o l e c u l a r d i s tances, i t s h o u l d be k e p t i n mind t h a t t h e atoms a r e designed w i t h o u t t h e i r Van der Waals r a d i i . metric

e * g . 1R,3R-trans-chrysanthemic

a c i d , O-lauroyl-mandel i c a c i d o r N-

l a u r o y l -L-pro1 i n e ( r e f s . 33,34) and Thumm ( r e f . 3 5 ) has i n t r o d u c e d t h e amino m o i e t y i n t o a P o l y s j l i c o n e backbone.

Except f o r r a i s i n g thermal s t a b i l i t y ,

these phases have p e r m i t t e d t h e e x t e n t i o n o f r e s o l u t i o n t o new c l a s s e s of compounds, such as n i t r i l e s and a l c o h o l s (e.g. m e n t h o l ) . Condensation o f an a-amino a c i d e s t e r w i t h phosgene l e d t o t h e f o r m a t i o n o f type

g solvents. R

-e

R

0 I

H

H

I,

0

122

The p a r t i c u l a r phase which we s t u d i e d was d e r i v e d f r o m L - v a l i n e i s o p r o p y l e s t e r , and was found t o be s t e r e o s e l e c t i v e f o r N-TFA d e r i v a t i v e s o f amines, amino a l c o h o l e s t e r s and a-, 6- and

y-amino a c i d e s t e r s .

The a v a l u e s r e p o r t e d

f o r t h e s e s o l u t e s a r e , i n g e n e r a l , o f t h e o r d e r o f 1.02-1.05

a t 12OoC ( r e f . 361,

except f o r t h e N-acyl d e r i v a t i v e s o f 2-aminoethyl benzene and t h e 4-phenyl-2aminobutane, which show much h i g h e r r e s o l u t i o n c o e f f i c i e n t s ( r e f . 37, see a l s o be1 ow)

.

The phase has two o u t s t a n d i n g f e a t u r e s :

The o r d e r o f emergence o f t h e s o l -

u t e s i s n o t u n i q u e l y determined by t h e c o n f i g u r a t i o n , as i s t h e case f o r a l l other solvents studied, b u t

i s a l s o i n f l u e n c e d by t h e r e l a t i v e s i z e o f t h e sub-

s t i t u e n t s a t t h e asymmetric carbon.

Thus, t h e m e t h y l e s t e r s o f t h e a-amino

a c i d s i n t h e L - s e r i e s change t h e i r o r d e r o f e l u t i o n w i t h t h e s i z e o f t h e s i d e group i n t h e a - p o s i t i o n . empirical rule:

These f i n d i n g s have been summarized by t h e f o l l o w i n g

I f t h e s u b s t i t u e n t s a t t h e asymmetric carbon have a c l o c k w i s e

L

arrangement ( + L A ) * i n o r d e r o f d e c r e a s i n g s i z e , t h e c o r r e s p o n d i n g compound emerges l a s t on t h e L-phase.

T h i s i s a u s e f u l r e l a t i o n s h i p which g i v e s more

w e i g h t t o t h e d e t e r m i n a t i o n o f c o n f i g u r a t i o n by t h e chromatographic b e h a v i o u r . 2-Amino-3-methyl butane and 2-amino-4-methyl pentane, on t h e s t e r e o c h e m i s t r y o f which t h e r e were c o n f l i c t i n g r e p o r t s , have been p r o p e r l y c o r r e l a t p d w i t h t h e i r s i g n o f r o t a t i o n by t h i s procedure ( r e f . 38). The o t h e r unusual f e a t u r e i s t h a t below t h e m e l t i n g p o i n t , t h e r e i s s t i l l a r e l a t i v e l y l a r g e r e t e n t i o n o f t h e s o l u t e which i s accompanied by c h i r a l d i f f e r e n t i a t i o n w i t h r e s o l u t i o n c o e f f i c i e n t s l a r g e r t h a n found i n t h e m e l t . behaviour was f i r s t r e p o r t e d by C o r b i n and Rogers ( r e f . 3 9 ) .

This

Subsequently,

Lochmueller and S o u t e r ( r e f s . 37,40) have produced evidence f o r t h e f o r m a t i o n o f smetic phases i n t h e p e r t i n e n t temperature range, and have l i n k e d t h e above phenomena w i t h t h e occurrence o f mesophases.

I t i s noteworthy t h a t a r e s o l u t i o n

c o e f f i c i e n t as h i g h as 2.205 has been observed on t h e i s o p r o p y l v a l i n e phase bel o w t h e m e l t i n g p o i n t ( a t 93OC).

Furthermore, t h e r e i s a p a r t i c u l a r l y l a r g e

d i f f e r e n c e i n t h e shape o f t h e e n a n t i o m e r i c peaks i n t h e mesophase r e g i o n .

Such

d i f f e r e n c e s have sometimes been observed f o r i s o t r o p i c phases; f o r t h e mesophases, t h e y are, however, much more pronounced, w i t h t h e more r e t a i n e d compound showing a c o n s i d e r a b l y reduced t h e o r e t i c a l p l a t e number. Lochmueller and S o u t e r ( r e f s . 37, 40) have i n v e s t i g a t e d a s e r i e s o f t y p e g compounds w i t h R=iBu and R'=Me, E t and t-Bu.

The c o r r e s p o n d i n g d e r i v a t i v e s be-

have s i m i l a r l y t o t h e i s o p r o p y l v a l i n e compound, though t h e v a r i o u s e f f e c t s mentioned a r e l e s s marked.

*L, M and S symbolize, as u s u a l , t h e l a r g e , medium and small s u b s t i t u e n t , respectively.

123 S e l e c t o r s w i t h charqe t r a n s f e r i n t e r a c t i o n

R( - ) - 2 - ( 2,4,5,7-Tetrani

L)

t r o - 9 - f l u o r y l ideneami nooxy) p r o p i o n i c a c i d (TAPA)

i s a r e a g e n t which has been engineered by Newman ( r e f . 41) f o r t h e

(type r e s o l u t i o n o f hexahelicene.

TAPA has been w i d e l y used f o r t h e s p l i t t i n g o f

racemates by c r y s t a l l i z a t i o n .

P a r t i a l r e s o l u t i o n o f 1-naphthyl-2-butyl

ether

by LC on an alumina column has been r e p o r t e d by Klemm and Reed ( r e f . 42) a l ready i n 1960 ( s e e a l s o r e f . 4 3 ) .

P a r a l l e l w i t h o u r experiments combining t h e

use o f TAPA w i t h modern HPLC, s i m i l a r work has been c a r r i e d o u t by H. Wynberg ( r e f . 44). Our c o n t r i b u t i o n t o t h e t o p i c c o n s i s t e d o f d e m o n s t r a t i n g t h e e f f i c i e n t r e s o l u t i o n o f a range o f p o l y a r o m a t i c compounds, i n c l u d i n g a l l t h e known carboh e l i c e n e s , p a r t i a l l y hydrogenated h e l i c e n e s , 1 , 1 2 - s u b s t i t u t e d benzo[c]phenanthrenes, 3,4,5,6-tetramethylphenanthrene,

-

1- m e t h y l c h o l a n t h r e n e ,

epoxides of p o l y a r o m a t i c hydrocarbons ( r e f s . 45,46).

and t h e d i o l

We a l s o l i n k e d t h e TAPA

m o l e c u l e c h e m i c a l l y t o s i l i c a g e l , which p e r m i t s o p e r a t i o n w i t h p o l a r s o l v e n t s as e l u e n t s . *

f -

/ '

io*

HEXAHELICENE

No2

Furthermore, we examined a s e r i e s o f r e a g e n t s , where t h e m e t h y l group o f TAPA was r e p l a c e d by a l o n g e r a1 k y l r a d i c a l (R=Et, i P r , n-Bu)

.

The s t e r e o s e l e c -

t i v i t y of t h e s e l a t t e r compounds was, however, l e s s t h a n t h a t o f TAPA. S e l e c t o r s o f t y p e f a r e e l e c t r o n w i t h d r a w i n g and t h e above p o l y a r o m a t i c s o l u t e s , which a r e r e s o l v e d b y them, a r e e l e c t r o n - d o n a t i n g . electron-affinity o f selector reagents.

Reversal o f t h e

and s e l e c t a n d s h o u l d g i v e complementary t y p e s o f

W i t h t h i s i d e a i n mind, we have s y n t h e s i z e d t h e P - ( + ) - 7 , 7 ' - h e x a h e l i -

cene d i c a r b o x y l i c a c i d ( t y p e 9). T h i s a c i d , when coated on s i l i c a g e l i n t h e form o f i t s sodium s a l t ( f o r r e d u c i n g s o l u b i l i t y i n t h e e l u e n t ) p e r m i t s t o r e s o l v e t h e 2,4-dinitrobenzene-a-amino

a c i d e s t e r s by HPLC.

Many o t h e r c l a s s e s

o f compounds which a r e e q u a l l y e l e c t r o n w i t h d r a w i n g s h o u l d show c h i r a l d i f f e r e n t i a t i o n by t h i s approach.

*Columns coated w i t h TAPA o r i t s s a l t s a r e a l s o e f f e c t i v e .

124

9

The pertinent work of Pirkle ( r e f . 47) should be mentioned. His point of departure was e n t i r e l y d i f f e r e n t from ours , and he developed d i f f e r e n t s e l e c t o r s . According t o P i r k l e and coworkers I chiral charge t r a n s f e r reagents capable of hydrogen bonding, such a s 2,2,2-trifluoro-l-[(lO-methyl)-9-anthryl] ethanol a r e incorporated i n t o the s t a t i o n a r y phase ( s i l i c a g e l ) . Substances a r e resolved which e i t h e r contain a CT complexing moiety i n t h e i r molecule, e.g. aromatic sulphoxides and lactones, o r posses a function through which such a moiety can be introduced, e . g . alcohols, mercaptans, amines, amino alcohols, amino acids and e s t e r s . Several types of phases have been introduced, and t h e v a r i e t y of molecules separated, as well the efficiency of t h e i r r e s o l u t i o n , i s impressive. CT complexation plays an important r o l e i n biology. We have undertaken some preliminary s t u d i e s on c h i r a l recognition i n p a r t i t i o n i n g systems by appropriate biological molecules, taking he1 icenes as a probe.

CH20H

H-l-OH

"-C-ai

I

H-C--OH

H J - H

I

I t was found t h a t r i b o f l a v i n resolves t h e aromatic hydrocarbons, when coated on s i l i c a g e l , ( r e f . 48). I t can be seen t h a t t h e f l a v i n has some f e a t u r e s analogous t o TAPA, namely, a t r i c y l i c aromatic ring system ( i s o a l l o x a z i n e ) and an asymmetric s u b s t i t u e n t ( 0 - r i b i t y l ) i n the middle r i n g . These experiments have a l s o been extended t o nucleosides and nucleotides and i t was found t h a t purines, b u t not t h e monocyclic pyrimidines showed c h i r a l d i f f e r e n t i a t i o n of he1 i cenes ( r e f . 49).

125

S e l e c t o r s i n t e r a c t i n q by c o o r d i n a t i o n w i t h m e t a l s . The r e s o l u t i o n o f o l e f i n s was t a c k l e d t o g e t h e r w i t h V.F.

At

S c h u r i g i n 1971.

t h e s t a r t , a search was made f o r a c o o r d i n a t i n g compound capable of r a p i d and r e v e r s i b l e complexation w i t h an o l e f i n and c o n t a i n i n g a c h i r a l l i g a n d c l o s e enough t o t h e u n s a t u r a t e d hydrocarbon f o r s t e r i c i n t e r a c t i o n w i t h i t .

I n the

f i r s t step, t h e aim was t o achieve e f f i c i e n t s e p a r a t i o n o f n o n - c h i r a l

isomeric

olefins.

I t was found t h a t s e l e c t o r

k, t h e

dicarbonyl-Rh~-3-fluoroacetyl-1R (Or

1s)-camphorate, d i s s o l v e d i n squalane and used as a gas chromatographic phase, had t h e d e s i r e d p r o p e r t i e s ( r e f . 5 0 ) .

The s e p a r a t i o n o f t h e f i r s t racemic o l e f i n

( 3 - m e t h y l c y c l o p e n t e n e ) was, however, achieved o n l y many y e a r s l a t e r by S c h u r i g ( r e f . 51), a f t e r he had p e r f e c t e d t h e c a p i l l a r y column p r e p a r a t i o n t e c h n i q u e s . U n f o r t u n a t e l y , r e s o l u t i o n s remained l i m i t e d t o o n l y one o t h e r o l e f i n ,

3-ethyl-

cyclopentene ( r e f . 5 2 ) , and t h e r e a r e a l s o d i f f i c u l t i e s i n r e p r o d u c i n g Column performance. On t h e o t h e r hand, S c h u r i g and h i s coworkers have extended, w i t h g r e a t success, t h e use o f m o d i f i e d t y p e d i f f e r e n t metals.

h

selectors, consisting o f bis-6-diketonates

of

In t h e l o n g s e r i e s o f papers t h e y have p r e s e n t e d , most i m -

p r e s s i v e r e s u l t s on t h e s e p a r a t i o n o f enantiomers o f d i v e r s e c l a s s e s of h e t e r o c y c l i c compounds, as w e l l as o f a l c o h o l s and ketones ( f o r a r e v i e w , see r e f . 53). Another s e l e c t o r t y p e , based on c o o r d i n a t i o n w i t h m e t a l s , which g i v e s v e r y e f f i c i e n t r e S O l u t i Ons a r e t h e N ,N ' - d i a l k y l -a-amino a c i d s :

0.

R'

R

126

These compounds have been used i n HPLC as a d d i t i v e s t o t h e e l u e n t ; a v a l u e s as h i g h as 2.75 (non-optimized) have been r e p o r t e d f o r c e r t a i n s o l u t e s w i t h these s e l e c t o r s .

A r e c e n t r e v i e w o f t h i s procedure and o f t h e v a r i o u s c h i r a l

a d d i t i v e s r e p o r t e d i n t h e l i t e r a t u r e appears i n r e f . 54. has been undertaken on t h e i n f l u e n c e o f t h e s u b s t i t u e n t s ectivity (ref. 54).

A systematic study

R and R 1 on s t e r e o s e l -

The copper complexes o f N,N'-di-n-propyl-L-alanine

and o f N , N ' - d i m e t h y l - L - v a l i n e

(DPA)

(DMV) have been found t o be e s p e c i a l l y u s e f u l .

A

procedure has been developed f o r t h e e n a n t i o m e r i c a n a l y s i s o f m i x t u r e s o f a1 1 common p r o t e i n amino a c i d s , i n v o l v i n g c a t i o n exchange f o r i s o l a t i n g t h e f r a c t i o n s c o n t a i n i n g , r e s p e c t i v e l y , t h e a c i d i c , n e u t r a l , and b a s i c components, p r i o r t o t h e c h i r a l r e s o l u t i o n s t e p ( r e f . 56) ( F i g . 7 ) .

F i g . 7. S e p a r a t i o n o f t h e amino a c i d s e l u t e d f r o m t h e cation-exchange c o l umn i n groups, on r e v e r s e d phase column. M o b i l e phase was t h e c h i r a l a d d i t i v e CU-DPA i n water, r e v e r s e d phase column ( 1 5 x 0 46 cm, S p h e r i s o r b LC-18, 5u). ( a ) F r a c t i o n 1, a c i d i c , f l o w r a t e 0.17 m l min-i; ( b ) f r a c t i o n 2, n e u t r a l , 1% a c e t o n i t r i l e added, f l o w r a t e 0.25 m l min-1; ( c ) f r a c t i o n 3, a r o m a t i c , 14% acet o n i t r i l e added, f l o w r a t e 0.5 m l min-1; ( d ) f r a c t i o n 4, b a s i c , f l o w r a t e 0.21111 min-1. Furthermore, i n m i x t u r e s of s i x t e e n Dns p r o t e i n amino a c i d s , t t l i r t e e n components were s p l i t i n t o t h e i r e n a n t i o m e r i c peaks w i t h Cu(DPA)2 i n aqueous aceton i t r i l e , and s o l v e n t programming.

The o t h e r t h r e e components were g l y c i n e

p r o l i n e ( w h i c h cannot be r e s o l v e d by t h i s s e l e c t o r ) and a r g i n i n e , one peak o f which seems t o o v e r l a p w i t h d a n s y l i c a c i d , a l s o p r e s e n t i n t h e sample ( r e f . 57).

127

Cu (DPA)2 shows s t e r e o s e l e c t i v i ty a l s o towards such compounds as a-amino a c i d e s t e r s , a-hydroxy a c i d s , and a-amino-e-caprolactame.

On t h e o t h e r hand, DMV

has been found u s e f u l f o r t h e s e p a r a t i o n o f enantiomers o f a-hydroxy a c i d s ( r e f . 58) and a-methyl-a-amino

acids ( r e f . 59).

R e c e n t l y , i t has been demonstrated

t h a t TLC p l a t e s impregnated w i t h Cu (DPA)2 p e r m i t t h e r e s o l u t i o n o f Dns-amino acids (except pro) ( r e f . 60).

By u s i n g b i d i m e n s i o n a l development, a l l t h e com-

ponents of complex m i x t u r e s can be s e p a r a t e d ( r e f . 6 1 ) . The mechanism o f r e s o l u t i o n by t h e s e s e l e c t o r s may i n v o l v e t h e f o r m a t i o n o f d i a s t e r o m e r i c i n n e r o r , a1 t e r n a t i v e l y , o u t e r sphere complexes.

Davankov ( r e f .

62) has produced evidence f o r t h e i n t e r m e d i a c y o f i n n e r sphere c o o r d i n a t i o n compounds o f t y p e [ s e l e c t o r - C u - s e l e c t a n d ] .

The a p i c a l p o s i t i o n s occupied b y

water, a s o l v e n t m o l e c u l e o r a t h i r d f u n c t i o n a l group o f t h e l i g a n d s , p l a y s an I t s h o u l d be n o t e d t h a t

e s s e n t i a l r o l e i n t h e process o f r e c o g n i t i o n .

Davankov's work r e f e r s t o c h i r a l c o m p l e x a t i o n r e a g e n t s c h e m i c a l l y l i n k e d t o a p o l y m e r i c backbone. W e i n s t e i n and L e i s e r o w i t z , on t h e o t h e r hand, suggested t h a t c h i r a l r e c o g n i t i o n i s due t o o u t e r sphere c o o r d i n a t i o n .

C.P.K.

models o f Cu (DPA)2 show

t h a t t h e r e i s a " c l e f t " i n t h e m i d d l e o f t h e m o l e c u l e above t h e m e t a l . s o l u t e , when p l a c e d i n t h i s " c l e f t " ,

The

i n t e r a c t s w i t h t h e p o s i t i v e l y charged

metal and by hydrogen bonding w i t h t h e l i g a n d .

The f i t o f t h e s e l e c t a n d w i t h

t h e s e l e c t o r w i l l be more o r l e s s good a c c o r d i n g t o i t s c o n f i g u r a t i o n .

The

r i g i d i t y i m p a r t e d t o t h e complex by t h e a l k y l groups ( N , N ' - d i - n - p r o p y l )

i s an

i m p o r t a n t f a c t o r i n d e t e r m i n i n g t h e s t e r e o s e l e c t i v i t y o f t h e system.

Evidence

f o r t h e o u t e r sphere c o o r d i n a t i o n has been produced by c r y s t a l e t c h i n g s t u d i e s ( r e f . 63).

Time (min)

F i g . 8. Chromatogram o f t h e p l a t i n u m complexes o f 3-methylpent-1-ene mobile phase: n-hexane/CH2ClZ/Z-butanol, 60/40/1.

(11);

128 An i n t e r e s t i n g development o f t h e l a s t mentioned s t u d i e s l i e s i n a d i f f e r e n t field.

Indeed, i n p u r s u i n g t h e a t t e m p t s t o r e s o l v e o l e f i n s ( s e e s e l e c t o r

k),

i t was decided t o t r y t o s o l v e t h i s problem by t h e HPLC o f a p p r o p r i a t e s t a b l e

d i a s t e r e o m e r i c metal complexes.

A f t e r experimenting w i t h diverse p o s s i b i l i t i e s ,

i t was found t h a t P t complexes, c o o r d i n a t e d t o a l i g a n d o f t h e t y p e

namely t r a n s - c h l o r o (N,N' - d i m e t h y l - 0 - p h e n y l g l y c i n e ) l y s u i t a b l e f o r t h i s purpose ( r e f . 64, 6 5 ) .

selector,

(01 e f i n ) P t I I , a r e eminent-

An example o f such a s e p a r a t i o n ,

showing peaks corresponding t o a l l f o u r p o s s i b l e P t complexes, i s g i v e n i n F i g . 8. Aromatic s u l p h o x i d e s , t o o , c o u l d be s p l i t i n t o d i a s t e r e o m e r s by t h i s approach.

T h i s method o f i n d i r e c t r e s o l u t i o n o f o l e f i n s was subsequently a p p l i e d t o a d d i t i o n a l hydrocarbons and some oxygenated compounds, c o n t a i n i n g one o r more double bonds, i n t h e l a b o r a t o r i e s o f Schomburg ( r e f . 66) and S c h u r i g ( r e f . 6 7 ) .

CONCLUSIONS The v a r i o u s s e l e c t o r s f o r chromatographic r e s o l u t i o n o f o p t i c a l isomers, developed i n t h e l a b o r a t o r y o f t h e a u t h o r , have been reviewed.

I t i s apparent

t h a t many types o f molecules e x i s t whose i n t e r a c t i o n w i t h a n t i p o d e s a r e s u f f i c i e n t l y d i f f e r e n t f o r b r i n g i n g about s e p a r a t i o n i n p a r t i o n i n g systems.

Once a

molecule i s discovered, which m a n i f e s t s c h i r a l r e c o g n i t i o n , i t i s p o s s i b l e by t h e c l a s s i c a l approach o f s y s t e m a t i c s t r u c t u r e change, t o i n c r e a s e i t s effect i v e n e s s and range of a p p l i c a t i o n .

Considerable research i n t h i s area i s going

on i n many l a b o r a t o r i e s t h r o u g h o u t t h e w o r l d .

I t i s t o be expected t h a t i n t h e

f u t u r e many more s e l e c t o r t y p e s w i l l be d i s c o v e r e d and t h e i r performance and scope f u r t h e r improved.

REFERENCES E. G i l - A v and D. Nurok, i n G.C. Giddings and R.A. K e l l e r (Eds.), Advances i n Chromatography, Marcel Dekker, New York, 1974, p. 99. B. Feibush, Ph.D. Thesis, s u b m i t t e d t o t h e F e i n b e r g Graduate School o f The 2 Weizmann I n s t i t u t e o f Science, Rehovot, I s r a e l , 1967. 3 E. G i l - A v , B. Feibush and R . C h a r l e s - S i g l e r , Tetrahedron L e t t . (1966) 1009. E. G i l - A v , B.Feibush and R . C h a r l e s - S i g l e r , i n A.B. L i t t l e w o o d ( E d . ) , Gas 4 Chromatography 1966, I n s t . o f Petroleum, London. 1967, P. 227. 5 E . G i l - A v and B . F e i b u j h , Tetrahedron L e t t . (1967), 3345. 6 E. G i l - A v , R. Charles and S.-C. Chang, i n G.W. Gehrke, K.C. Kuo and R.W. Zumwalt ( E d s . ) , Amino A c i d A n a l y s i s by Gas Chromatography, CRC Press I n c . , Boca Raton, F l o r i d a , 1984, i n p r e s s . 7 E.B. Kyba, K. Koga, L.R. Sousa, M.G. Siege1 and D.J. Cram, J. Amer. Chem. Soc., 95 (1973) 2696. 8 B. Feibush and E. G i l - A v , Tetrahedron, 26 (1970) 1361. 9 S. Nakaparksin, P. B i r r e l l , E. G i l - A v and J. Oro, J. Chromatographic S c i . , 8 (1970) 177. 10 J.A. Corbin, J.E. Rhoad and L.B. Rogers, A n a l . Chem., 43 (1971) 327.

1

129 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 40 41 42 43 44 45 46 47 48 49 50 51 52 53

S. Weinstein, G. Jung and E. G i l - A v , Proceedings 4 1 s t Meeting, I s r . Chem. SOC., October, 1971, p. 202. W. P a r r , J. P l e t e r s k i , C. Yang and E. Bayer, J. Chromatogr. S c i . , 9 (1971)

141. W. P a r r and P.Y. Howard, Anal. Chem., 45 (1973) 711. W.A. Koenig and G.J. Nicholson, i b i d . , 47 (1975) 951. K. S t o e t l i n g and \(.A. Koenig, Chromatographia, 9 (1976) 331. G.G. Smith and D.M. Wonnacott, Anal. Biochem., 109 (1980) 414. N . O i , T. Doi, H. K i t a h a r i and Y. Inda, J . Chromatogr., 208 (1981) 404. E. G i l - A v and B. Feibush, U.S. P a t e n t 3,494, 105, (1970); Japanese p a t e n t 567367. B. Feibush, Chem. Commun., 1971, 544. R. Charles, U. B e i t l e r , B. Feibush and E. G i l - A v , J. Chromatogr., 112 (1975) 121. R. Charles and E. G i l - A v , i b i d . , 195 (1980) 317. H. Frank, G.J. N i c h o l s o n and E. Bayer, Angew: Chem. I n t . Ed., 17 (1978) 363. T. Saeed, P. Sandra and M. Verzele, J. Chromatogr., 186 (1980) 611. W.A. Koenig, S. S i e v e r s and I . Benecke, i n R.E. K a i s e r (Ed.) Proceedings o f t h e F o u r t h I n t e r n a t i o n a l Symposium on C a p i l l a r y Chromatography, I n s t i t u t e o f Chromatography, Bad Durckheim, 1981, p. 703. V.F. S c h u r i g , Angew. Chem., 96 (1984) 1984. S.-C. Chang, E. G i l - A v and R. Charles, J. Chrornatogr., 289 (1984) 53. A. Dobashi, K . Oka and S. Hara, J. Amer. Chem. SOC., 102 (1980) 7122. A. Dobashi and S. Hara, J. Chromatogr., 267 (1983) 11. S.-C. Chang, Ph.D. Thesis, s u b m i t t e d t o t h e F e i n b e r Graduate School o f The ljjeizmann I n s t i t u t e o f Science, Rehovot, I s r a e l (198 1. S. Weinstein, B. Feibush and E. G i l - A v , J. Chromatogr.; 126 (1976) 97. K. Watabe and E. G i l - A v , i b i d . , 1984, i n p r e s s . S. Weinstein, L. L e i s e r o w i t z and E . G i l - A v , J . h e r . Chem. SOC., 102 (1980) 2768. N . - O i , H. K i t a h a r a , Y . Inda and T . Doi, J. Chromatogr. 237 (1982) 297. idem., i b i d . , 213 (1981) 137. D. Thumm, Ph.D. Thesis, U n i v e r s i t y o f Tubingen, 1980. B. Feibush, E. G i l - A v and T. Tamari, J. Chew. SoC., P e r k i n 11 (1972) 11g7 C.H. Lochmueller and R.W. S o u t e r , J . Chromatnar.. 88 (1974) 41. H. R u b i n s t e i n , B. Feibush and E. G i l - A v , J. Chem. S O C . (19731, 2094. J.A. Corbin and L.B. Rogers, Anal. Chem., 42 (1970) 974. C.H. L o c h m u e l l e r and R.d. S o u t e r , J . Chromatogr., 87 (1973) 243. M.S. Newman and 0 . L e d n i c e r , J. Amer. Chem. SOC., 78 (1956) 4765. L.H. Klemm and 0. Reed, J . Chromatogr., 3 (1960) 364. '4. Haenel and H.A. Staab, Chem. Ber. 106 (1973) 2203. H. Newman, R. H e l d e r and H. Nynberg, Rec. T r a v . Chim. Pays Bas, 95 (1976) 211. F. Mikes, G. B o s h a r t and E. G i l - A v , J. Chromatogr., 122 (1976) 205. Younq Hwam K i m , Ph.D. T h e s i s , s u b m i t t e d t o t h e F e i n b e r g Graduate School, The Weizmann I n s t i t u t e of Science, Rehovot, I s r a e l , 1981. W.H. P i r k l e , D.W. House and J.M. Finn, J. Chromatogr., 192 (1980) 143, and references c i t e d therein. Young Hwam K i m , A. Tishbee and E. G i l - A v , J . Amer. Chem. SOC., 102 (1980) 591 5. idem., Science, 213 (1981) 1379. E. G i l - A v and V . S c h u r i g , Anal. Chem., 43 (1971) 2030. V. S c h u r i g , Angew. Chem., 89 (1977) 113. V. S c h u r i g and E. G i l - A v , I s r . J. Chem., 15 (1976/77) 96. V . Schurig, i n J.D. M o r r i s o n (Ed.), Asymmetric S y n t h e s i s , V o l . I, Academic Press, London, 1983, p.59.

8

130 54 55 56 57 58 59 60 61 62 63 64 65 66 67

E. G i l - A v and S. W e i n s t e i n i n W.S. Hancock ( E d . ) , Handbook o f HPLC f o r t h e S e p a r a t i o n o f Amino A c i d s , P e p t i d e s and P r o t e i n s , V o l . I , CRC Press I n c . , Boca Raton, F1 ., p. 429. S. Weinstein, Angew Cheni. Suppl., (1982), 425; Angew. Chem. I n t . Ed. Engl, 21 (1982) 218. S. Weinstein, M.H. Engel and P.E. Hare, Anal. Biochem., 121 (1982) 370. S. W e i n s t e i n and S. Weiner, J . Chromatogr., 303 (1984) 244. I . Benecke, J. Chromatogr., 291 (1984) 155. S. W e i n s t e i n and N. G r i n b e r g , i b i d . , 1984, i n p r e s s . S. W e i n s t e i n , Tetrahedron L e t t . , 25 (1984) 985. N. G r i n b e r g and S. W e i n s t e i n , J. Chromatogr., 303 (1984) 251. V.A. Davankov, i n J.C. G i d d i n g s , E. Grushka, J . Cazes and P.R. Brown (Eds.), Advances i n Chromatography, V o l . 18, M. Dekker I n c . , N.Y., 1980, p.139. S. W e i n s t e i n and L . L e i s e r o w i t z , t o be s u b m i t t e d t o I s r . J. Chem. M. Goldman, Z. Kustanovich, A. Tishbee and E. G i l - A v , P o s t e r , Vth I n t e r n a t i o n a l Symposium on Column L i q u i d Chromatography, Avignon, 1981 P.36. M. Goldman, Z. Kustanovich, S. Weinstein, A. Tishbee and E. G i l - A v , J. Am. Chem. SOC., 104 (1982) 1093. J. Koehler and G. Schomburg, Chrornatographia, 14 (1981) 559. H. Retzbach, D i p l o m a r b e i t , U n i v e r s i t y Tubingen, 1982.

131

UNE

NOWELLE SIMUTATION NUMERIQUE DE LA PROPAGATION D'UN SOLUTE DANS UNE C O M N N E DE C H R O m W H I E EN REGIME NON LINEAIRE : SCHEMA DE GODOUNOV ET SCHEMA ANTIDIFFUSE

P. ROUCAON (I.), M. SCHOENAUER (2), P. VALENTIN (3), G. GUIOCHON (4)

RESUME : Un d e l e d e p r o p a g a t i o n d ' u n e zone d e c o n c e n t r a t i o n f i n i e d a n s une colonne d e chromatographie e s t e t u d i e d a n s l e c a d r e d e la t h h r i e moderne d e s s y s t e m s h y p e r b o l i q u e s non l i n e a i r e s . On p r e n d e n compte B la f o i s les e f f e t s non l i n e a i r e s d u s B l ' i n f l u e n c e d e s t r a n s f e r t s r a d i a u x s u r l'hydrodynamique e t a u x i n t e r a c t i o n s s H c i f i q u e s e n phase a b s o r b e e , a i n s i que les g r a d i e n t s d e p r e s s i o n d a n s la c o l o n n e .

Nous p r e s e n t o n s e n s u i t e des r e s u l t a t s numeriques o b t e n u s e n a p p l i q u a n t cette t h e o r i e a u modele B un composant : le schema d e Godounov c o n s i s t e , apres d i s c r e t i s a t i o n d e l a c o l o n n e , a r e s o u d r e e n chaque noeud d ' e s p a c e une s u i t e d e problemes d e Riemann ( e c h e l o n d e c o n c e n t r a t i o n e n chaque noeud d e t e m p s ) . On montre l ' i m p o r t a n c e d u rapport Az/At s u r la s t a b i l i t e du schema ; Le schema obtenu p a r a n t i d i f f u s i o n du p r e c e d e n t donne une m e i l l e u r e approximation l o i n d e s chocs, mais est 4 h 6 f o i s p l u s collteux.

une s i t u a t i o n p r e a l a b l e d e chocs, o b t e n i r d a n s l e cas d ' u n i s o t h e r m e complexe. C e s schemas n e n e c e s s i t e n t pas

impossible B

L ' e f f i c a c i t e d e cette s i m u l a t i o n est demontree par comparaison a v e c les r e s u l t a t s elcperimentaux dans l e cas d e l ' a d s o r p t i o n du n - h e m e SUT du n o i r d e c a r b o n e g r a p h i t i s e , B looo C . ABSTRACT : W e s t u d y a model for the p r o p a g a t i o n of a f i n i t e c o n c e n t r a t i o n band i n

a chromatographic column, o n the background of modern theory o f n o n - l i n e a r h y p e r b o l i c s y s t e m s . This m o d e l treats both the non l i n e a r e f f e c t s due t o t h e i n f l u e n c e of r a d i a l t r a n s f e r on hydrodynamics t o t h e specific i n t e r a c t i o n i n t h e absorbed p h a s e , and the p r e s s u r e g r a d i e n t i n t h e column. W e t h e n p r e s e n t n u m e r i c a l r e s u l t s o b t a i n e d by a p p l y i n g t h i s theory t o the one-

component model : t h e Godounov scheme, h a v i n g d i s c r e t i z e d t h e column, s o l v e s a t e v e r y space mesh-point a sequence of Riemann problems ( a c o n c e n t r a t i o n rung a t e v e r y time mesh p o i n t ) . W e have proven the s t a b i l i t y of the scheme h e a v i l y depends o n the n u m e r i c a l rate Az/At : the scheme w e g e t from that o n e b y "antidiffusion" g i v e s better a p p r o x i m a t i o n f a r from shocks, b u t needs 4 t o 6 time more computing t i m e . One d o s e n ' t need here t o locate the s h o c k s b e f o r e computing, i m p o s s i b l e w i t h a complex i s o t h e r m .

which is

quite

W e p r o v e the a c c u r a c y of s u c h a n u m e r i c a l s i m u l a t i o n by comparing it w i t h e x p e r i m e n t a l r e s u l t s , i n t h e case o f n - h e m e a d s o r p t i o n on g r a p h i t i z e d carbon, a t a t e m p e r a t u r e of loo0 C.

(1) ECOLE POLYTECHNIPUE - 91128 PALAISEAU E O E X ( 2 ) Centre de Hathematiques Appliquees - ECOLE POLYTECHHIPUE - 91128 Pslaisesu Cedex (3) Centre de Recherche de SOlaiZe - ELFIERAP - 8.P. 22 - 69360 St Symphorien d'0zon ( 4 ) Lab. de Chlmie Analytique Physique - ECOLE POLYTECHNIPUE - 91128 Palaiseau Cedex

132 L ' o b j e t d e cet article est la p r e s e n t a t i o n d e s i m u l a t i o n s colonne d e chromatographie e n phase gazeuse.

numeriques

d'une

Le chromatographie est un p r o c e d e d e separation q u i e x p l o i t e la d i f f e r e n c e d e v i t e s s e d e m i g r a t i o n des especes chimiques i n d u i t e par l e u r s a f f i n i t e s d i s t i n c t e s pour un m i l i e u a d s o r b a n t s t a t i o n n a i r e ( l i q u i d e ou s o l i d e ) finement g r a n u l e . La m i g r a t i o n est p r o d u i t e par le mouvement d ' u n g a z i n e r t e ( v e c t e u r ) s ' e c o u l a n t h t r a v e r s les g r a i n s .

La p r i n c i p a l e o r i g i n a l i t e d e ce t r a v a i l reside d a n s l ' u t i l i s a t i o n d e schemas numeriques s p c i f i q u e m e n t adaptes a u d e l e mathematique u t i l i s e : un system hyperbolique non l i n e a i r e ( v o i r Rouchon [ l ] , Rouchon e t a l . [ Z ] ) . Dans les d i x d e r n i e r e s a n n e e s , la s i t u a t i o n a beaucoup Bvolue, notamment quant aux methodes numeriques d e r e s o l u t i o n d e s systemes h y p e r b o l i q u e s non l i n e a i r e s , ce q u i j u s t i f i e l a mise a u p o i n t r e l a t i v e m e n t d e t a i l l e e e t g e n e r a l e que nous donnons. La t h h r i e d e s systemes h y p e r b o l i q u e s non l i n e a i r e s avait d e j h

ete

appliquee h

d e s modeles d e chromatographie I a i n s i par Jacob [ 3 ] , q u i u t i l i s e l a theorie d e s c a r a c t e r i s t i q u e s . e t par Rhee [ 4 ] , q u i c a l c u l e analytiquement une s o l u t i o n du probleme d e Riemann ( c r e s t h d i r e a v e c donnees c o n s t a n t e s h l ' e n t r e e d e

la colonne a u x temps t

>

0).

Enfin, il f a u t mentionner l ' o u v r a g e d e Aris e t Amundson ( 1 9 7 3 ) 1 5 1 , q u i e x p l i q u e e n d e t a i l la m4thode d e s c a r a c t e r i s t i q u e s e t c o n t i e n t nombre d'exemples relatifs a l a chromatographie. Mais le c a l c u l d e Rhee n e s ' a p p l i q u e qu'h un isotherme d e Langmuir, e t c e l u i d e qui sont traitees Jacob, e n d i s t i n g u a n t "parties c o n t i n u e s " e t "chocs", differemtent, ne r e s o u t pas l a d i f f i c i l e q u e s t i o n d e la l o c a l i s a t i o n d e s chocs

.

D a n s la s i m u l a t i o n proposee, a u c o n t r a i r e , les c h o c s a p p a r a i s s e n t n a t u r e l l e m e n t e t il n ' e s t nullement n e c e s s a i r e d e c o n n a i t r e leur p o s i t i o n a p r i o r i . D e meme,

nous ne c a l c u l o n s p a s d e s o l u t i o n a n a l y t i q u e . mais approchons numeriquement une s o l u t i o n physiquement acceptable ( e n un s e n s que nous p r e c i s e r o n s ) , e t ce q u e l que s o i t l'isotherme, e t p o u r t o u t e s donnees i n i t i a l e s . Remarquons h ce s u j e t , que l a r e s o l u t i o n du probleme de Riemann (chromatog r a p h i e f r o n t a l e ) est i n s u f f i s a n t e pour l ' o p t i m i s a t i o n d ' u n e s e p a r a t i o n oh la longueur d e l ' i n j e c t i o n est un d e s p a r a m e t r e s . On n o t e r a e n f i n que l ' i n t e r a c t i o n e n t r e l e p r o f i l d e p r e s s i o n l e l o n g d e la colonne e t les effets non l i n e a i r e s , i n t e r d i t pratiquement t o u t e s o l u t i o n "analytique"

.

133 D ' a u t r e part, on p e u t s ' a t t e n d r e a ce que le debit e n d e h o r s du s i g n a l ne s o i t p l u s l e meme d e ce f a i t , d e part e t d ' a u t r e de c e l u i - c i . E t , e n chromatog r a p h i e p r e p a r a t i v e gazeuse, l e g r a d i e n t d e p r e s s i o n d a n s l a colonne est t o u j o u r s i m p o r t a n t , pour d e s r a i s o n s d e p r o d u c t i v i t e b i e n d v i d e n t e s . Or il est tres s i m p l e d ' a d a p t e r l e schema n m e r i q u e p o u r t e n i r compte du g r a d i e n t d e pression. Nous donnons le p r i n c i p e g e n e r a l d e cette methode, a i n s i que l e s d e t a i l s d e s schemas d a n s l e cas s i m p l e du probleme a deux c o r p s ( u n s o l u t e + le g a z cas p o u r l e q u e l nous comparons r e s u l t a t s numeriques e t r e s u l t a t s vecteur), exp&imentaux (chromatographie gaz-solide. s o l u t e n-hexane, v e c t e u r azote). Cependant, a u vu d e s r e s u l t a t s d e Kvaalen, N e e 1 et Tondeur [ 6 ] s u r le p r o b l e m m u l t i c o r p s , l a forme tres s i m p l e d u schema d e Godounov que nous obtenons se g e n e r a l i s e immediatement a un nombre quelconque d e composants, la difficult6 &ant alors l ' i d e n t i f i c a t i o n d e s i s o t h e r m e s d ' a d s o r p t i o n . E n f i n il est b i e n clair que l e p r e s e n t t r a v a i l ne c o n s t i t u e q u ' u n e premiere approche du probleme d e l a s e p a r a t i o n multi-composant e n chromatographie non l i n e a i r e , que nous a b o r d e r o n s u l t e r i e u r e n t . Nous i n s i s t e r o n s s u r t o u t i c i s u r la partie s i m u l a t i o n numerique,

renvoyant

a

pour les d e t a i l s s u r l a partie e x p e r i m e n t a l e du travail a s a v o i r l ' o b t e n t i o n d e s d i f f e r e n t s parametres du modele, e n p a r t i c u l i e r d e l ' i s o t h e r m e d ' a b s o r p t i o n d u s o l u t e par l a M t h o d e Echelon-Impulsion. [Z]

134 I

- LE

I1

MODELE UTILISE

s ' a g i t du modele de p r o p a g a t i o n chromatographique a c o n c e n t r a t i o n

finie,

propose p a r P. V a l e n t i n ( [ 7 ] , ch. 2 ) .

Nous e n r a p p e l o n s les h y p o t h e s e s p r i n c i p a l e s , r e n v o y a n t a [ 7 ] ou [ 8 ] p o u r les d i s c u s s i o n s r e l a t i v e s a ces hypotheses : - I1 s ' a g i t d ' u n modele monodimensionnel, c'est a dire q u e t o u t e s les g r a n d e u r s p h y s i q u e s s o n t c o n s t a n t e s s u r une s e c t i o n de l a c o l o n n e . - Les gaz s o n t parfaits, e t l a l o i de D a r c y est v e r i f i e e . - Le gaz v e c t e u r n ' e s t pas adsorbe. - La temperature e s t c o n s t a n t e dans l a c o l o n n e e t a u c o u r s du temps. - Les echanges e n t r e les phases s o n t i n s t a n t a n e e s e n p a r t i c u l i e r , l a p h a s e gazeuse mobile e t l a p h a s e g a z e u s e adsorbee s o n t e n equilibre t h e r m i q u e . - La p r e s s i o n dans l a c o l o n n e n e varie pas a u c o u r s du temps, mais depend de l'abscisse dans l a c o l o n n e i nous n e n e g l i g e o n s pas le g r a d i e n t de p r e s s i o n (cf r91). n e g l i g e o n s les "effets d u second ordre", c'est a dire c e k r e s u l t a n t s de l a c i n e t i q u e d ' e c h a n g e , par rapport aux " e f f e t s d u p r e m i e r ordre" q u i p r o v i e n n e n t de l'equilibre thermodynamique, ce q u i c o n s t i t u e i c i une bonne a p p r o x i m a t i o n ( c f [ 7 ] , ch. 2 , p o u r u n e d i s c u s s i o n s u r ce p o i n t ) .

- Nous

L ' e q u a t i o n de b i l a n d i f f e r e n t i e l des masses p o u r un c o r p s A d a n s s ' e c r i t alors :

la

colonne

l'abscisse dans l a c o l o n n e

(modble

z(4.4 a )=-&4)

z 2 0, t > 0.

Les variables s o n t t. monodimensionnel)

l e temps,

.

et z ,

A

i n c o n n u e s s o n t NG, nombre de moles gazeux d u corps A par u n i t e A l o n g u e u r , Ns, nombre de moles adsorb& d u corps A par u n i t e de l o n g u e u r u, la v i t e s s e locale de transport a p p a r e n t d ' u n t r a c e u r i n e r t e (cf [ 7 ] ) .

Les

de

et

Notons que cette e q u a t i o n est v a l a b l e tant p o u r les s o l u t e s q u e p o u r les corps vecteurs. i

~ a n sl e cas oh n s o l u t e s s o n t e n p r e s e n c e , s i N: e t N~ representent r e s p e c t i v e m e n t l e nombre de m o l e s g a z e u s e s e t adsorbees du iCme s o l u t e , il existe des f o n c t i o n s ki ( i = l , n ) , t e l l e s q u e '

1

NA = k'(NG,

et

$+l s = O

...

,N:)

pour i

=

l,...,n

p o u r l e gaz

come n+lem c o r p s ( p u i s q u ' i l n ' e s t pas adsorbe).

i

vecteur,

considere

135 i

Faisant interrenir les fractions molaires Xi des especes i, reliees aux NG par i

N G = X p X i , X dependant de la colonne, et de la temperature, il vient pour chaque solute

...,

Nous utiliserons en fait, plutOt pour le systeme ((l), (2) (n), (n+l)) le systeme equivalent (l), ( 2 ) , (1) + (2) +...+ (n) + (n+l)), c'est a dire que nous remplaqons l'equation de bilan du gaz vecteur par l'equation d'evolution du debit total dans la colonne, que nous ecrivons sous la forme conservative suivante :

...,

L(up)

(n+l)'

+a [p + C" k i(pX1,....PX,)] at

aZ

Notons F = u p

= 0

1=1

( P est proportionnelle au debit molaire dans la colonne).

3

et en D'autre part, la pression p est donnee par la loi de Darcy, u = - C supposant alors up constant, ce qui donne, puisque la sortie de la colonne est libre ( p = 1 en z = L) :

oh

et

P L

est la pression imposee a l'entree de la colonne, est la longueur de la colonne.

En particulier, at desormais est donc

=

0,

et

Les inconnues sont F et les F X i

le

, i

systeme

=

.

1,. ,n.

sur

lequel

nous

travaillerons

136

Effet de sorption

l e cas d e l a chromatographie e n phase gazeuse, la vitesse n ' e s t pas c o n s i d e r e e come c o n s t a n t e . E l l e depend des transferts e n t r e phase mobile e t phase s t a t i o n n a i r e : e n effet, l ' a d s o r p t i o n d ' u n e q u a n t i t e d N d e s o l u t e e q u i v a u t a l a d i s p a r i t i o n pure e t s i m p l e du volume p a r t i e l occupe par cette q u a n t i t d d e phase mobile, p u i s q u e l e volume occup6 par la meme q u a n t i t e e n phase a d s o r b e e est e n v i r o n 200 f o i s moindre, donc n e g l i g e a b l e .

Dans

On p u t se faire une i d e e d u phenomene d e s o r p t i o n suivant :

a l ' a i d e du mod6le s i m p l e

membranes s e m i permeables

F i g u r e 1-1 1

Remarque 1 : Les e q u a t i o n s (P) o n t ete d t a b l i e s e n supposant les variables (NG et Nl, e t donc Xi , i - l , . . , n , e t u ) contincunent d e r i v a b l e s , par r a p p o r t aux variables z e t t . En f a i t , si l ' o n e t e n d l a n o t i o n de s o l u t i o n d ' u n t e l systeme a d e s f o n c t i o n s d i s c o n t i n u e s ( c o m e nous a l l o n s l e faire au 5 s u i v a n t ) , on v o i t q u e (P) c o n t i e n t Bgalement les h a b i t u e l l e s e q u a t i o n s q u e l ' o n B c r i t l e l o n g d e s d i s c o n t i n u i t e s ( c f . [3], par exemple). est d u a l a fonne c o n s e r v a t i v e des e q u a t i o n s (P).

Notons qye ceci

Conditions i n i t i a l e s e t co n d i ti o n s aw l i m i t e s

Nous c o n n a i s s o n s l ' e t a t i n i t i a l du systeme, c ' e s t a d i r e t o u t e s l e s inconnues a u temps t = 0 , a i n s i que les q u a n t i t e s i n j e c t e e s a l ' e n t r e e d e l a colonne ( e n = 0 ) p o u r t o u t temps t > 0 ( f i g . 1-2). I1 ne s ' a g i t p a s d e s c o n d i t i o n s a w limites u s u e l l e s du f a i t que s e u l l e q u a r t p o s i t i E du plan ( z , t ) existe physiquement. M a i s nous v e r r o n s ( c f IV) q u e l ' o n p e u t se ramener a un problerne d e Cauchy s t a n d a r d ( f i g . 1-3). car, come nous l e verrons p a r l a s u i t e , t o u t e s les v a l e u r s p r o p r e s du systeme s o n t p s i t i v e s : il n ' y a "transport d ' i n f o n n a t i o n " que d a n s l e s e n s d e s t c r o i s s a n t s .

z

137

z

4/

r\

/ / /

3/

u ?

u ?

u ?

x. ?

x. ?

x. ?

f

,/ / /

/

//,//////

////////////"//'/

//>

1

Remarque 2 : Notons enfin que, par rapport a un systeme hyperbolique non lineaire sous fome classique, les variables z et t sont inversees (voir egalement [ 5 ] (p. 357/358) sur ce sujet). Si, d'un point de w e mathernatique, cela ne change que les notations, cela perturbe par contre les significations physiques des fonctions, le gradient longitudinal des flux s'echangeant avec le gradient temporel de la quantite accumulee. Ainsi, le terme de vitesse usuel dans la theorie du systeme hyperbolique non lineaire correspond ici a un terme en dt/dz, soit un inverse de vitesse. C'est pourquoi, dans toute la suite, nous parlerons plutOt de taur de r&ention, qui s'exprime donc en sec/cm. Nous retrouverons ce probleme lors de mathematique et de son "'flux".

l'interpretation de

l'entropie

Remarque 3 : Une consequence importante de cette inversion est que l'espace vectoriel le plus approprie pour etudier les equations d'evolution est l'eapace des debits (oh les vecteurs ont pour coordonnees les debits des esp&ces l,..,n et D'un point de vue systeme n'est pas en phase mobile. le systeme est stationnaire.

le debit total), et non l'espace dea quantites (cf. [lo]). physique, lorsque les quantites sont connues localement, le entierement determine, il reste un arbitraire : la vitesse Par contre. lorsque les debits en phase mobile sont connus, entierement determine, y compris la composition en phase

138

I1

-

LES SYSTEMES HYBERBOLIQUES NON LINEAIRES

t 1 N i Notons w l e v e c t e u r ( F , .,F ), avec F = FXi, i On p e u t alors ecrire (P) s o u s les formes s u i v a n t e s

..

I %+

a at [

H(w)]

= 0

= -aaz +w

=

1..N.

aw at

D H(w) -

w

a v e c ~ ( w =)

H

est

derivee

Nt1 Ni donc une f o n c t i o n d e R d a n s R , e t DW ( H ) r e p r e s e n t e d e H par rapport a c h a c u n e d e s c o o r d o n n e e s d e w.

la

matrice

D e f i n i t i o n : Le systeme d ’ e q u a t i o n s a u d e r i v e e s partielles ( I ) est d i t hyperbolique ( r e s p e c t i v e m e n t h y p e r b o l i q u e s t r i c t ) si, p o u r t o u t v e c t e u r w, les v a l e u r s p r o p r e s de la m a t r i c e D H ( w ) s o n t reelles ( r e s p e c t i v e m e n t reelles W toutes distinctes). On e n d e d u i t e n p a r t i c u l i e r u n e propriete tres importante des systemes h y p e r b o l i q u e s non lineaires, la p r o p a g a t i o n “a vitesse f i n i e “ c ‘ e s t a d i r e i c i ( c f . I , remarque 3). a d t / d z f i n i , soit e n c o r e 21 t a u x de r e t e n t i o n f i n i : s i l a d o n n e e i n i t i a l e ( e n z = 0 ) est n u l l e e n d e h o r s d ’ u n b o r n e , l a s o l u t i o n e n tout point z

>

0 possede la meme

propriete ( v o i r f i g . 11-1). z

solution

‘A

I

’

solution nulle

7/ / / // // I / / / /

donnee i n i t i a l e n u l l c

donnee i n i t i a l e nulle

Figure 11-1

139 Par exemple, dans le cas du problem? a un solute, l e systeme devient :

( taux

1t( 1 - X ) k ' Ddns ce cas particulier les valeurs propres de D H sont 0 et W U de retention particulier) et le systeme est donc hyperbolique strict.

Mise a part la theorie des caracteristiques, calquee sur le cas lineaire, la theorie mathematique des systemes hyperboliques non lineaires est relativement recente (Lax [11],[12] dans les annees 50). et tres incomplete : ainsi il n'y a pas de resultat general d'existence de solution, et donc bien sar pas de resultat general de convergence de schemas d'approximations. Ceci provient du fait que ce type de probleme a la desagreable particularite de ne pas forcement posseder de solution reguliere, meme avec des donnees tres regulieres : des "singularit6s" peuvent apparaitre en tout z

>

0.

Le problem de l'apparition des singularites dans la solution est difficile, tant mathematiquement que physiquement (comprehension des phenomenes). I1 est d'autre part fondamental en ce qui concerne l'aspect "separation" en chromatographie. C'est pourquoi nous allons detailler les phenomenes, sur un exemple simple : Cas particulier : non lineaire :

Prenons le cas le plus simple d'une equation hyperbolique

t

(11)

E

R, z

>

0 ;

t E R.

f est une fin de R dans R et uo exemple OP

.

une fonction donnee de

classe

C"

par

Remarque : La matrice D A du probleme d e l e ( I ) est ici reduite au scalaire W f'(u) : le probleme est toujours hyperbolique (strict). Supposons qu'il existe une solution u de classe C

1

I1 est facile de voir que l e long des couches du demi plan ( (z,t) : z definies par :

on a

>

0 }

140 ce q u i e n t r a i n e

d t --= dz

f'(U0(tO))

.

c o u r b e s e n q u e s t i o n , q u e l ' o n dppelle c o u r b e s caracteristiques d u probleme, s o n t des droites. On d i t s o u v e n t q u e la donnee i n i t i a l e se propage l e l o n g des c a r a c t e r i s t i q u e s . Les

t r e s simple, p o u r t o u t temps t t e l t o u t e c o u r b e c a r a c t e r i s t i q u e i s s u e de d e u x p o i n t s d i s t i n c t s n e se c o u p e n t pas a v a n t l ' i n s t a n t t . La s o l u t i o n est alors d e t e r m i n e e de maniere

que

Par contre, plus exist-

des q u e d e u x c a r a c t e r i s t i q u e s d i s t i n c t e s se c o u p e n t ,

il ne p u t

de s o l u t i o m r&quli&rea.

P r e n o n s l'exemple classique de l ' e q u a t i o n de B u r g e r s :

t E t E (Modele simplifie de c i n e t i q u e de gaz

-

R,

2

>

=

uo(tO)z+tO.

0 ;

R.

e n e c h a n g e a n t x e t t ).

La c a r a c t e r i s t i q u e i s s u e d ' u n p o i n t ( t O , O ) est alors l a droite t

= t, o n p e u t r e p r e s e n t e r l a s o l u t i o n dans l e plan ( z , t ) : Avec %(t)

z

F i g u r e 11-2 u(t,z)

=

t l+z est

solution classique

u,(t) = - t 2 , par c o n t r e , les c a r a c t e r i s t i q u e s i s s u e s de -to e t to se c o u p e n t sur l'axe t = 0 , e t il n ' e x i s t e a u c u n z > 0 " a v a n t " l e q u e l u n e s o l u t i o n c l a s s i q u e serait d e f i n i e :

Avec

\z

F i g u r e 11-3

141 I1

f a l l a i t d o n c e l a r g i r le c o n c e p t d e s o l u t i o n s ,

discontinues

;

e n a u t o r i s a n t les s o l u t i o n s

o n cherche d o n c les s o l u t i o n s a u s e n s d e s d i s t r i b u t i o n s

:

D e f i n i t i o n : Une f o n c t i o n u sera d i t e s o l u t i o n faible d e ( I ) s i

1'- I [WE+ -1ao at +m

H(w)

0

d t dz

-

-m

m

pour t o u t e fonction v e c t o r i e l l e

d e classe C

I

+m

w,

@(O,t) d t

=

0

-m

.

On v o i t q u e , d a n s les d o m a i n e s oh u est c o n t i n c l m e n t d e r i v a b l e , o n r e t r o u v e ( I ) par i n t e g r a t i o n par partie. P a r c o n t r e . supposons u, courbe ( C ) ( t = s ( z ) ) .

e t de classe C1

s o l u t i o n de (111).

P o u r un o u v e r t R d u p l a n (z,t) r e n c o n t r a n t ( C ) , o n d e f i n i t R- et R + come c i - c o n t r e . et (C-) et ( c + ) come les parties des frontieres de R- e t R+ i n t e r i e u r e s a n. S o i e n t w- e t w + les l i m i t e s de w le long d e la courbe (respectivement limite a g a u c h e e t l i m i t e 2, d r o i t e ) .

sauf

sur

O G t = s(2)

F i g u r e 11-4

P r e n o n s d a n s I11 u n e f o n c t i o n

a support dans

[wg+

+m

0

--

H(w)

R . On a :

a@ ] d t d z at

= 0

En i n t e g r a n t par partie d a n s R - e t R + r e s p e c t i v e m e n t ( p u i s q u e w y est reguliere). on obtient, en notant v + = (u; , v ; ) la n o r m a l e e x t e r i e u r e a R + ( a v e c les m e w s n o t a t i o n s p o u r n - ) ( F i g u r e 111-4) :

- [

aw

+ a

H(w)

] 0 d t dz

+

une

142

Ceci devant

etre v r a i p o u r t o u t e f o n c t i o n 4~ a s u p p o r t dans fl , o n e n d e d u i t

*+a t

+H(w)

a2

=

0

d a n s fl+ e t d a n s fl- , e t (w+

- w-

) U:

+

- H(w-))

(H(Wt)

L a c o u r b e ( C ) e t a n t d e f i n i e par t = s ( z ) ,

(R.H.)

H(W+)

-

H(W-)

=

S'(2)

(w'

V t = 0

il v i e n t

-

W-

)

est l e taux d e r e t e n t i o n du choc. relation, d i t e r e l a t i o n de Rankine EIugoniot, est u n e c o n d i t i o n n e c e s s a i r e e t s u f f i s a n t e p o u r q u ' u n e f o n c t i o n v e r i f i a n t (11) e n dehors d ' u n e + s ( z ) , s o i t s o l u t i o n faible de ( I ) d a n s R x A l i g n e de d i s c o n t i n u i t e ( c h o c ) z ( i . e . v e r i f i e (111)).

s'(z)

Cette

-

Remarque : On r e t r o u v e , d a n s l e cas de l a chromatographie, les r e l a t i o n s q u i , u s u e l l e m e n t , e t a i e n t a j o u t e e s e n a p p a r e n c e arbitrairement, aux e q u a t i o n s (I). En f a i t , avec l a definition d ' u n e s o l u t i o n faible de ( I ) par (111), ces r e l a t i o n s s o n t i m p l i c i t e m e n t c o n t e n u e s dans (I)..

Mais le remede est p r e s q u e pire q u e l e mal, c o r n l e m o n t r e l'exemple s u i v a n t , oh une e q u a t i o n h y p e r b o l i q u e non lineaire p e u t a v o i r une i n f i n i t e de s o l u t i o n s faibles, ce q u i est p h y s i q u e m e n t ( e t p h i l o s o p h i q u e m e n t ) i n a c c e p t a b l e . C o n s i d e r o n s l ' e q u a t i o n de B u r g e r s , avec d o n n e e i n i t i a l e n u l l e : t € R , z > O ;

1

u(0,t)

= 0

Une s o l u t i o n est evidemment l a f o n c t i o n n u l l e I s u i v a n t e . p o u r to e R e t a > 0 d o n n e s : u(t.2) = 0 u(t.2)

=

-a

u(t.2)

=

a

u(t,z)

= 0

t E

R.

M a i s considerons la fonction

a s i t e ] - , t , , - - z2 [ , a s i t e ] t o - Z z , to [ , a s i t E ] t o ,t , + z z [ , a s i t e ] tO+;iz, +-[.

une s o l u t i o n faible de ( I ) p u i s q u e s o l u t i o n c l a s s i q u e l h oh e l l e est continue, les r e l a t i o n s (RH) & a n t v e r i f i e e s l e l o n g des c o u r b e s de discontinuite. C'est

143

Notion d'entropie mathematique On

est

donc

amen6

a restreindre la classe des solutions admissibles par

rapport A la classe des solutions faibles en imposant une contrainte supplementaire : c'est ainsi que l'on definit la notion de solution entropique d'un systeme hyperbolique non lineaire.

Definition : Une fonction 4 de R dans R est appelee entropie (mathematique) (appelee flux d'entropie) telle que du systeme ( I ) s'il existe une fonction

w

S'il

existe une solution reguliere w de (I),

en multipliant (I) a gauche par

@ ' ( w ) on obtient

mais cela devient faux si w n'est pas reguliere.

Une premiere maniere de selectionner la solution entropique du system (I) est d'imposer

pour toute entropie @ convexe du systeme, de flux associe V (cf Lax [13] ).

Remarque 1 : historiquement, provenaient de

L'emploi du terme "entropie" ne doit pas surprendre : les premiers problemes hyperboliques non lineaires etudies la cinetique du gaz, et le terme mathematique adopt4

correspondait A la notion physique d'entropie (Lax [12]). Le mot est rest&, meme si, sur d'autres problems, il n'a plus aucune signification physique. Nlammoins, il est important de relier l'entropie physique aux entropies mathematiques : ce sera la seule possibilite de "choisir" la bonne solution falble. Mais ce probleme, lie h la reversibilite du phenornene de separation, d6passe le cadre de la presente etude.

Remarque 2 : La terme "flux", en toute rigueur, represente en fait ici le gradient temporel de l'accumulation d'entrople, en raison de l'inversion de z et t ( v o i r I, remarque 3).

144

Une maniere p e u t - e t r e p l u s n a t u r e l l e d ' i n t r o d u i r e cette n o t i o n d e s o l u t i o n e n t r o p i q u e est l a s u i v a n t e . On c o n s i d e r e que l a s o l u t i o n e n t r o p i q u e ( o n espere a v o i r e x i s t e n c e e t u n i c i t e p o u r l a s o l u t i o n e n t r o p i q u e ) , d o i t etre l a limite des s y s t e m s avec un petit t e r m e d e v i s c o s i t e o u d i f f u s i o n l o r s q u e c e l u i - c i t e n d v e r s 0 , c'est h d i r e d e

Le terme d e d i s s i p a t i o n d u s e c o n d m e m b r e a un effet r e g u l a r i s a n t . Le p r o b l e r n e (P ) a u n e u n i q u e s o l u t i o n r e g u l i e r e w e . ~e p l u s , s i $I est u n e e n t r o p i e c o n v e x e d u system d e f l u x fl'. o n a p o u r t o u t E > 0, a&wE)

az

+-

awe) < O

at

ce q u i d o n n e s i o n p u t passer a la limite

En f a i t , ce t e r n existe, e t a ete n e g l i g e p o u r l ' o b t e n t i o n d u systeme ( I ) : d ' o h le f a i t q u ' i l s e l e c t i o n n e la "bonne" s o l u t i o n p h y s i q u e . Une

etude

detaillee

de l ' i n t r o d u c t i o n d u t e r m e d i f f u s i o n n e l a

ete

effectuee

par Rhee, B o d i n , Amundson [14] p o u r la r e s o l u t i o n d u p r o b l b m e d e Riemann. Une a u t r e methode p o u r s e l e c t i o n n e r 2 "bonne" s o l u t i o n pami les s o l u t i o n s faibles c o n s i s t e h i n t e r d i r e c e r t a i n s c h o c s . E l l e a ete i n t r o d u i t e par Lax ([lz]), e t a B t e l a premiere a p p r o c h e d e l a s o l u t i o n e n t r o p i q u e . C ' e s t e g a l e r n e n t celle q u e n o u s u t i l i s e r o n s d a n s l a s u i t e . C o n d i t i o n d e Lax d i t q u ' u n choc est "admissible" s ' i l n ' e x i s t e "sortant d u choc".

On

pas

de

caracteristiques

Plus p r e c i s e m e n t . o n appelle un k-choc u n choc t e l que les c o u r b e s c a r a c t e r i s t i q u e s de l a k-ieme famille r e n t r e n t d a n s l e choc, e t les a u t r e s familles d e c o u r b e s c a r a c t e r i s t i q u e s n e r e n c o n t r e n t pas l e choc. Voyons comment cela se t r a d u i t : S o i t w une s o l u t i o n f a i b l e d e ( I ) . (C) (

c o n t i n u e s a u f le l o n g d ' u n e c o u r b e d e choc

t = s(2) ) .

C ' e s t u n e c o u r b e d e k-choc s i

Ak(w-) Ak-q(W-)

l e long d e la courbe.

> <

s'(z) S ' ( z )

>

<

kk(w+) Ak+q(w+)

145

En effet, les valeurs propres &ant rangees par ordre croissant, Xj(w(z,t)) est le taux de retention de la j+me courbe caracteristique au point (2.t). et s ' ( z ) est le taux de retention de choc sur le point de la courbe ( z , s ( x ) ) . I1 existe d'autres manieres de selectionner les "bons" chocs (cf. Dafemos, Liu ... [15] [ 1 6 ] ) , et la encore, la theorie generale est tres incomplete : On ne sait montrer l'equivalence de toutes ces conditions que dans des cas particuliers (cas de l'equation scalaire, ou cas de chocs de faible amplitude).

peut donner une idee succinte de l'avancement de la theorie des hyperboliques non lineaires.

On

systemes

Resultats theoriques

p=l

:

Le cas d'une equation scalaire est a peu pres theoriquement resolu

-

Tous les criteres d'entropie sont equivalents

-

I1

y

a existence et unicite de la solution entropique pour

toute

:

donnee

initiale uo bornee ( [ 171 , [le 1 1. p-2 : Un resultat tres recent (Di Perna [19]) d'existence d'une solution pour certains systemes particuliers avec donnee initiale bornee, par la methode de viscosite artificielle en utilisant des resultats tres fins d'analyse non-lineaire.

Cas qeneral : - Pas d'equivalence des conditions d'entropie.

- Un resultat d'existence d'une solution entropique (critere de Lax), dans le cas d'une donnee initiale uo a variation bornee, et s'eloignant peu d'une valeur constante (Glinnn [ Z O ] ) . La demonstration est basbe sur discretisation du probleme, et sur une etude des interactions des chocs.

une

146

111- SCHEMks NUMERIQUES N o u s a v o n s d o n c v u q u e l a methode des c a r a c t e r i s t i q u e s , simple e t precise, d e c a l c u l d e s o l u t i o n s d e problemes h y p e r b o l i q u e s non l i n e a i r e s , e s t e n f a i t i n a p p l i c a b l e d e s q u ' a p p a r a i t u n choc ( i . e . u n e d i s c o n t i n u i t e ) . NOUS a l l o n s d o n c u t i l i s e r d e s sch&nas d e type d i f f e r e n c e s f i n i e s .

Le

principe

g e n e r a l d e t e l s schemas est le s u i v a n t :

t e m p s A t e t un pas d ' e s p a c e Az, e t le d e m i - p l a n z

>

0

o n choisit un pas d e est a i n s i d i s c r e t i s e :

2

F i g u r e 111-1

On

va alors,

par r e c u r r e n c e s u r n ,

c a l c u l e r me s o l u t i o n approchee a u p o i n t

nAz, q u i sera c o n s t a n t e par morceaw s u r c h a q u e i n t e r v a l l e I i A t , ( i + l ) A t [ . On n o t e u:

l a v a l e u r de l a s o l u t i o n c a l c u l e e a u pas nAz s u r ] i A t , ( i + l ) A t [ .

recurrence exemple ) La

sera

amorcee l o r s q u e l ' o n a u r a discretise u

ui0 =

1

(it1)At

IIAtu,,(t)

en

posant

(par

dt

F i g u r e 111-2 ntl

I1 exlste d e n o m b r e u s e s m a n i e r e s d e d e f i n i r e n s u i t e l a r e l a t i o n d o n n a n t u,

f o n c t i o n d e s u:,

, E ~ ,

en

147 P a r exemple,

[ l l ] , le premier a avoir hyperbolique non l i n e a i r e

l e schema d e L a x - F r i e d r i c h

s'ecrit, pour resoudre l ' e q u a t i o n n

u

n+l

-

u,' it1

+

e te

utilise,

n

u"

1-1

2

C ' e s t l a rnethode l a p l u s n a t u r e l l e d e d i s c r e t i s e r par d i f f e r e n c e s f i n i e s (c'est a d i r e s a n s p r i v i l e g i e r d e d i r e c t i o n p a r t i c u l i e r e d e centrees p r o p a g a t i o n ) l ' e q u a t i o n (1). Conditions d e

stabilite

L ' e t u d e d e s "vitesses de p r o p a g a t i o n " ( t a w de r e t e n t i o n s ) n o u s d o n n e immediatement u n e c o n d i t i o n n e c e s s a i r e de s t a b i l i t e : e n e f f e t , n o u s a v o n s vu q u e les p r o b l h e s h y p e r b o l i q u e s non l i n e a i r e s se c a r a c t e r i s e n t par u n t a u d e l a d o n n e e " i n i t i a l e " est n u l l e lors r e t e n t i o n f i n i . En p a r t i c u l i e r , s i uo,

-

d ' u n intervalle d e t e m p s [ a , b ] , la s o l u t i o n ( e n t r o p i q u e ) a u p o i n t z est n u l l e sur l ' i n t e r v a l l e d e t e m p s [a-Mz, b+Mz], oh

M

= SUP

UER

De p l u s ,

I

xk(u)

k€[l,n]

I

l e t a u d e r e t e n t i o n n u m e r i q u e p o u r un schema t e l que ( S l ) schema

a

un pas ) e s t A t / A z . Par c o n s e q u e n t , il y a u r a f o r c e m e n t "perte d'infonration" s i l e t a u x de r e t e n t i o n n u m e r i q u e est i n f e r i e u r e a u t a u x d e r e t e n t i o n reel d u probleme i n i t i a l : z

m

jt

F i g u r e 111-3 La s o l u t i o n approchee c a l c u l e e sera f o r c e m e n t n u l l e d a n s l e domaine h a c h u r e

Cela

se

abrege)

t r a d u i t par la c o n d i t i o n d i t e d e C o u r a n t F r i e d r i c h s Lewy ( i n t r o d u i t e d a n s [ 2 1 ] ).

P a r e x e m p l e , s u r (Sl), p o u r l ' e q u a t i o n (11). cela d o n n e A2 -

At

sup yER

I f'(Y)

I

<

1

(Cne n

148 Schema de Godounov Nous utiliserons en fait le schema de Godounov (cf. [ Z Z ] ) , construit sur la remarque suivante : au point nAz, nous disposons en fait d'une solution approchee constante par morceaux. Or, nous savons plus ou moins resoudre le probleme (I ) avec, pour donnee "initiale" une fonction du type 0

t

<

0

u (t) = u+, t

>

0

l o

u(t)

=

u-,

Ce probleme est apple prrsblde Rieaann pour l'equation (I). Donc, nous pouvons esp6rer resoudre localement au point nAz, une suite de problemes de Riemann (en chaque iAt), puis, en "recollant les morceaux", obtenir une solution approchee au point nAz + Az.

( n i l ) A~

l+1/2

1-1/2

-

n Az (i-1)At

t

(ii1)At

i A t

I Figure 111-4 En chaque point iAt, (iaz) on rdsout un probleme de Riemann : soit wYtllZ la solution sur la verticale AB (en effet, du fait de l'homogeneite de l'equation comme de la condition initiale, il est h d d i a t de voir que la solution du probleme de Riemann est constante s u r toute droite passant par le point de discontinuite de la donnee initiale). En integrant l'equation (1) sur (ABCD), il vient alors :

''ABCD

Soit (SZ) U a i s ceci en supposant que la valeur wYt112 n'est pas "perturbee" par les problemes de Riemann adjacents. Or, la solution du probleme de Riemann est

constante sous les droites de pentes XI

pour x

w

Figure 111-5

<

0,

A n pour x

>

0.

149 Donc, e n i m p o s a n t A z a s s e z petit p o u r que les d r o i t e s i s s u e s d e i A t d e p e n t e 11(wn1 1 1 et 1p( w "1) n e r e n c o n t r e n t pas r e s p e c t i v e m e n t les s e g m e n t s EF e t DC, o n j u s t i f i e l a f o r m u l e ( 5 2 ) . Or ceci s ' e c r i t :

et

Az X,,c~f)

6 At

On remarque que ces d e w c c o n d i t i o n s s o n t d e C o u r a n t F r i e d r i c h Levy (CFL).

realisees si

o n a impose la c o n d i t i o n

C o n v e r g e n c e d e s s c h e m a s de type Godounov

sar ete p r o u v e s p o u r l ' e q u a t i o n scalaire ([22], [23]). Le r G s u l t a t d e p a s s e d ' a i l l e u r s l e cadre d u schema d e Godounov, p u i s q u ' i l a ete p r o u v e p o u r u n e classe b e a u c o u p p l u s large d e schemas : les schemas m o n o t o n e s s o u 8 forme c o n s e r v a t i v e e t c o n s i s t a n t s avec (11), c o n v e r g e n t vers l ' u n i q u e s o l u t i o n e n t r o p i q u e de (11) ( C r a n d a l l M a j d a [ 2 4 ] ) . Nous ne d e t a i l l e r o n s pas ces r e s u l t a t s i c i , n o u s c o n t e n t a n t d e r e m a r q u e r qu'ils s ' a p p l i q u e n t a u schema de Godounov. Les p r e m i e r s r e s u l t a t s de c o n v e r g e n c e o n t b i e n

Les r e s u l t a t s s u r les schemas approximant les systemes ( I ) s o n t e n c o r e t o u s p a r t i e l s , ce q u i est nonnal compte t e n u d e s " t r o u s " de l a theorie des systemes hyperboliques non lingaires

.

C i t o n s les principaux : l e r e s u l t a t de G l h [ Z O ] b i e n sar, q u i f u t l o n g t e m p s l e s e u l r e s u l t a t d ' e x i s t e n c e p o u r les systemes : apres avoir r e s o l u les ntl d i f f e r e n t s problemea d e Riemann, a u l i e u de p r e n d r e p o u r ui la moyenne d e

l a s o l u t i o n exacte s u r ] i A t , ( i t l ) A t [ , G l h choisit un t e m p s de cet i n t e r v a l l e a u h a s h e t fixe u": come l a v a l e u r exacte d e l a s o l u t i o n e n ce temps. La methode converge, mais h l ' i n c o n v e n i e n t de n e c e s s i t e r l e c a l c u l d e

l a s o l u t i o n exacte a u point ( n + l ) Az des problemes de Riemann d u p o i n t n A z , ce q u i p u t etre d i f f i c i l e . N o t o n s e n f i n que l e s c h e m a d e Glirmn n ' e s t pas s o u s forme c o n s e r v a t i v e . Les r e s u l t a t s de Lax Wendroff [ 2 5 ] c o n c e r n e n t p l u s d i r e c t e m e n t les s c h e m a s d e type Godounov : s i u n schema s o u s forme c o n s e r v a t i v e c o n v e r g e , alors l a l i m i t e

est

s o l u t i o n faible de ( I ) . S i d e p l u s u n e r e l a t i o n d ' e n t r o p i e d i s c r e t e h c h a q u e pas, a l o r s l a Limite est s o l u t i o n e n t r o p i q u e .

est

verifiee

Enfin, d a n s l e cas p a r t i c u l i e r d u schema d e Godounov, la c o n d i t i o n d ' e n t r o p i e d i s c r e t e est t o u j o u r s v e r i f i e e : s ' i l c o n v e r g e , c'est v e r s l a ( u n e ) s o l u t i o n e n t r o p i q u e (ce q u i n e p r o u v e n i l ' e x i s t e n c e d ' u n e s o l u t i o n e n t r o p i q u e , n i la c o n v e r g e n c e de l a s u i t e d e s o l u t i o n c o n s t r u i t e par ce s c h e m a ) . C e s o n t s u r lesquels o n d i s p o s e d u maximum d ' i n f o r m a t i o n s neammoins les schtheoriques.

150 Mais on sait aussi de ces schemas qu'ils sont d'ordre 1, c'est a dire que l'erreur commise a chaque passage d'un nAz a (n+l)Az est de l'ordre de Az (cf [ 2 6 ] ). En particulier, ils ont tendance a "arrondir" les chocs. D'autre part, les schemas d'ordre 2 existant par exemple c o m e approximation de problemes hyperboliques lineaires (de type Lax Wendroff), oscillent fortement en presence de chocs, d'oQ l'idee de construire des schemas quasi d'ordre 2 (cf Leroux [23]), c'est a dire d'ordre 2 le plus souvent possible, mais qui sont d'ordre 1 pres des chocs.

Le schema antidiffuse

L'idBe generale est donc, partant d'un schema convergeant d'ordre 1, de le modifier dans les zones oQ la solution est reguliere (c'est a dire loin des chocs ), pour en faire un schema plus precis (d'ordre 2 ou plus )

.

schema d'ordre 1 (ou schema predicteur) sera par exemple Godounov decrit plus haut :

Le

le schema de

Le schema d'ordre 2 sera un schema de type Lax Wendroff, c'est a dire obtenu en ecrivant un developpement limit6 a l'ordre 2 de win t l suppose fonction repliere des w; (jEZ). On ecrit le schema obtenu sous la forme : (53)

ntl

wi

-

- wy

-

Q

H(W7t,/2

)

- H(Wy-1/2

US termes A:,,,~ sont appeles termes "correcteurs". pas dans le cas general.

1 1

-

+

et nous ne les ecrirons

Le schema quasi d'ordre 2 s'obtient alors en testant pour chaque i si on se trouve pres d'un choc, par exemple en regardant si la solution calculee au point nAz par (53) a tendance a osciller, (mais d'autres choix de test sont possibles) et, dans l'affirmative seulement, a utiliser le schema ( 5 2 ) . On sait (cf Leroux 1231) que ce type de schema, applique a l'equation scalaire, converge vers la solution entropique, avec une condition CFT, deux fois plus contraignante. Pour les systemes, on a le meme type de resultats partiels que pour le schema de Godounov.

151 IV -LE SYSTEME DE LA CHROMAMCRAPHIE POUR LE PROBLEME A 1 CORPS

L'equation est alors (on a pose F

=

up)

On a donc

DwH

=

I

(l+k'(PX))

I$

k'( PX)

- - (l+k'(PX))

qui a pour valeurs propres 1

+

(1-X) k'(PX1 U

Le systeme est donc hyperbolique strict, et les vecteurs propres associes sont

1 =

[Fl

;

L'etude du second champ passe par l'etude de la quantite DwXz.wz Glimm [ 2 0 ] ) . Ici, DwXz.Wz

=

(P/F2) [l+(l-X)k'] [P(l-X)k"-Zk']

(cf p.ex.

.

Tout depend donc de la forme de l'isothenne. L'etudo de la quantite p( l-x)k"-2k' (s'annule-t-elle et combien de fois ? ) est necessaire pour une etude fine de la resolution du problem de Riemann.

Mais, pour la construction du schema de essentielles,mais elementaires suffisent. Probleme de Riemann On a la situation suivante :

Figure IV-1

Godounov, quelques

remarques

152 La s o l u t i o n v a u t w- d a n s l a r e g i o n 1 de l a F i g u r e IV-1, Wl

2

W+

4

l a r e g i o n 3, on a ( e v e n t u e l l e m e n t ) une s u c c e s s i o n de 2-ondes simples : s o i t chocs, soit "ondes de d e t e n t e " , c'est ti d i r e morceaux de s o l u t i o n s r e g u l i e r e s par rapport a l a v a r i a b l e t / z ( r a p p e l o n s que l e v o c a b u l a i r e est d i r e c t e r n e n t i s s u de la dynamique des g a z ) , e t wl est d e t e r m i n e par :

Dans

-

meme c o u r b e i n t e g r a l e de premiere famille que wp e u t passer de w1 a w+ par une ou p l u s i e u r s ( s u i v a n t l e s i g n e d e p( l-X)k"-Zk' ) Z-ondes admissibles. On sait que cette s o l u t i o n existe s i w- e t w + s o n t assez proches, e t s i p ( 1 - X ) k"-2k' n e s ' a n n u l e pas dans un v o i s i n a g e d e w- e t w+. wl se t r o u v e s u r l a

- On

dans t o u s les cas, et c'est la le point emsentiel, cette v a l e u r w 1 s t a t i o n n a i r e s u r la droite v e r t i c a l e k = 0 , verifie l a r e l a t i o n de Rankine Hugoniot :

Mais

[ H(w1)

c'est a dire H(W1)

-

-

] = 0

H(W-)

.

[ W1

-

W-

1

B(W-)

En p a r t i c u l i e r , les c o u r b e s i n t e g r a l e s de l a premiere famille s o n t les c o u r b e s X = c o n s t a n t e , e t s u r t o u t , sans c o n n a i t r e w1 eltactement, o n c o n n a i t H(wl), q u i est la mule quantiM par l a q u e l l e w1 i n t e r v i e n t dans le schema de Godounov. C o n d i t i o n d e CFL E l l e est i c i p a r t i c u l i e r e m e n t s i m p l e a ecrire ( e t ti verifier), p u i s q u e une des deux v a l e u r s propres est i d e n t i q u e m e n t n u l l e ; il reste :

soit e n c o r e , apres m a j o r a t i o n s Az At

- a

u

min

8

<

- l+k' max

oh unin est le minimum de vitesse d ' u n t r a c e u r i n e r t e , kImex la p e n t e maximum d e

l ' i s o t h e r m e d ' a b s o r p t i o n , p u i s q u e la v i t e s s e varie a u passage d u pic ( v o i r 5 I de s o r p t i o n ) .

- Effet

C o n d i t i o n s aux limites Nous sommes e n mesure, h l a lumiere de ces r e s u l t a t s , de t r a n s f o r m e r l e systeme reel e n un systeme avec c o n d i t i o n s aux limites standard (cf F i g u r e s 1-2 e t 1-3).

153 D'une p a r t , le probleme &ant h y p e r b o l i q u e , il y a p r o p a g a t i o n a t a u x d e r e t e n t i o n f i n i : il existe d o n c u n temps f i n i T a u d e l a d u q u e l l'etat i n i t i a l de la c o l o n n e ( t = 0, z > 0 ) n ' i n t e r v i e n t p l u s a la sortie de l a c o l o n n e (cf Fig. IV-2). On p e u t d o n c e t u d i e r l e system e n s u p p o s a n t l ' e t a t i n i t i a l c o n s t a n t , ce qui d o n n e un s y s t b m e " e x p 8 r i m e n t a l e m e n t B q u i v a l e n t " ( c ' e s t a

d i r e , e n s o r t i e d e c o l o n n e , apres B v a c u a t i o n de

l'etat

initial).

D ' a u t r e part, les t a u de r e t e n t i o n propres ( v a l e u r s propres de l a . m a t r i c e DwH(w)) s o n t positifs ou n u l s : l e systeme avec c o n d i t i o n a u x limites s t a n t a r d ( F i g . 1-3) a donc p o u r s o l u t i o n cette v a l e u r constante d a n s l e q u a d r a n t z>O, e t est B g a l d a n s l e e m r i m e n t a l ( Fig. IV-3 )

t
.

q u a d r a n t z>O, t > O

a la s o l u t i o n d u

problhe

sortie

L

ex p erim en t a l e

>t

0

T

Figure I V - 3

Fiqure 1\1-23

S i g n a l o n s q u e cette derniere &ape n ' a q u ' u n i n t e r e t t h e o r i q u e p u i s q u e , p r a t i q u e m e n t , n o u s n ' a v o n s pas r e c a l c u l e les v a l e u r s ( c o n s t a n t e s ) des u7 pour i

<

0 (c'est

a

d i r e s i t u e e s dans l e q u a d r a n t t

<

0, z

>

0) I

La schema d e Godounov

Compte t e n u des remarques p r e c e d e n t e s , il

w"+' 1

=

w"

-

s'ecrit

az At [ WwY) - E(w?-,

)

1

En f a i t , la f o n c t i o n p d e p e n d a u s s i de z : n o u s l ' a v o n s j u s q u ' a p r e s e n t n e g l i g e . M a i s il s u f f i t de p r e n d r e d a n s l e s c h e m a p = pn = p ( n A z ) : On o b t i e n t l a forme f i n a l e d u schema :

avec

154 Le schema antidiffuse

On ecrit donc, pour trouver le terme correcteur, un developpement de Taylor h l'ordre 2 d'une eventuelle solution au problem :

awn az i

wntl = w:

+

A z (-)

=

-

AZ

wn

1

+

a Ha ( w )t ) (

(2); +

(Az)~

~ ( A Z ) ~

2

( A z ) ~a z

(v) +

o(Az)z

On a, puisque H depend de z :

=

[

a

DwH(w)

a

aW

+ DzH(w)

] a

Plusieurs choix sont alors possibles d'approximation des termes en Ici nous les ecrirons naturellement ':

6[

[ 2%):a ]

D ~ H

1-

a/at.

[ D ~ H ;. ( q t 1 - ~7 ) - D,H:-,.(H:-

avec bien sbr

et

On obtient finalement le sch6ma d'ordre 2 de type Lax-wendroff :

H:-+

3

155 Le schema q u a s i d'ordre 2 c o n s i s t e alors h u t i l i s e r (LW) l o i n des c h o c s et ( C )

au v o i s i n a g e de ceux-ci. Une manibre d ' y p a r v e n i r est d'ecrire a n t i d i f f u s 4 s o u s l a forme s u i v a n t e :

qt1-<

le

schema

A i n s i , par exemple, s i et s o n t des s i g n e s c o n t r a i r e s , l e s t e r m e s a ~ + l / ze t Byt1,2 s o n t n u l s : s i la s o l u t i o n a u p o i n t n Az t e n d h osciller, lea termes c o r r e c t e u r s ne s o n t paa p r i s e n compte, e t le c a l c u l des ntl a p a r t i r d e s wy par l a sch4ma ( A ) e s t alors i d e n t i q u e a u c a l c u l par l e wi schema de Godounov ( G ) Ce schema est, d ' u n p o i n t de vue t h e o r i q u e , p l u s precis que l e schema de Godounov. Neanmoins, e t conme o n s ' e n d o u t e h voir les formules le defin i s s a n t , il sera beaucoup p l u s coateux e n temps c a l c u l . Le probleme est alors de decider si ceci j u s t i f i e c e l h .

.

156 IV - RESULTATS Nous presentons ici une serie de simulations numeriques de l'adsorptoin de n-hexane sur du noir de carbone graphitisd a 100" C. Nous avons ainsi pu obtenir l'isotherme par lissage par d w fonctions splines de points experimentaw obtenus par la d t h o d e Echelon-Impulsion (cf [I] et [Z]), ce qui nous pennet ensuite de comparer quantitati-nt resultats e x p e r m e n t a u et resultats thhriques. Signalons tout de suite qua le schema antidiffuse n' pas donne d'amelioratron sensible par rapport au schema de Gcdounov dans le cas ob nous avons pu comparer resultats thbriques et experimentaw. C'est pourquoi nous ne presenterons pas de rdsultats de ce schici. Dans tous les cas, nous avons choisi Q = At/Az (le t a w numerique) constant au cours du calcul.

de

retention

Description des resultats Les figures V-1 h V-8 concernent la modelisation de l'elution de divers creneaux de n-herane dans une colonne de chromatographie gaz-solide. Nous renvoyons encore a [ Z ] pour une description detaillee des conditions experimentalen.

- Discretisation de

la condition initiale : On tire des conditions experimentales les valeurs (stationnaires) initiales : debit constant, concentration du solute nulle. h i s , h l'instant t 0, on injecte en z = 0 un creneau de concentration que l'on discretise c o r n suit

-

X

Injection d'un creneau de hauteur % durant un temps to. On en deduit alors le creneau de debit. connaissant le temps d'injection, la section gazeuse de la colonne et le volume inject6 (cf [I] ou 1 2 1 ) .

- Etalonnage satisfaisants, maximum d'un

:

L'etalonnage du catharometre n'ayant pas donne de resultats leS Concentrations ont ete obtenues a partir du calage du pic calcule sur un pic experimental (il s'agit du pic

151 lo6 ; 4888

PICS CALCULES

4

3800

2088

2

I 008

8

\

188. 8

128. B

14a8

160.0

'

F i g u r e V-1

o5 x

3888

COURBES 1 X =0,183% 0

II

'I' I

lo6

x COURBES 2 X =2,83 % 0

2888

1900

t en s e c F i g u r e V-2

158 6 10

x

4888

COURBES 3 X = 8% 0

3888

2000

I I I

ma

a

lea a

I

l a0

iaa a

I&

149.0

1.a

a

148 a

16&0

t en sec. Figure V-3

COURBE 3 X = 8%

COURBE 1 Xo= 0 , 1 8 3 5

0

= ’

max

---

F=O. 17699

0,1776

= 0,17704

F=F =O, 1 7 7 0

L i i aa

12B 0

ia a

im a

t cn zec.

lea a

Figure V - 4

1ZB 0

I4a 0

t en see.

159

fa 6 x S

7088

E V O L U T I O N DU P I C D A N S L A COLONNE AU COURS DU TEMPS

( e n t r e Is e t 4 0 s ) 6889

5088

4088

2s

3088

2888

lW

.I

I

ZEN

4aee

6888

use

ieaa

1288

14aa

m a

i i a

z08.a

z en mrn Figure V-5

E V O L U T I O N DU P I C D A N S LA COLONNE AU COURS DU TEMPS

( e n t r e 10s e t 100s)

9.5

I

lea a

a0.B

3888

trra u

588 a

z en mrn

Figure V-6

160

2888

lRHB

\L

\! t en see.

In

'"

F i g u r e V-7

x

\

Avec CFL = 3

I

5 0

1

I00

F i g u re V-8

...

t en sec.

I

150

Th,,

161 c o r r e s p o n d a n t h un c r e n e a u i n i t i a l de 8 % ) . Les a u t r e s p i c s e n o n t ete d e d u i t s d ' a p r e s l a c o n s e r v a t i o n des aires, alors p r o p o r t i o n n e l l e s a u x c o n c e n t r a t i o n s . : La f i g u r e V-1 montre la serie d e pics modelises. h s c o n c e n t r a t i o n s i n j e c t e e s s o n t r e s p e c t i v e m e n t de 0.183%. 2.83%. 8% e t 1 1 . 7 3 % . Les deux f i g u r e s s u i v a n t e s r e p r e n n e n t ces r e s u l t a t s e n s u p e r p o s a n t les c o u r b e s t h e o r i q u e s ( e n traits pleins ) e t e x p e r i m e n t a l e s ( e n p o i n t i l l e s ) p o u r chaque cas ( a t t e n t i o n a u changement d'echelle p o u r les c o u r b e s 1). La f i g u r e V-4 donne, d a n s les cas 1 e t 3, l a s u p e r p o s i t i o n des c o u r b e s d e c o n c e n t r a t i o n e t d e d e b i t h l a s o r t i e d e la c o l o n n e . Les f i g u r e s V-5 e t V-6 m o n t r e n t , a u c o n t r a i r e , l ' e v o l u t i o n d ' u n pic, c o r r e s p o n d a n t h une i n j e c t i o n i n i t i a l e d e 8 % . l e l o n g de l a colonne ( i l f a u t v o i r chacune de ces c o u r b e s conune la p h o t o g r a p h i e d e l a s i t u a t i o n dans l a colonne h un i n s t a n t d o n n e ) . La f i g u r e V-7 montre l ' i n f l u e n c e du g r a d i e n t de p r e s s i o n s u r le pic c a l c u l e . Quant h la d e r n i e r e c o u r b e , e l l e montre ce q u i p e u t a d v e n i r lorsque la c o n d i t i o n d e Courant-Friedrichs-Lewy e s t violee...

- Courbes p r e s e n t e e s

Comentaires

Lea courbes 1 h 4 m o n t r e n t que l e schema propose se comporte f o r t b i e n l o r s q u e compare h d e s c o n d i t i o n 8 r d e l l e s d'e-rience. Les temps de sortie, ainsi q u e la s i t u a t i o n des chocs s o n t tres proches des g r a n d e u r s p h y s i q u e s q u ' e l l e s v e u l e n t representer. Lea r e s u l t a t s sur l e maximum des pics s o n t merlleurs p o u r des c o n c e n t r a t i o n s p l u s i m p o r t a n t e s ; mais n ' o u b l i o n s pas q u e l e modele theorique que n o w a v o n s u t i l i s e n e g l i g e les effets d u second ordre, e f f e t s d o n t l'importance r e l a t i v e est d ' a u t a n t p l u s grande que l a c o n c e n t r a t i o n est f a i b l e , ce q u i p u t expliquer l a c o u r b e 1, par exemple. D ' a u t r e part, il ne f a u t pas oublier que l a methode de c a l c u l de l ' i s o t h e r m e h partir de p o i n t s e w p e r i m e n t a w est Bgalement susceptible d ' i n t r c d u i r e des e r r e u r s . La figure V-4 montre qua l ' o n observe Bgalement n d r i q u e m e n t l e c r e n e a u de debit e n s o r t i e d e colonne, awc d e s reserves cette f o i s d'ordre numerique e n ce q u i c o n c e r n e les faibles c o n c e n t r a t i o n s ( l d g e r e s o s c i l l a t i o n s du d e b i t e n

.

a v a l du choc ) f i g u r e s V-5 e t V-6 o n t un interet e s s e n t i e l l e m e n t academique. On n o t e r a t o u t e f o i s l a d d c r o i s s a n c e e x p o n e n t i e l l e d u maximum du pic a u c o u r s du temps. E n f i n , la f i g u r e V-7 j u s t i f i e h posteriori n o t r e choix de la dependance de l a p r e s s i o n par rapport h l'abscisse dans l a c o l o n n e , p u i s q u e l e pic c a l c u l e h p r e s s i o n constante est p l u s e l o i g n e d u pic e x p e r i m e n t a l . Mais, i c i e n c o r e , nous avons f a i t une apppro-tion p o u r c a l c u l e r cette dependance, e t une a u t r e methode pourrait p u t - e t r e dOMer de meilleurs r e s u l t a t s . S i g n a l o n a e n f i n que l a legere "bosse" e n amont d e t o u s les p i c s nous semble purement n w r i q u e . En e f f e t , n o t r e c a l c u l d e l ' i s o t h e r m e est e f f e c t u e de la manibre suivante : celle-ci est s u p p o s e e lineaire p o u r les tres petites valeurs de l a c o n c e n t r a t i o n , puis est raccordee a u l i s s a g e des p o i n t s e r p e r i m e n t a u x par f o n c t i o m splines c u b i q u e s , l a raccord, p o u r t a n t d e classe 1 C , a y a n t preciserm?nt l i e u p o u r la v a l e u r d e l a c o n c e n t r a t i o n p o u r l a q u e l l e l a bosse apparait.

Les

162

CONCLUSION

&thode d e r e s o l u t i o n quo nous proposons p o u r le modele h y p e r b o l i q u e non l i n e a i r e d ' u n e colonne de chromatographie p e r m e t donc de faire v a r i e r t o u s les parmetres elrperimentaux. La

- Nature

de l ' i s o t h e n n e : ainsi que nous l ' a v o n s vu, t o u t e i s o t h e r m e donnee analytiquement p e u t etre u t i l i s e e dans l e schema. Les d i f f i c u l t e s s u r g i s s e n t l o r s q u e l ' o n souhaite u t i l i s e r d e s dOMeeS exp&rimentales, q u ' i l eSt i m p e r a t i f d e "lisser", l e problhe d e l a d t h o d e optimale restant o u v e r t ( [ 2 1 ).

- Po=

de l ' i n j e c t i o n initiale : i c i e n c o r e , il f a u t , d a n s l e cadre d ' u n e s i m u l a t i o n d ' u n e w r i e n c e , a j u e t e r au mieux l a d i s c r e t i s a t i o n du s i g n a l h l ' e n t r e e d e l a colonne a v e c les c o n d i t i o n s e l r p e r i m e n t a l e s - jamais ideales.

- Gradient

de p r e e e i o n dans la c o l o n n e : nous avons i c i c h o i s i de c o n s i d e r e r g r a d i e n t de p r e s s i o n connne donne par l a l o i de 0a.~Cy, i n t e g r e s o u s l'hypothese de debit constant, e t avons observe une faible v a r i a t i o n de debit dans l a colonne. I1 est e v i d e n t q u e le schema serait i d e n t i q u e p o u r t o u t g r a d i e n t de p r e e e i o n I1 est Bgalement possible de c o n s i d e r e r l a p r e s s i o n cornme UM inconnue suppl&nentaire, mais l e system o b t e n u n ' e s t p l u s h y p e r b o l i q u e s t r i c t , a t lee sch-s employ& i c i s o n t i n a p p l i c a b l e s .

le

e.

S i g M l o n s q u e les SchemaS proposes (Godounov ou schema a n t i d i f f u s e ) , s o n t des achemas standard en theorie des s y s t e m s h y p e r b o l i q u e s non l i n e a i r e s , e t q u ' i l eriste d e tres nombreux a u t r e s schemaa d ' a p p r o x i m a t i o n s qui r e s t e n t h tester ( a t e v e n t u e l l e m n t h adapter) sur ce d e l e . N o t o n s egalement l e faFble volume de c a l c u l s n e c e s s i t d s p a r n l a mise e n oeuvre du schema de Godounov, dana ce caa precis il est p a r f a i t e m e n t e n v i s a g e a b l e d ' e f f e c t u e r t o u t e la partie eimfmlation numerique s u r un micro-ordinateur.

Enfin, nous n'avons f a i t q u ' e f f l e u r e r l e p r o b l h e h p l u s i e u r s corps : si l a theorie est, du p o i n t de vue des schemas, h peu prbs independante du nombre de corps ( > l), l a mise e n o e u v r e de ces schemas n ' e s t sarement pas a u s s i s i m p l e p o u r p l u s d ' u n corps q u ' e l l e ne l ' e s t i c i p o u r un corps. M a i s ceci f e r a l'objet d'une a u t r e etude...

163

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L. NEEL, D. TONDEUR D i r e c t i o n s of r e v e r s i b l e mass and e n e r g y i n multicomponent e q u i l i b r i a . Implications i n separation science. P r e p r i n t . E.

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These d ' E t a t . U n i v e r s i t d d e P a r i s VI (1971).

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Symp. U n i v e r s i t y o f Wisconsin, [l3] P.D. LAX Shock waves and e n t r o p y , P r o c . e d i t e d by E.A. Z a r a n t o n e l l o , pp 603-634 (1971). 1141 H.K. W E , BODIN, N . R . AMUNDSON

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164 [ I S ] C.H. D ~ R E C O S The e n t r o p y rate admissibility c r i t e r i o n f o r s o l u t i o n o f J. D i f f . Equ. 14, pp 202-212 h y p e r b o l i c systems o f c o n s e r v a t i o n l a w s . ( 1976).

[ 1 6 ] T.P. LIU The e n t r o p y c o n d i t i o n and t h e admissibility of shocks. J. Math. Anal. and Appl. 53 pp 369-388 (1976). [17] O.A. OLEINIK Uniqueness and s t a b i l i t y o f t h e g e n e r e l i z e d s o l u t i o n o f t h e Cauchy problem f o r a q u a s i - l i n e a r e q u a t i o n . Amer. Math. SOC. Trans. S e r . 2, 33, p p 285-290 ( 1 9 6 3 ) .

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KRUSKOV F i r s t o r d e r q u a s i - l i n e a r e q u a t i o n i n several v a r i a b l e s . Math. USSR Sb. pp 217-243 51970).

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pp 297-322 (1976).

165

HISTORICAL INTRODUCTION AND GEL PACKING MATERIALS FOR HPLC SEPARATION OF PROTEINS AND NUCLEIC A C I D S

H i r o y u k i HATANO Department o f Chemistry, F a c u l t y o f Science, Kyoto U n i v e r s i t y , Kyoto 606

Japan

1. HISTORICAL OVERVIEW OF LIOUID CHROMATOGRAPHY I N JAPAN. Column l i q u i d chromatography and paper chromatography was i n t r o d u c e d succ e s s f u l l y i n t o chemicel r e s e a r c h and r e l a t e d f i e l d s i n Japan, a f t e r t h e development o f p a r t i t i o n chromatography by A.J.P.

M a r t i n , i n s e v e r a l books by S. Ku

wata ( 1 ) and by K. Satake ( 2 ) ( 1 9 5 2 ) . A book on i o n exchange r e s i n was p u b l i shed by M. Honda, H. Kakihana, and Y . Yoshino ( 3 ) i n 1955, and one on t h i n - l a y e r chromatography by Y . Hashimoto ( 4 ) i n 1962. S a t i s f a c t o r y r e s u l t s on i o n - e x change s e p a r a t i o n o f f i s s i o n p r o d u c t s ( r a d i o a c t i v e c o n s t i t u e n t s ) o f e x p l o s i v e ash f r o m n u c l e a r bombs i n B i k i n i I s l a n d s were p u b l i s h e d by K. Kimura and h i s c o l l e a g u e s ( 5 ) i n 1954. The Research Group of Automatic L i q u i d Chromatography i n Japan was o r g a n i zed i n 1958 b y H. Hatano. The Group has been m e e t i n g once a y e a r s i n c e 1958 and h o l d i n g workshops g i v e n by l e a d i n g r e s e a r c h e r s i n l i q u i d chromatography e v e r y summer s i n c e 1963, i n o r d e r t o modernize l i q u i d chromatographic t e c h n i q u e s and t o promote l i q u i d chromatographic r e s e a r c h . A book o f a u t o r a t i c amino a c i d a n a l y s i s was p u b l i s h e d i n 1959 by H. Hatano ( 6 ) , and a s e r i e s o f books on a u t o m a t i c and modern l i q u i d chromatography has been p u b l i s h e d s i n c e 1964 ( 7 - 1 3 ) . Data books on h i g h performance l i q u i d chromatography were p u b l i s h e d f r o m 1978 t o 1983 by Hatano's Research Croup ( 1 4 ) , under sponsorships o f t h e Japanese Soc i e t y f o r Promotion o f Science and o f The Promotion Bureau o f Science and Technology. These a c t i v i t i e s and e f f o r t s have been a c o n t i n u i n g and e f f i c i e n t c o n t r i b u t i o n t o t h e development o f modern l i q u i d chromatography. The f i r s t U.S.-Japanese

Seminar on Advanced Techniques o f L i q u i d Chrornato-

graphy was h e l d i n 1978 a t B o u l d e r , Colorado 115), and t h e second i n 1982 a t Honolulu, Hawaii ( 1 6 ) . They were o r g a n i z e d b y H. Walton and H. Hatano, and M. Novotny and D. I s h i i , r e s p e c t i v e l y , under sponsorships o f t h e N a t i o n a l Science

166

Foundation, and The Japanese Society for Promotion of Science. The 18th International Symposium : Advances in Chromatography, was held in 1982 in Tokyo (17), chaired by A. Zlatkis and L. Ettre, and organized by 6 . Muto, H. Hatano, N . Ikekawa and S. Hara. The International Symposium on High Performance Liquid Chromatography was held in 19

at Tokyo (18) and was organized by H. Hatano, un-

der sponsorships of The Japanese Society for Promotion of Science and of The Science Council of Japan. These meetings were also co-sponsored by The Chemical Society o f Japan, The Japanese Society for Analytical Chemistry, The Japanese Biochemical Society, The Agricultural Chemical Society of Japan, The Pharmaceutical Society of Japan and The Japanese Analytical Instruments Manufacturers' Association.

A historical review of the development

o f liquid chromatography in Japan,

written in 1981 by G. Muto (19), was included in "History of Analytical Chemistry in Japan" published by The Japanese Society for Analytical Chemistry. Recently, books on modern liquid chromatography have been pub1 ished by several authors (20-28). Instrumentation of an automatic 1 iquid chromatograph" equipped with a two-wavelength spetrophotometric detector was reported by

s. Egashira,

K. Ozawa,

S. Ganno and H. Hatano in 1961 (29) and 1962 (30). The liquid chromatograph consisted of a column chromatographic separation system equipped with a post-column chemically-derivatizing system that utilized an automatic fractionation mode, a two-wavelength spectrophotometric detector with a self-recording system, and an automatic operating system. The column was made from mantled glass, usually 0.9 cm in dia. by 100 cm in length, and equipped with stepwise elution reservo-

irs. Various sizes of column (dia, length) and packing materials (silica, ion-exchange resin, or size exclusion gel, etc.) could be used alternatively. It was easily replaceable with another column in the desirable separation mode. The two-wavelength spectrophotonetric detector enables the selection of two wavelengths from 200 to 750 nm for simultaneous spectrophotometric detection after colour-developing (chemically derivatizing) and self-recording. Between the column and the colour-developing vessel, a pH-adjusting system was equipped for regulating the pH of the eluent for the subsequent colour-developing (chemically derivatizing) of the eluted colourless compounds (for measuring other properties O

Hitachi Model KLF-1 Automatic Liquid Chromatograph produced by Hitachi Ltd. in 1961.

167 o f t h e e l u e n t s ) . The post-column d e v i c e c o n s i s t e d o f a m a n t l e d v e s s e l i n t o which a c o l o u r - d e v e l o p i n g o r c h e m i c a l l y d e r i v a t i z i n g r e a g e n t was i n t r o d u c e d a u t o m a t i c a l l y f r o m a r e a g e n t r e s e r v o i r . The a u t o m a t i n g r e g u l a t i n g system c o n s i s t e d o f e l e c t r o m a g n e t i c v a l v e s , e l e c t r i c t i m e r s and r e l a y s , and a c o n t r o l l i n g e l e c t r o n i c system which enables t h e o p e r a t i o n o f a l l p a r t s o f t h e chromatograph. Quant i t a t i v e o p e r a t i o n depending upon d r o p - c o u n t e r s was performed t h r o u g h o u t c h r o matographic s e p a r a t i o n and t h e s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n . P r e p a r a t i o n o f separated components was c a r r i e d o u t by f r a c t i o n c o l l e c t o r s b e f o r e t h e c o l o u r - d e v e l o p i n g o r a f t e r t h e s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n . Simultaneous two-wav e l e n g t h measurements p e r m i t t e d q u a l i t a t i v e s p e c i a t i o n o f t h e separated compounds, o f w h i c h r a t i o s o f absorbance a t t h e two wavelengths o r o f absorbance and anot h e r p r o p e r t y , were r e c o r d e d s i m u l t a n e o u s l y . The i n s t r u m e n t was e s p e c i a l l y usef u l b o t h f o r q u a n t i t a t i v e l y d e t e r m i n i n g t h e amounts o f t h e separated components and f o r q u a l i t a t i v e l y examining o t h e r p r o p e r t i e s s p e c t r o p h o t o m e t r i c a l l y .

For

example, simultaneous d e t e r m i n a t i o n o f an enzyme p r o t e i n and i t s enzymatic a c t i v i t y o f y e a s t a l c o h o l i c dehydrogenase was s u c c e s s f u l l y performed by t h e i n s t r u ment ( 3 1 ) . An amino a c i d a n a l y z e r " , which was equipped w i t h t h r e e c o n s t a n t - f l o w d e l i -

v e r y pumps and a t h r e e - w a v e l e n g t h f l o w p h o t o m e t r i c d e t e c t o r u t i l i z i n g a c o n t i -

nuous f l o w i n g mode, was produced by H. Hatano and h i s c o l l e a g u e s i m m e d i a t e l y a f t e r t h e above work. T h i s i n s t r u m e n t d i f f e r e d f r o m t h e well-known amino a c i d a n a l y z e r , d e s c r i b e d by S. Moore and h i s c o l l a b o r a t o r s , i n t h e f o l l o w i n g t h r e e p o i n t s : t h e a n a l y z e r by Hatano was equipped w i t h c o n s t a n t f l o w d e l i v e r y pumps, a t h r e e - w a v e l e n g t h f l o w - p h o t o m e t r i c d e t e c t o r and a new c y l i n d r i c a l programmer, w h i l e t h e a n a l y e e r used by Moore had a d j u s t a b l e d e l i v e r y pumps, a two-wavelength d e t e c t i o n system and mechanical t i m e r s . The t h r e e - w a v e l e n g t h p h o t o m e t r i c d e t e c t o r f o r t h e amino a c i d a n a l y s i s was used a t 570, 440, and 640 nm. The o r d i n a r y 570-nm-wavelength was f o r most e f f e c t i v e d e t e c t i o n o f n i n h y d r i n c o l o u r i n g o f o r d i n a r y alpha-amino a c i d s , t h e 440-nm-wavelength was f o r i m i n o a c i d s , p r o l i n e and h y d r o x y p r o l i n e , and t h e a d d i t i o n a l 640-nm-wavelength was f o r measurements o f o n e - t h i r d absorbance o f s t r o n g c o l o u r - d e v e l o p i n g by e x c e s s i v e amounts of e l u t e d amino a c i d s . The 640-nm d e t e c t i o n i s an a l t e r n a t i v e t o t h e a d d i t i o n a l c e l l w i t h o n e - t h i r d l i g h t - p a t h w h i c h O

H i t a c h i Model KLA-2 Automatic Amino A n a l y z e r produced b y H i t a c h i L t d . i n 1962.

168

was used i n Moore's amino a c i d a n a l y z e r . T h i s three-wavelength d e t e c t i o n , however, 'vas r o t o n l y s u c c e s s f u l f o r 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 o f t h e e x c e s s i v e amounts o f t h e e l u e n t s , b u t was a l s o e f f e c t i v e f o r q u a l i t a t i v e s p e c i a t i o n o f t h e separated c o n s t i t u e n t s by means o f t h e absorbance i n t e n s i t y r a t i o s a t t h e t h r e e wavelengths. F u r t h e r development o f a c o u l o m e t r i c d e t e c t o r was r e p o r t e d i n 1973 by Y. Tak a t a and G. Muto ( 3 2 ) , and o f a s p e c t r o f l u o r i m e t r i c d e t e c t o r i n 1973 by H. Hatano and h i s co-workers ( 3 3 ) f o r h i q h Derformance l i q u i d chromatography.

The

s p e c t r o f l u o r i m e t r i c d e t e c t o r enables t h e d e t e r m i n a t i o n o f e x t r e m e l y m i n u t e amounts o f f l u o r e s c e n t c o n s t i t u e n t s , and t h e d e t e c t i o n , b o t h u n i v e r s a l l y and s e l e c t i v e l y by means o f s e l e c t i v e l y chosino t h e e x c i t a t i o n and e m i s s i o n wavel e n g t h s , o f t h e e l u e n t s . The minimum d e t e c t i o n l i m i t a l l o w e d by t h e h i g h sens i t i v i t y o f t h i s d e v i c e was 1 x

O.D.

i n a c e l l volume o f 8

~ a1t l i g h t -

- p a t h o f 10 mm, and t h e s e l e c t i v e d e t e c t i o n p r o v i d e d q u a l i t a t i v e examination effectively

.

Concerning r e c e n t developments o f h i g h performance l i q u i d chromatography i n Japan, t h e a u t h o r has r e p o r t e d on an e l e c t r o n s p i n resonance s p e c t r o m e t r i c det e c t o r combined w i t h a h i q h performance l i q u i d chromatograph, which enables t h e s e p a r a t i o n of s t a b l e f r e e r a d i c a l s and trapped s p i n adducts o f u n s t a b l e r a d i c a l s ( 3 4 ) , used f o r t h e d e t e c t i o n o f f r e e r a d i c a l s . A r e v i e w paper on l i q u i d chromatographic d e t e c t o r s ( 3 5 ) was r e a d i n 1983 i n Melbourne and w i l l be p u b l i shed i n t h e Proceedings o f t h e I n t e r n a t i o n a l Conference on D e t e c t o r s and Chromatography. A new development i n m i c r o - i o n chromatoqraphy ( 3 6 ) was r e p o r t e d a t t h e 8 t h I n t e r n a t i o n a l Synymnosium on Column L i q u i d Chromatoqraphy i n 1984 i n New York. Several good works on m i n i a t u r i z a t i o n o f columns and i n s t r u m e n t s have been r e p o r t e d by D. I s h i i and h i s c o l l a b o r a t o r s s i n c e 1977 ( 3 7 ) , and by T. Tsuda and M . Novotny f o r m i c r o b o r e packed columns ( 3 8 ) . New developments i n i o n chromatography have been r e p o r t e d by Y. Hanaoka and h i s coworkers (39), and by S. Rokushika and h i s c o l l a b o r a t o r s ( 4 0 ) . Hyphenated systems o f a h i a h performance l i q u i d chromatograph w i t h a mass spectrometer have been d e s c r i b e d by T. Takeuchi and h i s c o l l a b o r a t o r s ( 4 1 ) and by S. Tsuqe ( 4 2 ) ; w i t h i o n s e l e c t i v e e l e c t r o d e s by N. I s h i b a s h i and h i s c o l l a b o r a t o r s ( 4 3 ) ; w i t h a l a s e r - f l u o r e s c e n c e d e t e c t o r by T. Imasaka and h i s c o l l a b o r a t o r ( 4 4 ) ; and w i t h a s p e c t r o s c o p i c d e t e c t i o n system by K. J i n n o and h i s c o l laborators (45).

169 2. HPLC SEPARATION OF PROTEIN AND NUCLEIC A C I D 2.1 Gel p a c k i n g m a t e r i a l and column t e c h n o l o g y Enormous e f f o r t s have been made i n p r e p a r i n g v a r i o u s porous p o l w e r g e l s f o r s i z e e x c l u s i o n and ion-exchange chromatography b y T. Yamabe ( 4 6 ) , by S. Tak a i (47-48), and by S. R o k u s h i l a and H. Hatano ( 4 9 ) . These aqueous and non-aque-

ous porous pol.mer g e l s have been oroduced and t h e p r o d u c t s have been a v a i l a b l e c o m m e r c i a l l y f r o m t h e teams o f S . Ganno, H i t a c h i L t d . , as H i t a c h i - q e l s e r i e s , from S. Nakamura, Showa Denko Co., as Shodex-gel s e r i e s , f r o m Y. i s h i d a , Shimadzu L t d . , as Shimadzu-qel s e r i e s , f r o m T . Hashimoto, Toyo Soda Co.,

as TSK-gel

s e r i e s , and f r o m K. Noquchi, Asahi Kasei Co., as Asahi-gel s e r i e s , and f r o m Jasco Co., as Finepak-gel s e r i e s , and f r o m M i t s u b i s h i Kasei Co., ries

as M C I g e l se-

000

The p o l y s t y l e n e t y p e g e l s a r e copolymers o f p o l y s t y r e n e - d i v i n v l benzene, E s t e r - t y p e s o f g e l s a r e p o l y a c r y l a t e , p o l y m e t h a c r y l a t e o r p o l y v i n y l a c e t a t e . The o t h e r g e l s a r e hydroxyrnethyl, c a r b o x y l , q u a r t e r n a r y ammonium; and s u l f o n a t e d d e r i v a t i v e s o f t h e copolymers. These q e l s have l a r g e s u r f a c e areas g r e a t e r t h a n

300 m2 p e r 9, and i n ranges o f p o r e s i z e s f r o m 40 t o 1000

a and o f

particle

s i z e s f r o m 5 t o 2 0 p m . A l l o f these q e l s , employing b o t h aequous methanol and non-aequous hexane-methanol, d i s p l a y m o b i l e phases i n t h e normal o r r e v e r s e d phase. Important c h a r a c t e r i s t i c s o f the packing m a t e r i a l s for separation o f b i o l o g i c a l l y a c t i v e molecules a r e n o t o n l y h i q h performance on t h e s e p a r a t i o n b u t a l s o t h e complete r e c o v e r a b i l i t y o f t h e a c t i v i t y d u r i n q t h e s e p a r a t i o n . Moderat e s e p a r a t i o n and e f f e c t i v e r e c o v e r y of t h e a c t i v i t y can be o b t a i n e d on s i z e e x c l u s i o n g e l columns. Ion-exchange r e s i n i s a t r a d i t i o n a l column-packing m a t e r i a l f o r s e p a r a t i o n of r e l a t i v e l y small m o l e c u l e s such as amino a c i d s and n u c l e i c a c i d c o n s t i t u e n t s . Recently, t h e p r o p e r t i e s o f t h e ion-exchangers have been improved f o r s e p a r a t i o n

o f l a r g e molecules such as p r o t e i n s and n u c l e i c a c i d s . Progress i n t h e separat i o n of biomolecules has been r e p o r t e d a l s o i n r e v e r s e d phase l i q u i d chromatog r a p h i c techniques. Developments o f h i g h performance columns ( 5 0 ) and p a c k i n g m a t e r i a l s (51) were d e s c r i b e d . 2.2 Packing m a t e r i a l s and columns f o r HPLC s e p a r a t i o n o f p r o t e i n and n u c l e i c acid. P r o t e i n s and n u z l e i c a c i d s a r e p o l y - v a l e n t i o n i c compounds w i t h l a r g e mole-

170 c u l a r w e i g h t s and t h e y sometimes show o l i q o m e r i c a s s o c i a t i o n b e h a v i o u r s i n s o l u t i o n . These p r o p e r t i e s o f p r o t e i n and n u c l e i c a c i d molecules make i t p o s s i b l e t o separate c h r o m a t o g r a p h i c a l l y on t h r e e modes o f i o n exchange by t h e i o n i c d i s s o c i a b l e p r o p e r t y , o f s i z e e x c l u s i o n b y t h e l a r g e m o l e c u l a r s i z e s , and o f r e v e r s e d phase i n t e r a c t i o n by t h e hydrophobic p r o p e r t y o f l a r g e m o l e c u l a r ske1eton. Packing m a t e r i a l s o f porous Dolymer g e l f o r h i g h performance l i q u i d c h r o matographic columns can be c l a s s i f i e d i n t o t h r e e k i n d s o f g e l ; w h o l e - i n o r g a n i c s i l i c a , whole-organic s y n t h e t i c Dolymer, and s i l i c a bonded c h e m i c a l l y w i t h o r g a n i c molecules. High r e s o l u t i o n a b i l i t y o f t h e s e g e l packings i s caused by nomogeneous s p h e r i c a l f i n e p a r t i c l e s w i t h e x t r e m e l y small p a r t i c l e diameter, w i t h an homogeneously d e s i r a b l e p o r e - s i z e , and a l s o w i t h homogeneous pore-depth. Among these p r o p e r t i e s o f t h e g e l packinqs, i t i s r ' e s i r a b l e t h a t t h e p o r e - s i z e i s r e l a t i v e l y l a r g e f o r s e p a r a t i o n o f l a r g e b i o m o l e c u l e s and t h a t t h e l o a d i n g c a p a c i t y i s as l a r g e as p o s s i b l e f o r p r e p a r a t i o n o f p o s s i b l e l a r g e amounts o f t h e b i o m o l e c u l e s (e.g. l o a d i n g c a p a c i t y o f t h e g e l p a c k i n g s w i t h a p o r e - s i z e o f 300

1

i s about 50-100 k i l o - d a l t o n ) . I t seems t h a t column-size ( i . e . l e n g t h

and i n n e r d i a m e t e r )

, which

i s i m p o r t a n t f o r s e p a r a t i o n o f s m a l l molecules w i t h

l o w m o l e c u l a r weiqhts, such as amino a c i d s and n u c l e i c bases, i s o f r e l a t i v e l y l e s s importance f o r t h e s e p a r a t i o n o f l a r g e b i o m o l e c u l e s . G e n e r a l l y

, gel

pa-

c k i n q m a t e r i a l s f o r s i z e e x c l u s i o n chromatograohy can be c l a s s i f i e d i n t o t h r e e c a t e g o r i e s , depending upon t h e i r mechanical s t r e n g t h : s o f t g e l , semi-hard g e l and hard g e l . The polystyrene-divinyl-benzene copolymer i s i n a semi-hard g e l group and u s e f u l f o r h i g h performance s i z e e x c l u s i o n chromatography. A l l TSK- g e l H type, Shodex g e l A t y p e and Shimadzu-gel HSG and H i t a c h i GELKO GL s e r i e s a r e p o l y s t y r e n e g e l and can be used f o r non-aqueous g e l permeation chromatography o f s y n t h e t i c polymer compounds, m o l e c u l a r w e i g h t s o f w h i c h a r e below about 10'.

TSK-gel I4 t y p e ( p o l y e t h y l e n e g l y c o l d i m e t h a c r y l a t e )

, LS

type (sulphonated

p o l y s t y r e n e g e l ) and PW t y p e ( p o l y a c r y l a m i d e ) , Shodex OH-pak ( g l y c e r y l methac r y l a t e ) , and Asahi-pak GS t y p e ( p o l y v i n y l a l c o h o l ) and Shim-pak W type, H i t a c h i GL-W520, can be used f o r aqueous g e l f i l t r a t i o n chromatography o f b i o l o g i c a l , h i g h m o l e c u l a r w e i g h t compounds such as p r o t e i n s and n u c l e i c a c i d s , e x c l u s i o n l i m i t s of which a r e below about lO'.TOYO-PEARL

HW t y p e i s a h y d r o p h i l i c v i n y l -

-polymer and c a n ' b e used i n wide ranges o f e x c l u s i o n l i m i t s f r o m 1 0 3 t o

lo7,

for

171 p r e p a r a t i o n o f t h e biopolymers. B i o l o g i c a l a c t i v i t i e s such as t h o s e o f enzymes s h o u l d n o t be l o s t d u r i n g t h e s e p a r a t i o n procedures. The whole-organic s y n t h e t i c polymer g e l m i g h t have most d e s i r a b l e p r o p e r t i e s f o r t h e s e p a r a t i o n o f t h e b i o l o g i c a l l y a c t i v e compounds. 2.3 Gel packings f o r s i z e e x c l u s i o n chromatography o f p r o t e i n s and n u c l e c acids. Porous s y n t h e s i z e d p o l y m e r g e l s o f m e t h a c r y l a t e , amide, and p o l y v i n y l r e s i n , and porous s i l i c a g e l s have been u s i n g f o r aqueous g e l f i l t r a t i o n o f b i o l o g i c a l l y a c t i v e molecules. P r e s s u r e - r e s i s t a n t and l e s s a b s o r p t i v e aqueous polymer g e l s a r e l i s t e d i n Table l. TABLE 1 Gel packings f o r aqueous s i z e e x c l u s i o n chromatography o f b i o m o l e c u l e s (comm e r c i a l l y a v a i l a b l e i n Japan).

L

E

Trade name

Hydrophilic methacrylate

M C I GEL C Q P l O 30 Shodex OH-pak B-804 TSK GEL PW300 PW400 T O Y 0 PEARL HW-series

10

Polyvinyl alcohol

Asahi -pak 1x31 0,320 GS510,520

9

Glycerylpropyl s i l i c a

Shodex AQ-pak R403 R404 Shim-pak D i o l - 1 5 0 Diol-300 M C I GEL C Q S l O CQS30 TSK GEL SW-series

; ; Glyceryl methacrylate L V

P o l y a c r y l amide

.r

CI

2 c,

2s

Gel m a t e r i a l

Hydrophil i c vinylpol-mer

gz‘g ‘ijiit loo# 300

[r

>> v,

m

V

.r .r

Hydroxyl s i l i c a

Ln

50

Hydrophilic s i l i c a

L

0

a

Glyceryl ether s i l i c a

10 10 5 5 9-10 9-10 10-30

100 300

100 300 130 240 450

SUPPl

ier

M itsubishi -Kasei 5x105 Showa Denko 3x1 O’wwToyo Soda 1X l O 6 5x1 O3 5x1 07 3x105 Asahi Kasei

9x104 8x105 2x10’ 2x1 06

Showa Denko Shimadzu M itsubi shi -Kasei

6x104 3x105 1x106

The most d e s i r a b l e p o r e - s i z e seems t o be 100-500 A f o r t h e s e p a r a t i o n o f p r o t e i n s and t h e e x c l u s i o n l i m i t s o f d i f f e r e n t k i n d s o f g e l m a t e r i a l s should be chosen a c c o r d i n g t o t h e k i n d o f sample m i x t u r e s . G l y c e r y l - p r o p y l s i l i c a , w h i c h has a s l i o h t l y a b s o r p t i v e p r o p e r t y and shows d i s c r e p a n c i e s from 1 i n e a r r e l a t i o n s h i p s between r e t e n t i o n volumes and l o g a r i t h m s o f m o l e c u l a r w e i g h t s , has been improved f o r more e f f i c i e n t r e c o v e r i e s

172 o f b i o l o g i c a l a c t i v i t i e s o f t h e biomolecules by t r e a t m e n t s w i t h g l y c e r y l coating. One i n t e r e s t i n g example o f p r o t e i n s e p a r a t i o n on t h e TSK g e l columns was r e p o r t e d f o r l i p o p r o t e i n s , comparing t h i s method w i t h t h e r e s u l t s o f u l t r a c e n t r i f u g a l s e p a r a t i o n ( 5 3 ) , and m e d i c a l l y s i g n i f i c a n t components o f t h e l i p o p r o t e i n s r e l a t e d t o l i v e r d i s e a s e have been c l a r i f i e d i n t h e f i e l d o f c l i n i c a l medicine. A p p l i c a t i o n o f t h e polymer packings t o s e p a r a t i o n o f o l i g o n u c l e o t i d e s has been r e p o r t e d ( 5 4 ) u s i n g t h e Asahi-pak GS320 columns. Separat i o n s o f o l i g o n u c l e o t i d e s i n a m i x t u r e o f t h r e e k i n d s o f deoxyhexamers (dCGTCCA, dTGTCCA, and dGGTCCA), and o f ribosomal r i b o n u c l e i c a c i d s (rRNA) i n a m i x t u r e o f 23s (HW:1,100,000),

16S(550,000)

and 5s (PIW:160,000)

rF?NA p a r t i -

c l e s were performed s u c c e s s f u l l y on t h e Asahi-pak GS320 columns r e s p e c t i v e l y (55), and i t has been shown t h a t t h e s e g e l packings a r e v e r y u s e f u l i n t h e newly d e v e l o p i n g f i e l d s o f g e n e t i c s and b i o - t e c h n o l o g y . 2.4 I o n exchange chromatography o f p r o t e i n s and n u c l e i c a c i d s I o n exchange chromatography u s i n g sulphonated p o l y s t y r e n e columns has been t h e t r a d i t i o n a l method f o r a n a l y s i s o f amino a c i d c o n s t i t u e n t s o f p r o t e i n s , and n u c l e i c bases, nucleosides, and n u c l e o t i d e s o f n u c l e i c a c i d s . The f i r s t amino a c i d a n a l y s i s on t h e ion-exchange r e s i n column a t S. Moore's L a b o r a t o r y i n 1958 was a memorial e v e n t i n t h e f i e l d s o f i n s t r u m e n t a l l i q u i d chromatography and p r o t e i n c h e m i s t r y . P r o t e i n s e p a r a t i o n had been c a r r i e d o u t by i o n exchange chromatography s i n c e 1954. However, t h e s t r o n g l y a c i d i c c a t i o n exchangers a r e i r r e v e r s i b l y a b s o r b a b l e on p r o t e i n s and n u c l e i c a c i d s owing t o h y d r o p h o b i c i t y o f t h e i r p o l y s t y r e n e s t r u c t u r e s . Porous g l a s s , which bonds chem i c a l l y w i t h aminopropyl t r i m e t h o x y s i l a n e ,

i s a1 so absorbable n o n - s p e c i f i c a l l y

owing t o r e s i d u a l s i l a n o l groups on t h e s u r f a c e o f t h e porous g l a s s . H i q h p e r formance s e p a r a t i o n by ion-exchanqe i s performed by u s i n g ion-exchangers coat e d w i t h epoxy o r polyamine. Three k i n d s o f ion-exchangers a r e now a v a i l a b l e : p e l l i c u l a r coated ion-exchanger on i n a c t i v e c a r r i e r s , whole porous s i l i c a i n troduced ibn-exchangeable groups, and s y n t h e t i c polymer g e l s i n t r o d u c e d i o n -exchangeable groups. The ion-exchangers f o r r a p i d and h i g h performance separ a t i o n s h o u l d be m e c h a n i c a l l y s t r o n g enough t o o b t a i n a l i n e a r v e l o c i t y o f 1 mm/sec; i r r e v e r s i b l y non-absorbable f o r h i g h e r r e c o v e r i e s , have l a r g e exchange c a p a c i t y f o r p r e p a r a t i o n (30 t o 100 nm i n p o r e d i a m e t e r and 0.5 t o 1.0

173

m l / g i n pore d e p t h ) , be s t a b l e f o r wide ranges o f pH, show homogeneously spher i c a l shape w i t h 5 t o 1 0 y m i n d i a m e t e r , and o f course, be l e s s expensive even f o r l a r g e p r e p a r a t i v e columns. The a v a i l a b l e i o n exchangers f o r s e p a r a t i o n o f p r o t e i n s and n u c l e i c a c i d s a r e presented i n Table 2 ( 5 2 ) . Table 2 I o n exchange f o r h i g h performance l i q u i d chromatographic s e p a r a t i o n o f p r o t e i n s , n u c l e i c a c i d s and t h e i r c o n s t i t u e n t s . Ma t e r ia 1 Trade name

Functional group

dp Am

Whole TSK GEL DEAE 2SW -N(C,H,), porous 3 silica CM -COOH Shodex Axpak U424 Dolyamine Porous P o l y a c r y l a m i d e g e l qel TSK GEL SP5Pld -so DEAE -N(?2F5)8 PnlyHitachi resin -S03-(Na ) 2600 s e r i e s styrene DVB Shodex C X pak -S03-(Nat) Shim pack ISC07 -SO,M C I GEL CK -sop -SO, Jasco AA pak

5 10 10 10

Type

sw

Exchanqe Pore catzcjty size

Supplier

0.30.30.30.3-

130A 240.5 240j 300A

Toyo Soda

i n , i 5 0.310,15 0.313-17

ioood

~ o y oSoda

7 597 5

Showa Denko

l0OOA Hitachi Showa Denko S h imad zu F l i t s u b i s h i Kasei Jasco

The TSK GEL 2SW i s s u i t a b l e f o r s e p a r a t i o n of p e p t i d e s , n u c l e o t i d e s and o t h e r r e l a t i v e l y s m a l l molecules, and a l s o f o r o l i g o m e r s . The TSK GEL 3SW and Shodex GEL Axpak U424 a r e s u i t a b l e f o r s e o a r a t i o n o f p r o t e i n s and enzymes. F o r these s e p a r a t i o n s o f l a r q e molecules such as p r o t e i n s and enzymes, i t i s b e t t e r t o use t h e p o l y a c r y l a m i d e o r p o l p e t h a c r y l a t e polymer g e l s , which a r e l e s s abs o r b a b l e on p r o t e i n s , r a t h e r t h a n u s i n q t h e p o l y s t y r e n e d i v i n y l b e n z e n e c o p o l y mer g e l s . The TSK GEL and t h e Shodex Gel a r e q e l p a r t i c l e s w i t h s m a l l d i a m e t e r s of 5 t o 10,um. However, t h e macroDorous q e l w i t h l a r q e r p a r t i c l e d i a m e t e r s o f 30 t o 7 0 p m seems t o be b e t t e r f o r t h e p r e p a r a t i v e uses o f l a r g e r b i o m o l e c u l e s . The TSK GEL PI4 s e r i e s c o n s i s t s o f p o l y a c r y l a m i d e q e l p a r t i c l e s w i t h l a r g e 0

Dore s i z e s o f about 1000 A which a r e v e r y e f f i c i e n t i n t h e s e p a r a t i o n o f p r o t e i n s and n u c l e i c a c i d s . B e a u t i f u l s e p a r a t i o n p a t t e r n s o f m i x t u r e s o f o l i g o d e o x y n u c l e o t i d e s have been o b t a i n e d on t h e TSK GEL DEAE-PW5OO by K. Makino and h i s c o l l a b o r a t o r s ( 5 5 ) . The m i x t u r e s c o n t a i n e d e i q h t k i n d s o f o l i g o - d e o x y n u c l e o t i des f r o m hexamer t o 7 6 - r e s i d u a l o l i g o m e r ; dCPTGGT, dCTAAATC, dCGGGATTTGA, dCGACCCGGGT, dCATCTTCATGX, unknown s e q u e n t i a l 1 6 - r e s i d u a l oligomer,dCCIAAITCCATCCAI-

174 C C I T A I G C , dCCIAAITCCATCCAICCCATITAITC (D:deoxy, C : c y t i d i n e , ne, I : i n o s i n e ,

T: thymine). Some polystyrene-DVB

A:adenine,

G : guani

-

copolymers ( T a b l e 2), such as

t h e H i t a c h i 2600 s e r i e s e t c , a r e an e f f i c i e n t ion-exchanger f o r a n a l y s s o f amino a c i d s . 2.5 Reversed phase chromatography o f p r o t e i n s and n u c l e i c a c i d s . Hydrophobic and h y d r o p h i l i c i n t e r a c t i o n s among c h e m i c a l l y bonded r e v e r s e d phase packings, aqueous m o b i l e phase s o l v e n t s w i t h o r g a n i c m o d i f i e r s , and amp h o t h e l i c molecules of p r o t e i n s and n u c l e i c a c i d s , a r e l i k e l v t o be e x t r e m e l y c o m p l i c a t i n q f a c t o r s f o r e f f i c i e n t s e D a r a t i o n b e h a v i o u r s . However, i n p r a c t i c e , v a r i o u s k i n d s o f r e v e r s e d phase packinqs have been a v a i l a b l e f o r e f f i c i e n t sep a r a t i o n o f p r o t e i n s and n u c l e i c a c i d s . P r o p e r t i e s o f s e v e r a l p a c k i n q m a t e r i a l s f o r s e p a r a t i o n o f p r o t e i n s and n u c l e i c a c i d s a r e p r e s e n t e d i n T a b l e 3 ( 5 6 ) , and p r o d u c t s o f t h e packings a r e shown i n Table 4 ( 5 6 ) . S e p a r a t i o n of cytochromp c, myoglobin, r i b o n u c l e a s e , lyzozyme, alpha-chymot r i p s i n , and alpha-chymotripsinogen was c a r r i e d o u t - s u c c e s s f u l l y on t h e TSK g e l Phenyl-5PW column ( 5 7 ) . T h i s r e c e n t l y developed g e l p a c k i n g i s one o f var i o u s aqueous polymer t y p e s c h e m i c a l l y bonded w i t h phenyl groups and used e f f i c i e n t l y i n t h e f i e l d s o f p r o t e i n s e p a r a t i o n . However, t h e s e p a r a t i o n mechanism seems t o be r a t h e r more c o m p l i c a t e d than t h e s o l v o p h o b i c i n t e r a c t i o n i n r e v e r sed phase chromatography. TABLE 3 F u n c t i o n a l groups o f c h e m i c a l l y bonded r e v e r s e d phase p a c k i n g m a t e r i a l s ( 5 6 ) . Polarity

Functional group

Loading amount %w/w

Remark

none

CH3(CH,),,-

10-25

p a r t i c l e shape, s i z e , s u r f a c e area, p o r e s i z e a r e d i f f e r e n t f r o m each s u p p l i e r and others less hydrophobicity

CH,(CH,),C6H11C6H5-

5-15 -10 -10

CH, (CH, 12-

hiqh

CH3CH2-

1-10

CH,OOHCN NO,NH,-

5 5-10 -5 2-1 0

r e l a t i v e l y s m a l l p e p t i d e s and hormones containing aromatic r i n g s t r u c t u r e s weaker i n t e r a c t i o n t o n o n - p o l a r compounds t h a n C, s p e c i a l l y s e l e c t i v e t o some k i n d s o f proteins

175

TABLE 4 C h e m i c a l l y bonded r e v e r s e d phase p a c k i n g m a t e r i a l s ( a v a i l a b l e i n Japan) ( 5 6 ) Polarity

Trade name

Materials

dp

F u n c t i o n a l group

Supplier

octadecylsilane

Hitachi

octadecyl s i lane

Yanaco

o c t a d e c y l s i 1ane (capping) octadecyl s i l a n e o c t a d e c y l s i l ane

Jasco Showa Denko Nomura Kagaku

o c t a d e c y l s i 1ane

Nakarai Kagaku

octadecyl s i l a n e ( c a p p i n?)l o c t y l s i 1ane octylsilane o c t y l s i 1ane

Toyo Soda

PN none

H i t a c h i g e l #3050 #3053 #3056 #3063 ODs-N Yana pak ODs-A ODs-T Yanaco Pel ODs Fine-pak S I L C18 ODS Dak F Devei o s i 1

Cosmosil TSK GEL F i n e pak Cosmosil Devel o s i 1

Shim-pack Cosmosil F i n e pak Fime pak Yanapa k Cosmosil Shim-pak TSK GEL

4-6 4-6 5 5 7 10 30-40 5,lO 10 C18T series 5,10 ODs 3,5,7,10 10-20 15-30 ~CUI 5 5Ci8-P 5 ODs-120 5,lO 120T 5,lO 10 SIL c* 5C8 5 C8 3,5,7,10 10-20 15-30 Pc8 10 5PH 5 SIL c, 10 10 SIL c DYS 5 5TNS 5 TMS 5 TMS-250 10

Jasco Nakarai Kagaku Nomura Kagaku

octyl silane phenyl ethyl diethylsilane dimethylsilane trimethylsilane t r i m e t h y l s i l ane trimethylsilane

S h imadzu Nakarai Kagaku Jasco .Jasco Yanaco Nakarai Kagaku Shimadzu Toyo Soda

weak

F i n e pak TSK GEL

S I L OH OH-120

10 5,lO

hydroxyl hydroxyl

Jasco Toyo Soda

medi um

F i n e pak Yana pak Yanaco Pel Cosmosi 1

SIL CN CN CN 5CN-R

10 10 35

nitrile nitrile nitrile n i t r i 1e

Jasco Yanaco Yanaco Nakarai Kagaku

p o l yami de

Yanaco

amino amino amino amino aminopropyl

Shimadzu Yanaco

high

Yanaco Pel

PA

S him-pa k

PNH,

Yanaco Pel Yana pak Shodex F i n e pak

NH2 NH2

10 35 10

NH pak SIL NH,

10

Showa Denko Jasco

176

TSK GEL

NH,-60

B i l e pak Catechol Dak

5,lO

aminopropyl

Toyo Soda Jasco Jasco

10 5

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"

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9. H. Hatano (Ed.), New L i q u i d Chromatography, Nankodo Co., Tokyo, 1969. 10. H. Hatano (Ed.), High Speed L i q u i d Chromatography, Uankodo Co., 1973. 11. H. Hatano ( E d . ) , A p p l i e d L i q u i d Chromatography, Nankodo Co.,

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T. Yamabe (Ed.), I n t r o d u c t i o n t o High Performance L i q u i d Chromatography, Saiwai Book Co., Tokyo, 1978.

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Tokyo, 1983.

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239 (1982)

178 0

The H i t a c h i S c i . I n s t r . News, 15 (1972) 10.

"47.

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48.

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49.

S. Rokushika and H. Hatano, (1. Chromatoor.,

176 (1979) 456.

'50.

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"51.

Y. Kato and

"52.

H. Hatano ( E d . ) , Report o f t h e P h y s i c a l and Chemical P r o p e r t y Data, L i q u i d Chromatography Data, V o l . 5, p r o m o t i o n Bureau, Science and Technology Agency, Japan.

T.

Hashimoto, BUMSEKI, (10) (1984) 725.

53.

M. Okazaki e t a l . ,

54.

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55.

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"56.

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"57.

Toyo Soda Catalog, TSK q e l Phenyl-5PW, 1984.

Oa0

Correspond w i t h : Asahi Kasei Co., Yak0 1-3-2, Kawasaki-ku, Kawasaki 210 Japan H i t a c h i L t d . , Naka Works, I c h i q e 887, Katuta, I b a r a k i 312 JaDan Jasco Co., Ishikawa-cho, H a t h i o j i , Tokyo, 192, Japan M i t s u b i s h i Kasei Co., 5-2, Marunouchi 2-chome, Chiyoda-ku, Tokyo, Japan Shimadzu L t d . , Sanjo Works, N i s h i - n o - k y o Kuwabara-cho 1 , Nakagyo-ku, Kyoto 604 Japan Showa Denko Co., Shiba-Daimon 1-13-9, Yinato-ku; Tokyo 105 Japan Toyo Soda Co., Tohsoh Bldg., Akasaka 1-7-7; Minato-ku, Tokyo 107 Japan

i n Japanese

179

DISPLACEWENT CEEOHATOGEAPEY: YESTERDAY, TODAY AHD T0IK)BROU CSABA BORVATB

Department of Chemical E n g i n e e r i n g , Yale U n i v e r s i t y , New Haven, C o n n e c t i c u t 06520, U.S.A.

INTRODUCTION Gas chromatography, t h i n l a y e r chromatography and h i g h performance l i q u i d

chromatography i n t h e e l u t i o n m o d e have become t h e most w i d e l y used t e c h n i q u e s f o r c h e m i c a l a n a l y s i s of multicomponent m i x t u r e s o v e r t h e p a s t t h i r t y y e a r s .

As

a r e s u l t t h e t e r m c h r o m a t o g r a p h y h a s become synonymous w i t h l i n e a r e l u t i o n chromatography.

Concomitantly, most c h r o m a t o g r a p h i c t e x t s p u b l i s h e d d u r i n g t h e

l a s t t w e n t y y e a r s h a v e i g n o r e d d i s p l a c e m e n t d e v e l o p m e n t , t h e o t h e r mode of chromatography f o r s e p a r a t i n g m u l t icomponent mixtures according t o Tiselius’ c l a s s i f i c a t i o n (1,2). Indeed, t h e l i n e a r e l u t i o n mode of chromatography i s e m i n e n t l y s u i t a b l e f o r c h e m i c a l a n a l y s i s s i n c e t h e e l u i t e s t r a v e l down t h e column w i t h d i f f e r e n t v e l o c i t i e s a s i n d i v i d u a l quasi-Gaussian peaks which can be e a s i l y d e t e c t e d by h i g h s e n s i t i v i t y . Under c o n d i t i o n s of l i n e a r chromatography, which a r e a t t a i n e d w i t h s m a l l s a m p l e s , t h e r e t e n t i o n v a l u e s ahd peak s h a p e s a r e independent o f t h e sample l o a d i n g so t h a t r e p r o d u c i b l e r e s u l t s c a n be r e a d i l y o b t a i n e d i n q u a n t i t a tive analysis with the precision instrumentation available. As a result the v a r i o u s chromatographic t e c h n i q u e s u s i n g t h e l i n e a r e l u t i o n mode have grown i n t o m i c r o a n a l y t i c a l t o o l s of unsurpassed v e r s a t i l i t y , i n g A.J.P.

Martin’s

s e m i n a l w o r k s (3-5).

speed and s e n s i t i v i t y f o l l o w -

Furthermore, t h e t h e o r y of l i n e a r

chromatography has been developed t o a high l e v e l of s o p h i s t i c a t i o n and a g r e a t d e a l of e x p e r i e n c e h a s been accumulated over t h e p a s t decades t o a i d i t s a p p l i c a t i o n t o v a r i o u s a n a l y t i c a l problems. C o n d i t i o n s of l i n e a r a d s o r p t i o n chromatography, however, a r e a t t a i n e d o n l y when t h e c o n c e n t r a t i o n of t h e e l u i t e i s very low b o t h i n t h e m o b i l e phase and a t t h e s u r f a c e of t h e s t a t i o n a r y phase.

Consequently,

p r e p a r a t i v e chromatography

i n t h e e l u t i o n mode i s u s u a l l y a s s o c i a t e d w i t h a r a t h e r p o o r u t i l i z a t i o n o f column c a p a c i t y and equipment.

N e v e r t h e l e s s , t h e r e l a t i v e e a s e of s c a l i n g - u p an

a n a l y t i c a l s e p a r a t i o n led t o a predominant u s e of e l u t i o n chromatography a l s o i n p r e p a r a t i v e work today,

In contrast,

t h e d i s p l a c e m e n t mode of chromatography,

i n o b l i v i o n f o r so long, o f f e r s d e f i n i t e a d v a n t a g e s i n p r e p a r a t i v e s c a l e s e p a r a t i o n s due t o t h e r e l a t i v e l y h i g h column l o a d i n g and t h e r e s u l t i n g high concent r a t i o n of t h e feed components i n t h e e f f l u e n t .

180 Displacement development can be used i n a l l branches of i n t e r a c t i v e chromatography a l t h o u g h i n t h e p r e s e n t t r e a t m e n t it w i l l be d i s c u s s e d o n l y w i t h r e g a r d t o h i g h performance l i q u i d chromatography, HPLC.

For t h e development of chroma-

togram, t h e s o l u t i o n o f a d i s p l a c e r s u b s t a n c e , which h a s g r e a t e r a f f i n i t y t o t h e s t a t i o n a r y phase t h a n any of t h e f e e d components, i s i n t r o d u c e d behind t h e feed. The d i s p l a c e r f r o n t d r i v e s t h e f e e d c o m p o n e n t s t h r o u g h t h e c o l u m n w h i l e t h e y m u t u a l l y d i s p l a c e each o t h e r and s e p a r a t e i n t o a d j a c e n t bands. isotachic

Upon r e a c h i n g

c o n d i t i o n s t h e zones of a l l components move w i t h t h e v e l o c i t y of t h e

d i s p l a c e r f r o n t . I n p r i n c i p l e , t h e t e c h n i q u e i s analogous t o i s o t a c h o p h o r e s i s w h i c h was o r i g i n a l l y c a l l e d " d i s p l a c e m e n t e l e c t r o p h o r e s i s " by i t s i n v e n t o r A.J.P.

Martin.

The s e p a r a t i o n i s b a s e d o n c o m p e t i t i o n o f t h e c o m p o n e n t s f o r

a d s o r p t i o n s i t e s on t h e s t a t i o n a r y p h a s e and t h e p r o c e s s i s t h e r e f o r e nonlinear.

I n o r d e r t o o b t a i n a c l e a n s e p a r a t i o n of t h e components h i g h column

e f f i c i e n c y i s r e q u i r e d which had n o t been a v a i l a b l e b e f o r e t h e a d v e n t of HPLC. P r o g r e s s i n t h i s f i e l d has a l s o been impeded by t h e t h e o r y of n o n - l i n e a r chromatography b e i n g more c o m p l i c a t e d t h a n t h a t of l i n e a r e l u t i o n chromatography and by t h e p a u c i t y of e x p e r i m e n t a l support i n t h e l i t e r a t u r e f o r s e l e c t i n g o p e r a t i n g c o n d i t i o n s i n d i s p l a c e m e n t chromatography t h a t h a s been viewed a s a c u r i o s i t y r a t h e r t h a n a p r a c t i c a l method o v e r t h e p a s t t w e n t y years. Advances i n HPLC have brought on column m a t e r i a l s and columns f a r s u p e r i o r i n terms of e f f i c i e n c y t h a n t h o s e employed i n l i q u i d chromatography before. T h i s prompted r e c e n t work (6-8) i n o u r l a b o r a t o r y t o r e v i v i f y d i s p l a c e m e n t development by u s i n g advanced t o o l s of modern chromatography and e x p l o r e i t s p o t e n t i a l f o r p r e p a r a t i v e l i q u i d chromatography. I n t h i s r e p o r t a b r i e f h i s t o r i c a l overview and o u t l i n e of t h e t h e o r y a r e f o l l o w e d by a c o n c i s e e x a m i n a t i o n of r e c e n t developments i n h i g h performance displacement chromatography.

The r e a d e r i s a d v i s e d t o c o n s u l t t h e o r i g i n a l

publications given a s references f o r f u r t h e r d e t a i l s .

EISTOBICAL OVERVIEW A t h i g h column o v e r l o a d d i s p l a c e m e n t of one sample component by a n o t h e r may

o c c u r i n e l u t i o n c h r o m a t o g r a p h y a s was a l r e a d y n o t e d by T s w e t t i n 1906.

The

v a r i o u s modes o f c h r o m a t o g r a p h y , h o w e v e r , h a d n o t b e e n c h a r a c t e r i z e d b e f o r e T i s e l i u s (1) c l a s s i f i e d them a s e l u t i o n , d i s p l a c e m e n t and f r o n t a l a n a l y s i s i n 1943.

With r e s p e c t t o d i s p l a c e m e n t development T i s e l i u s r e c o g n i z e d t h a t "as t h e

c o u r s e of development i n p r a c t i c a l chromatographic work h a s n e v e r been analyzed i n d e t a i l , i t i s p o s s i b l e t h a t p r o c e d u r e s m o r e o r less b e l o n g i n g t o t h i s k i n d a r e q u i t e common."

Indeed i n many a d s o r p t i v e s e p a r a t i o n p r o c e s s e s t h e p r i n c i -

p l e s of d i s p l a c e m e n t chromatography have found a p p l i c a t i o n w i t h o u t r e c o g n i t i o n . Displacement chromatography was used by Spedding & t i o n of r a r e e a r t h complexes on ion-exchanger column.

&. (9) f o r

t h e separa-

The p r o c e s s was used f o r

t h e m a n u f a c t u r e of p u r e r a r e e a r t h s i n t h e 1950's and 1960's and t h e e f f e c t o f

181 c e r t a i n o p e r a t i n g p a r a m e t e r s on t h e t h r o u g h p u t was i n v e s t i g a t e d i n g r e a t d e t a i l . F i g u r e 1 s h o w s t h e s e p a r a t i o n o f s a m a r i u m , neodymium and p r a s e o d y m i u m , w i t h c i t r i c a c i d a s c h e l a t o r i n t h e m o b i l e p h a s e and ammonium c i t r a t e a s t h e d i s placer.

The chromatograms o b t a i n e d w i t h columns having i n c r e a s i n g l e n g t h s a r e

t y p i c a l f o r t h e s u c c e s s i v e development s t a g e s of t h e d i s p l a c e m e n t t r a i n . p r o c e s s was f u r t h e r r e f i n e d by u s i n g o t h e r c h e l a t o r s such a s EDTA.

The

The s u c c e s s -

f u l i m p l e m e n t a t i o n of d i s p l a c e m e n t ion-exchange chromatography gave r i s e t o a g r e a t d e a l of s i m i l a r work i n i s o t o p e s e p a r a t i o n s (10-13).

Volume o f e l u a t e , l i t e r s . S e p a r a t i o n o f samarium, neodymium and praseodymium on 30, 60 and 120 cm i o n - e x c h a n g e r c o l u m n s o f 2 2 m m i . d . C a r r i e r , 0.1% c i t r a t e s o l u t i o n , pH 5.30; f l o w v e l o c i t y , 0.5 cm/min. From Ref. 9.

F i g . 1.

Fig. 2. Displacement development o f o l i g o s a c c h a r i d e s on c h a r c o a l column w i t h 4% phenol i n w a t e r . Samples: A. sucrose; 6. sucrose and r a f f i n o s e , 1:2; C. sucrose and r a f f i n o s e , 1 : l ; D. g l u c o s e and r a f f i n o s e , 1 : l . F r o m R e f . 2 1 .

Displacement g a s chromatography was p i o n e e r e d by Claesson ( 1 4 ) and P h i l l i p s

(15) p r i o r t o i n t r o d u c t i o n of g a s - l i q u i d p a r t i t i o n chromatography by James and M a r t i n i n 1952.

S i n c e t h e n , g a s chromatography h a s been p r a c t i c e d a l m o s t e x c l u -

s i v e l y i n t h e l i n e a r e l u t i o n mode a n d m o s t l i k e l y t h i s i s r e s p o n s i b l e f o r t h e h i a t u s i n t h e f u r t h e r development of d i s p l a c e m e n t chromatography u n t i l very recently.

N e v e r t h e l e s s , P h i l l i p s and co-workers have made a n o t h e r a t t e m p t (16-

18) t o r e v i v e d i s p l a c e m e n t g a s chromatography by u s i n g a h e a t f r o n t moving down t h e column a s t h e d i s p l a c e r .

The c o n c e p t o f t h i s " h e a t e r d i s p l a c e m e n t " i s

s i m i l a r t o t h a t of t h e c o n t i n u o u s h y p e r s o r p t i o n p r o c e s s (19) which used a moving a d s o r b e n t bed and w a s p r a c t i c e d a f t e r World War 11.

I t h a s g i v e n p l a c e , how-

e v e r , t o t h e Sorbex p r o c e s s (20) which i s t h e most s u c c e s s f u l i n d u s t r i a l s c a l e chromatographic p r o c e s s and u s e s an i n g e n i o u s v a l v i n g arrangement t o s i m u l a t e

182 bed movement. Our f o c u s h e r e i s on b i o c h e m i c a l s e p a r a t i o n s where d i s p l a c e m e n t chromatography was employed i n t h e f o r t i e s and f i f t i e s u n t i l A.J.P.

Martin's

pioneering

work on p a r t i t i o n chromatography h a s changed t h e c o u r s e of e v e n t s i n t h e h i s t o r y of chromatography and made l i n e a r e l u t i o n t h e predominant mode of s e p a r a t i o n . B e f o r e t h a t t h e p o o r q u a l i t y o f a d s o r b e n t s a n d i n s t r u m e n t a t i o n by t o d a y ' s s t a n d a r d s had plagued t h e development of s o l i d - l i q u i d e l u t i o n c h r o m a t o g r a p h y . This explains t h e f a c t t h a t t h e introduction of displacement development f o r a n a l y t i c a l chromatography was h a i l e d i n 1948 by C a s s i d y

who n o t e d t h a t t h e u s e

of a d i s p l a c e r " i n one s t r o k e " gave r e p r o d u c i b l e chromatograms w i t h r e l a t i v e l y s h a r p b o u n d a r i e s t h u s e l i m i n a t i n g problems a r i s i n g from n o n - l i n e a r b e h a v i o r and poor column e f f i c i e n c y i n contemporary s o l i d - l i q u i d e l u t i o n chromatography. F i g u r e 2 shows t h e s e p a r a t i o n of s u g a r s by d i s p l a c e m e n t chromatography a s c a r r i e d o u t by Claesson (21) i n 1948.

The s c h o o l of T i s e l i u s p i o n e e r e d v a r i o u s

a p p l i c a t i o n s o f t h e t e c h n i q u e t o b i o c h e m i c a l s e p a r a t i o n p r o b l e m s (22-26) a n d i n t r o d u c e d s e v e r a l i n n o v a t i v e c o n c e p t s t o improve t h e e f f i c i e n c y of s e p a r a t i o n . A s e r i e s o f c o l u m n s h a v i n g s m a l l e r and s m a l l e r d i a m e t e r s was f o u n d t o b e o f

advantage a s t h e wide b o r e columns f a c i l i t a t e d t h e c o n t a c t of t h e f e e d w i t h a l a r g e amount of a d s o r b e n t whereas i n t h e narrow b o r e column a t t h e o u t l e t t h e v o l u m e o c c u p i e d by t h e m i x e d b o u n d a r i e s b e t w e e n z o n e s c o u l d b e e f f e c t i v e l y reduced.

Another approach t o improve r e s o l u t i o n was t a k e n by adding t o t h e f e e d

a m i x t u r e of "spacer" s u b s t a n c e s which occupied i n t e r c a l a t i n g p o s i t i o n s i n t h e d i s p l a c e m e n t t r a i n between t h e f e e d components.

This technique c a l l e d c a r r i e r

d i s p l a c e m e n t chromatography (21) was employed t o s e p a r a t e v a r i o u s m i x t u r e s of biochemical significance.

Q 8,

a

2.

r

F i g . 3. C h r o m a t o g r a m o f egg a l b u m i n h y d r o l y z a t e o b t a i n e d on a c a t i o n exchanger column by d i s p l a c e m e n t w i t h 0.15N NH40H. B r a c k e t e d f r a c t i o n s a r e f o r t r e a t m e n t on secondary columns. From R e f . 32.

S e p a r a t i o n o f p r o t e i n s (27-28) and p r o t e i n h y d r o l y z a t e s (24-31) by d i s placement chromatography was t h e s u b j e c t of e x t e n s i v e i n v e s t i g a t i o n .

Figure 3

183 i l l u s t r a t e s t h e r e s u l t s o b t a i n e d i n d i s p l a c e m e n t ion-exchange

chromatography of

t h e h y d r o l y z a t i o n p r o d u c t s of egg albumin by P a r t r i d g e i n 1950 (32).

I t i s seen

t h a t t h e i n d i v i d u a l components c o u l d n o t be o b t a i n e d i n pure form and t h e f r a c t i o n s had t o be s e p a r a t e d i n subsequent chromatographic runs.

The r e l a t i v e -

l y low column e f f i c i e n c i e s of t h o s e y e a r s d i d n o t a l l o w d i s p l a c e m e n t chromatog r a p h y t o become a s e p a r a t i o n p r o c e s s o f c h o i c e a f t e r t h e r a p i d a c c e p t a n c e o f M a r t i n ' s p a r t i t i o n c h r o m a t o g r a p h y i n t h e l i n e a r e l u t i o n mode. a t t e m p t s have been made t o e x p l o i t c e r t a i n a d v a n t a g e s of c a r r i e r

Nevertheless, displacement

development by u s i n g a m i x t u r e of a m p h o l i t e s developed f o r i s o e l e c t r i c f o c u s i n g a s spacer substances (33,341.

The u s e o f c a r b o x y m e t h y l d e x t r a n s o f v a r i o u s

d e g r e e s o f s u b s t i t u t i o n a s s p a c e r s i n c a r r i e r d i s p l a c e m e n t chromatography of p r o t e i n s on D U E - c e l l u l o s e h a s a l s o shown v e r y p r o m i s i n g r e s u l t s (35-37). Looking back t o t h e h i s t o r y of d i s p l a c e m e n t chromatography we can conclude t h a t a f t e r a s u c c e s s f u l s t a r t mainly due t o t h e e f f o r t s by T i s e l i u s and h i s coworkers,

t h e t e c h n i q u e could n o t develop i n t o a l a b o r a t o r y s e p a r a t i o n p r o c e s s

and c o m p e t e w i t h l i n e a r e l u t i o n c h r o m a t o g r a p h y w h i c h had become p r e d o m i n a n t a f t e r M a r t i n i n t r o d u c e d p a r t i t i o n chromatography.

Adsorbent p r o p e r t i e s , column

e f f i c i e n c y and equipment were s i m p l y i n a d e q u a t e t o o b t a i n s h a r p d e m a r c a t i o n of t h e a d j a c e n t bands.

Moreover r a p i d emergence of t h e t h e o r y of l i n e a r e l u t i o n

chromatography h a s provided a s t r o n g b a s i s t o d e a l w i t h t h e thermodynamic and dynamic a s p e c t s of t h i s chromatographic mode, whereas t h e t h e o r e t i c a l c o m p l e x i t y of n o n - l i n e a r c h r o m a t o g r a p h y was m o r e o f a d e t e r r e n t t o t a c k l i n g p r o b l e m s of d i s p l a c e m e n t chromatography.

A l l t h i s has changed when HPLC developed i n t o a

w i d e l y used s e p a r a t i o n t e c h n i q u e i n t h e l a t e s e v e n t i e s .

Novel m i c r o p a r t i c u l a t e

s t a t i o n a r y p h a s e s h a v i n g f a v o r a b l e thermodynamic p r o p e r t i e s and y i e l d i n g h i g h column e f f i c i e n c i e s have become a v a i l a b l e and i n s t r u m e n t a t i o n i n HPLC reached a high l e v e l of s o p h i s t i c a t i o n .

A s a r e s u l t a g r e a t d e a l of t h e physico-chemical

phenomena a s s o c i a t e d w i t h t h e r e t e n t i o n p r o c e s s have been e l u c i d a t e d . o t h e r hand, t h e t h e o r y of non-linear

On t h e

chromatography i n t h e d i s p l a c e m e n t mode has

become t r a c t a b l e mainly by t h e work of H e l f f e r i c h (38-40). Thus, t h e main o b s t a c l e s have been removed from e x p l o r i n g t h e p o t e n t i a l of h i g h p e r f o r m a n c e d i s placement chromatography i n t h e e i g h t i e s .

TEEORY Displacement chromatography t a k e s p l a c e when t h e c o n c e n t r a t i o n s of t h e f e e d c o m p o n e n t s a r e s u f f i c i e n t l y h i g h t o be i n t h e n o n - l i n e a r p o r t i o n o f t h e i r a d s o r p t i o n i s o t h e r m s , which should be concave toward t h e a b s c i s s a , and t h e i n t r o d u c t i o n of t h e f e e d i n t o t h e column i s f o l l o w e d by a s o l u t i o n of t h e d i s p l a c e r s u b s t a n c e t h a t i s more s t r o n g l y adsorbed t h a n any of t h e f e e d components, i.e.,

i t s a d s o r p t i o n i s o t h e r m o v e r l i e s t h o s e of t h e f e e d components a s shown i n F i g u r e

4.

The v e l o c i t y of t h e d i s p l a c e r f r o n t , uD, is g i v e n by

184

where uo i s t h e f l o w v e l o c i t y of t h e mobile p h a s e , @ i s t h e phase r a t i o i n

the

column and qD and CD a r e t h e r e s p e c t i v e c o n c e n t r a t i o n s of t h e d i s p l a c e r on t h e The r a t i o qD/CD i s shown i n Fig. 4 a s . t h e

s t a t i o n a r y and i n t h e m o b i l e phases.

s l o p e of t h e cord drawn from t h e o r i g i n t o t h e p o i n t on t h e d i s p l a c e r i s o t h e r m t h a t c o r r e s p o n d s t o CD.

The c o m p o n e n t s o f t h e m i x t u r e a r e d i s p l a c e d when t h e

s l o p e of t h e c o r d , which i s c a l l e d t h e o p e r a t i n g l i n e , i s s m a l l e r t h a n t h e i n i t i a l s l o p e of t h e t h e i r i s o t h e r m s . The d i s p l a c e m e n t t r a i n i s f u l l y d e v e l oped,

i.e.,

reached,

isotachic conditions are

when t h e f e e d c o m p o n e n t s f o r m

a d j a c e n t bands t h a t move w i t h t h e v e l o c i t y of t h e d i s p l a c e r f r o n t so t h a t

U D

= u2 = u3 =u4

(2)

w h e r e u 2 , u 3 , and u4 a r e t h e z o n e v e l o c i t i e s of t h e t h r e e f e e d components which a r e displaced.

In t h i s case

qD/cD’q2/c2*’q3/c3* where C 2

*,

C3

*,

=q4/c4*

(3)

and C4* a r e t h e c o n c e n t r a -

t i o n s o f c o m p o n e n t s 2,

3,

and 4 i n t h e

VOLUME OF EFFLUENl

m o b i l e p h a s e and q 2 , q3, a n d 9 4 t h e c o r r e F i g . 4. I s o t h e r m s o f t h e f e e d components end t h e d i s p l a c e r w i t h t h e and the c o r r B operating line sponding f u l l y d e v e l o p e d d i s p l a c e m e n t t r e i n (61.

sponding c o n c e n t r a t i o n s on t h e s t a t i o n a r y phase a t equilibrium.

The p o i n t s a r e i l -

l u s t r a t e d i n Fig. 4 where t h e o p e r a t i n g l i n e i n t e r s e c t s t h e i s o t h e r m s of each com-

ponent w i t h t h e e x c e p t i o n of component 1 which i s e l u t e d . The f i n a l s t a g e of d i s p l a c e m e n t development, t h e r e f o r e , i s u n i q u e l y d e t e r mined by t h e a d s o r p t i o n i s o t h e r m s of t h e d i s p l a c e r and t h e f e e d components a s

w e L l a s by t h e c o n c e n t r a t i o n o f t h e d i s p l a c e r .

From e q s . 2 a n d 3 i t f o l l o w s

t h a t i n t h e f u l l y developed d i s p l a c e m e n t t r a i n t h e c o n c e n t r a t i o n s of t h e components i n t h e a d j a c e n t bands, i.e.

zone h e i g h t s , a r e c o n t r o l l e d by t h e chroma-

t o g r a p h i c system and t h e p r o p e r t i e s of t h e i n d i v i d u a l components. v a t i o n r e q u i r e s t h a t t h e v o l u m e o f t h e z o n e s , i.e.

Mass c o n s e r -

bandwidth, t h e r e f o r e , i s

d e t e r m i n e d by t h e amount of components p r e s e n t , The r e s u l t s shown i n F i g . 4 r e p r e s e n t t h e f i n a l s t a g e o f d i s p l a c e m e n t

185 development under c o n d i t i o n s of i d e a l chromatography i n t h e absence of a x i a l d i s p e r s i o n and secondary c h e m i c a l e q u i l i b r i a between t h e components. High e f f i c i e n c y columns employed i n HPLC and t h e s e l f s h a r p e n i n g b o u n d a r i e s o b t a i n e d when t h e i s o t h e r m s a r e Langmuirian o r quasi-Langmuirian f a c i l i t a t e t h e a t t a i n m e n t of f i n a l r e s u l t s p r e d i c t a b l e from t h e t h e o r y of e q u i l i b r i u m chromatography.

The

s i m p l i c i t y a s s o c i a t e d w i t h t h e d e s c r i p t i o n of t h e f u l l y developed d i s p l a c e m e n t t r a i n i s i n s h a r p c o n t r a s t w i t h t h e t r e a t m e n t of t h e development s t a g e i n which t h e i n t e r f e r i n g a d s o r p t i o n b e h a v i o r of t h e components b r i n g s about t h e i r separation.

I n t h e f o r t i e s and f i f t i e s many a u t h o r s c o n t r i b u t e d t o t h e t h e o r y

of n o n - l i n e a r chromatography (41-46).

The t r e a t m e n t of d i s p l a c e m e n t development

f o r m u l t i c o m p o n e n t m i x t u r e s , h o w e v e r , was m a i n l y a d v a n c e d by t h e t h e o r y o f H e l f f e r i c h (38) which h a s been p u b l i s h e d i n t h e form of a monograph i n 1970

(39).

The k e r n e l of t h e t h e o r y i s t h e s o - c a l l e d h - t r a n s f o r m a t i o n which f a c i l i -

t a t e s t h e u s e of a set of a l g e b r a i c e q u a t i o n s f o r t h e c a l c u l a t i o n s i n s t e a d of t h e system of coupled n o n l i n e a r d i f f e r e n t i a l e q u a t i o n s which govern t h e concent r a t i o n b e h a v i o r i n mult i-component

chromatography.

The method i s a p p l i c a b l e

when t h e s e p a r a t i o n f a c t o r s a r e c o n s t a n t such a s i n t h e c a s e of s t o i c h i o m e t r i c ion-exchange o r Langmuirian a d s o r p t i o n behavior.

It should be n o t e d t h a t an

e q u i v a l e n t t h e o r e t i c a l approach was p u t f o r w a r d by Rhee &

(47,481 t o adsorp-

t i o n chromatography and a p p l i e d t o t h e c a l c u l a t i o n of c o n c e n t r a t i o n p r o f i l e s i n d i s p l a c e m e n t chromatography. I n h - t r a n s f o r m a t ion c o n c e n t r a t i o n variables a r e transformed t o variables c h a r a c t e r i s t i c of c o m p o s i t i o n s a s s o c i a t e d w i t h t h e boundaries.

The v a r i a b l e hi

h a s t h e p r o p e r t y t h a t a c r o s s a b o u n d a r y , t h e v a l u e o f o n l y o n e h i c h a n g e s and t h e o t h e r h i v a l u e s a r e c o n s t a n t , and i n t u r n , a c h a n g e i n t h e v a l u e o f a h i d e n o t e s a boundary between bands of c o n s t a n t composition.

The u s e of hi a s t h e

dependent v a r i a b l e g r e a t l y f a c i l i t a t e s t h e c a l c u l a t i o n of boundary v e l o c i t i e s o r t h e t r a j e c t o r i e s of c o n c e n t r a t i o n b o u n d a r i e s i n t h e column.

The r e s u l t s a r e

c o n v e n i e n t l y r e p r e s e n t e d by a d e v e l o p m e n t d i a g r a m i n w h i c h t h e m o b i l e p h a s e volume passed through t h e column i s p l o t t e d a g a i n s t t h e a x i a l d i s t a n c e i n t h e c o l u m n and t h e b o u n d a r y t r a j e c t o r i e s a r e shown. calculations a r e adsorption isotherms,

The d a t a r e q u i r e d f o r s u c h

column hold-up volume,

volume of t h e f e e d and d i s p l a c e r c o n c e n t r a t ion.

c o m p o s i t i o n and

For i d e a l c h r o m a t o g r a p h i c

c o n d i t i o n s and by u s i n g t h e a p p r o p r i a t e c o m p e t i t i v e i s o t h e r m s t h e development diagram e n t a i l s a c o m p l e t e d e s c r i p t i o n of t h e p r o c e s s and a l l o w s t h e c a l c u l a t i o n of t h e c o n c e n t r a t i o n p r o f i l e s i n t h e e f f l u e n t f o r a g i v e n column l e n g t h . The book by H e l f f e r i c h and K l e i n ( 3 9 ) i s an i n v a l u a b l e s o u r c e of informat i o n needed f o r a n a l y s i s of t h e t r a n s i e n t s t a g e o f t h e d i s p l a c e m e n t process. The a p p l i c a t i o n of h - t r a n f o r m a t i o n

i n t h e p r a c t i c e of displacement chromato-

graphy under c o n d i t i o n s of Langmuirian adsorption behavior is g r e a t l y f a c i l i t a t e d by r e c e n t t r e a t m e n t s o f t h e s u b j e c t ( 4 9 , 5 0 1 w h i c h p r o v i d e d e t a i l e d d e -

186 s c r i p t i o n of t h e p r o c e d u r e s f o r e v a l u a t i o n of boundary t r a j e c t o r i e s and concentration profiles.

Comparison of c a l c u l a t e d and e x p e r i m e n t a l l y o b t a i n e d d i s -

placement chromatograms r e p r e s e n t i n g i n t e r m e d i a t e s t a g e s of d i s p l a c e m e n t development were i n good agreement a l t h o u g h compound a d s o r p t i o n i s o t h e r m s were used i n s t e a d of c o m p e t i t i v e isotherms.

Whereas t h e t h e o r y n e g l e c t s t h e e f f e c t of

r e l a t i v e l y slow maas t r a n s f e r and d i s p l a c e m e n t k i n e t i c s , i t h a s developed t o t h e p o i n t where s i m p l e computer c a l c u l a t i o n s u f f i c e s t o p r e d i c t t h e column l e n g t h r e q u i r e d f o r t h e f u l l development of t h e d i s p l a c e m e n t t r a i n of a multicomponent m i x t u r e o r f o r t h a t s t a g e of development t h a t y i e l d s s a t i s f a c t o r y s e p a r a t i o n i n a particular practical situation.

The e f f e c t o f a x i a l d i s p e r s i o n may b e c o n -

s i d e r e d by u s i n g some e m p i r i c a l c o r r e c t i o n . The p r a c t i c a b i l i t y of t h e t h e o r e t i c a l approach h i n g e s on t h e a v a i l a b i l i t y

of i s o t h e r m d a t a a p p l i c a b l e t o t h e chromatographic system under c o n s i d e r a t i o n . Recent work (51) u s i n g column and equipment g e n e r a l l y employed i n HPLC f o r t h e measurement of a d s o r p t i o n i s o t h e r m s by f r o n t a l chromatography h a s d e m o n s t r a t e d t h a t t h e t e c h n i q u e i s s u i t a b l e f o r r a p i d measurements and o n l y a s m a l l amount of s o l u t e i s r e q u i r e d when narrow b o r e columns a r e used.

The d a t a o b t a i n e d w i t h a

v a r i e t y of s o l u t e s and a d s o r b e n t s employed i n HPLC l e n d s s u p p o r t t o t h e fundamental assumption of t h e t h e o r y t h a t t h e i s o t h e r m s a r e c l o s e t o Langmuirian. Only i n c a s e s of secondary e q u i l i b r i a was observed d e v i a t i o n from Langmuirian a d s o r p t i o n behavior.

F r o n t a l chromatography was a l s o extended r e c e n t l y t o t h e

r a p i d measurement of c o m p e t i t i v e i s o t h e r m s (52).

The r e s u l t s a r e encouraging a s

L a n g m u i r i a n b e h a v i o r was f o u n d t o p r e v a i l o v e r a w i d e r a n g e o f c o n d i t i o n s o f i n t e r e s t i n p r a c t i c a l a p p l i c a t i o n s of d i s p l a c e m e n t chromatography.

Easy a c c e s s

t o such d a t a i s d e s i r a b l e t o enhance t h e a c c u r a c y of t h e o r e t i c a l c a l c u l a t i o n s and t o f a c i l i t a t e p r o c e s s d e s i g n .

The a v a i l a b i l i t y of a s u f f i c i e n t l y l a r g e

l i b r a r y of a d s o r p t i o n i s o t h e r m s i s expected t o l e a d t o t h e development of t e c h n i q u e s f o r t h e e s t i m a t i o n of a d s o r p t i o n p a r a m e t e r s from t h e c h e m i c a l s t r u c t u r e of t h e s o l u t e and, t h u s , t o r e l a x t h e r e q u i r e m e n t s f o r e x p e r i m e n t a l d a t a i n t h e c a l c u l a t i o n of t h e c o u r s e of d i s p l a c e m e n t development. PUCTICE OF DISPLILCEMERT CBBOBIATOGEAPEY Stages of operation.

There a r e s e v e r a l sudden changes i n t h e m o b i l e phase

c o m p o s i t i o n i n d i s p l a c e m e n t development,

so t h a t t h e p r o c e s s c o n s i s t s of d i s -

t i n c t s t e p s u n l i k e t h a t of e l u t i o n chromatography.

The column i s f i r s t e q u i l i -

b r a t e d w i t h t h e s o - c a l l e d c a r r i e r which a l s o s e r v e s a s t h e s o l v e n t f o r t h e f e e d m i x t u r e t o be s e p a r a t e d and t h e d i s p l a c e r p r o p e r ,

Thus, t h e c a r r i e r can be

c o n s i d e r e d a s t h e mobile phase. F i g u r e 5 shows s c h e m a t i c a l l y t h e v a r i o u s s t e p s involved i n a d i s p l a c e m e n t r u n which s t a r t s w i t h t h e i n t r o d u c t i o n of t h e f e e d , t h e components of which a r e adsorbed on t h e s t a t i o n a r y phase and occupy a c e r t a i n l e n g t h of t h e column.

In

187 t h i s s t a g e some s e p a r a t i o n D I S P L A C E R

REGENERANT

CARRIER

a l r e a d y o c c u r s by f r o n t a l chromatography.

After

l o a d i n g of t h e column w i t h t h e m i x t u r e t o be s e p a r a t e d i s completed, t h e d i s p l a c e r s o l u t i o n i s pumped i n t o t h e boundary of t h e displacer, F i g . 5.

tography

which b i n d s more s t r o n g l y

.

S t a g e s o f o p e r a t i o n i n d i s p l s c e m e n t chroma-

t o t h e s t a t i o n a r y phase t h a n any of t h e f e e d compo-

n e n t s , moves down t h e column and c a u s e s them t o d e s o r b and t o move ahead o f t h e displacer front.

The mutual c o m p e t i t i o n of t h e f e e d components f o r t h e adsorp-

t i o n s i t e s b r i n g s about t h e s e p a r a t i o n so t h a t t h e y form a d i s p l a c e m e n t t r a i n of a d j a c e n t bands of t h e pure components i n t h e o r d e r of t h e i r a f f i n i t y t o t h e s t a t i o n a r y p h a s e a n d a l l b a n d s move w i t h t h e v e l o c i t y of t h e d i s p l a c e r f r o n t upon r e a c h i n g i s o t a c h i c c o n d i t i o n s .

After the displacement t r a i n is f u l l y

d e v e l o p e d no f u r t h e r s e p a r a t i o n o c c u r s , t h e r e f o r e , t h e l e n g t h o f t h e c o l u m n should n o t exceed t h a t r e q u i r e d f o r r e a c h i n g t h e i s o t a c h i c s t a t e .

Upon comple-

t i o n of t h e development t h e p r o d u c t s l e a v e t h e column i n t h e e f f l u e n t and c o l l e c t e d s e p a r a t e l y and t h e column i s f i l l e d up w i t h t h e d i s p l a c e r s o l u t i o n a s d e p i c t e d i n Fig. 5.

Before t h e n e x t r u n t h e d i s p l a c e r must be s t r i p p e d from t h e

column and r e e q u i l i b r a t e d w i t h t h e c a r r i e r . t i a l p a r t of t h e p r o c e s s .

The r e g e n e r a t i o n s t e p i s a n e s s e n -

Thus t h e t i m e r e q u i r e d f o r a d i s p l a c e m e n t r u n e n t a i l s

t h e t i m e of f e e d i n t r o d u c t i o n , d i s p l a c e m e n t development, product r e c o v e r y and column r e g e n e r a t ion.

Equipment.

I n most r e c e n t work on d i s p l a c e m e n t chromatography, t h e same columns

w e r e e m p l o y e d a s t h o s e i n a n a l y t i c a l HPLC.

C o n s e q u e n t l y , c o n v e n t i o n a l HPLC

i n s t r u m e n t a t i o n c o u l d be r e a d i l y m o d i f i e d f o r s e m i - p r e p a r a t i v e

s e p a r a t i o n s by

d i s p l a c e m e n t d e v e l o p m e n t a n d s e v e r a l a r r a n g e m e n t s h a v e b e e n d e s c r i b e d (6-

8,49,53-55).

F i g u r e 6 shows t h e f l o w s h e e t of a d u a l u n i t which c o n s i s t s of a

d i s p l a c e m e n t chromatograph denoted a s " f r a c t i o n a t o r " a n d a n a n a l y t i c a l l i q u i d chromatograph c a l l e d "on-line

analyzer."

The f r a c t i o n a t o r h a s a s i n g l e pump f o r t h e s e q u e n t i a l p e r f u s i o n o f t h e column w i t h t h e c a r r i e r , d i s p l a c e r s o l u t i o n and r e g e n e r a n t .

The f e e d v a l v e i s

s i m i l a r t o t h o s e of t h e sampling s y s t e m s employed i n a n a l y t i c a l chromatography but h a s a l o o p of l a r g e volume u s u a l l y exceeding one m i l l i l i t e r .

The c o l u m n

e f f l u e n t p a s s e s through t h e sampling v a l v e of t h e o n - l i n e a n a l y z e r t h e n t h r o u g h t h e f l o w c e l l of a d i f f e r e n t i a l r e f r a c t i v e index d e t e c t o r b e f o r e e n t e r i n g t h e

188 .

...

.....

.......

......

f r a c t i o n collector.

..~.. -

OIBPLACER APPIPB RIDINEBAWT

j

O N - L I N E ANALYZER

The d e t e c t o r m o n i t o r s t h e column e f f l u e n t

ELUENT

i n terms o f p r o d u c t c o n c e n t r a t i o n but i n most c a s e s i t cannot be used t o d e m a r c a t e t h e b o u n d a r i e s of t h e a d j a c e n t bands of t h e separated components which i s r e q u i r e d f o r ...

t h e i r recovery.

By

s a m p l i n g t h e column 6. Flow s h e e t of t h e combined f r a c t i o n a t o r and onl i n e analyzer f o r use in displacement chrometography. e f f l u e n t i n s h o r t From R e f . 53. t i m e i n t e r v a l s , e.g.

Fig.

every 15 o r 30 s e c , t h e o n - l i n e a n a l y t i c a l HPLC can p r o v i d e t h e i n f o r m a t i o n on t h e e f f l u e n t c o m p o s i t i o n so t h a t f r a c t i o n s c o n t a i n i n g t h e p u r e components a s w e l l a s t h e i r m i x t u r e s when t h e r e i s a n o v e r l a p o f t h e a d j a c e n t b a n d s , c a n b e recovered s e p a r a t e l y .

The u s e of a s h o r t column, 3 t o 5 cm i n l e n g t h , s u f f i c e s

f o r o n - l i n e a n a l y s i s a s i n most c a s e s n o t more t h a n two s u b s t a n c e s a r e p r e s e n t i n t h e e f f l u e n t sample. A l t e r n a t i v e l y s m a l l f r a c t i o n s of t h e column e f f l u e n t a r e c o l l e c t e d and s u b j e c t e d t o post-run a n a l y s i s by HPLC o r t h i n l a y e r chromatography.

The u s e o f TLC o f f e r s a r a p i d and e f f i c i e n t m e t h o d f o r s i m u l t a n e o u s

a n a l y s i s o f a l a r g e number o f f r a c t i o n s i n o r d e r t o s o r t t h e m o u t f o r p r o d u c t recovery. T y p i c a l f l o w r a t e of t h e d i s p l a c e r s o l u t i o n through coiumns having dimens i o n s o f 250 x 4.6 m and p a c k e d w i t h 5- o r 10- m p a r t i c l e s i s 0.1 m l l m i n , whereas t h e f l o w r a t e s d u r i n g r e g e n e r a t i o n and r e e q u i l i b r a t i o n w i t h t h e c a r r i e r s o l v e n t r a n g e from 1 t o 3 mllmin.

Stationary phase. S o r b e n t s employed i n HPLC can b e used i n d i s p l a c e m e n t development a s w e l l .

They should a l l o w s t r o n g r e t e n t i o n of t h e f e e d components, have

high c a p a c i t y and o f f e r r a p i d d s i p l a c e m e n t k i n e t i c s w i t h o u t i r r e v e r s i b l e adsorption or catalytic effects.

The a d s o r p t i o n i s o t h e r m s of t h e components on t h e

s t a t i o n a r y phase should be concave downward i n o r d e r t o meet t h e r e q u i r e m e n t s f o r a w e l l behaving d i s p l a c e m e n t system.

I n t h e f i n a l a n a l y s i s s e l e c t i v i t y and

c a p a c i t y d e t e r m i n e t h e e f f i c i e n c y of t h e s e p a r a t i o n p r o c e s s .

High column e f f i -

c i e n c y a s m e a s u r e d by e l u t i o n c h r o m a t o g r a p h y i s e s s e n t i a l t o m i n i m i z e b a n d o v e r l a p p i n g which can b e p a r t i c u l a r l y d e t r i m e n t a l t o t h e s e p a r a t i o n e f f i c i e n c y i n d i s p l a c e m e n t development.

Another r e q u i r e m e n t i s r a p i d r e g e n e r a b i l i t y and

l o n g e v i t y of t h e column under o p e r a t i n g c o n d i t i o n s . A l k y l - s i l i c a columns used i n r e v e r s e d phase chromatography were f r e q u e n t l y

189 used i n displacement chromatography w i t h aqueous c a r r i e r s .

Examination of

nu me rous a d s o r p t i o n i s o t h e r m s o b t a i n e d on s u c h s o r b e n t s showed q u a s i - L a n g m u i r i a n behavior.

M i c r o p a r t i c u l a t e s i l i c a g e l columns o f f e r a n a l t e r n a t i v e and t h e

c h o i c e i s made on t h e b a s i s o f t h e s o l u b i l i t y o f f e e d co mp o n en t s i n t h e s o l v e n t s c h o s e n a s c a r r i e r s , as w e l l as t h e co lu m n s e l e c t i v i t y and c a p a c i t y .

Carrier s o l v e n t . t i o n a r y p h a s e us ed .

The s e l e c t i o n of t h e m o b i l e p h a s e s i s d e p e n d e n t on t h e s t a A s i n a n y k i n d o f p r e p a r a t i v e c h r o m a t o g r a p h y , s o l u b i l i t y of

t h e f e e d c om ponen ts i s a k ey p a r a m e t e r .

T h i s i s p a r t i c u l a r l y so i n d i s p l a c e m e n t

c h r o m a t o g r a p h y , w h i c h i s most e f f i c i e n t a t h i g h c o n c e n t r a t i o n a n d t h e r e f o r e r e q u i r e s h i g h s o l u b i l i t y of t h e f e e d i n t h e m o b i l e p h ase.

An e m i n e n t f e a t u r e o f d i s p l a c e m e n t d e v e l o p m e n t i s t h a t s o m e o f t h e comp o n e n t s become more c o n c e n t r a t e d w i t h r e s p e c t t o t h e f e e d s o l u t i o n i n t h e c o u r s e of t h e s e p a r a t i o n p r o c e s s .

A s a r e s u l t p r e c i p i t a t i o n o f many c o m p o n e n t s may

o c c u r i n t h e column i f t h e i r m o b i l e phase becomes s a t u r a t e d and c o n c o m i t a n t c olumn p l u g g i n g may o c c u r .

In o r d e r t o a v o i d s u c h an u n t o w a r d s i t u a t i o n , e l e -

v a t e d c olumn t e m p e r a t u r e may b e n eed ed t o i n c r e a s e t h e s o l u b i l i t y . Other r e q u i r e m e n t s f o r t h e m o b i l e phase i n c l u d e c h e m i c a l i n e r t n e s s , low v i s c o s i t y and t o x i c i t y a 6 w e l l a s c o m p a t i b i l i t y w i t h t h e co l u mn m a t e r i a l and t h e equipment.

In o r d e r t o f a c i l i t a t e p r o d u c t r e c o v e r y t h e m o b i l e p h a s e s h o u l d b e

e a s i l y removable from t h e product w i t h o u t r e s i d u e .

With a l k y l s i l i c a column

n e a t water o r hydro-organic m i x t u r e s a r e u s u a l l y chosen whereas w i t h s i l i c a g e l c o l u m n s o r g a n i c s o l v e n t s a r e used. T h i n

cnpZ

cncil

CCI4

I I 1 i

.:I

n

..

i~<

c h r o m a t o g r a p h y may be o c c a s i o n a l l y used

r:;:c:

.I. .. I

D,lplarr

Spolllng L44"ld

lwei

6%

10%

2%

6%

I0 %

2%

6%

f o r t h e s e l e c t i o n of a c a r r i e r a s shown

~~

2%

l a y e r

10%

DEEDA

i n F i g . 7.

For t h e

s e p a r a t i o n of t h r e e c o r t i c o s t e r o i d s on s i l i c a gel (7) di-

F i g . 7.

TLC e x p e r i m e n t s w i t h c a r b o n t e t r a c h l o r i d e , c h l o r o f o r m and methylene c h l o r i d e t o s c o u t f o r c a r r i e r s o l v e n t . The c o n c e n t r a t i o n o f d i e t h y l e t h y t e n e d i a m i n e u s e d 8 s t h e d i s p l a c e r i s g i v e n a t t h e b o t t o m . S a m p l e : 0, d e o x y c o r t i c o s t e r o n e ; S, 1 1 - d e o x y - 1 7 - h y d r o x y c o r t i c o s t e r o n e ; H, c o r t i costerone. From R e f . 7 .

ethylethylenediamine

was c h o s e n a s t h e d i s p l a c e r and t h r e e s o l v e n t s w e r e examined

usefulness a s carrier.

for

their

Th in l a y e r p l a t e s w e r e s p o t t e d w i t h t h e t h r e e component

m i x t u r e and w e r e d e v e l o p e d w i t h t h e d i s p l a c e r i n t h e o r g a n i c s o l v e n t s a t d i f f e r e n t c o n c e n t r a t i o n s . A s s e e n i n Fig . 7 w i t h c a r b o n t e t r a c h l o r i d e no d i s p l a c e m e n t o c c u r e d and t h e co m p o n en ts e l u t e d b e h i n d t h e d i s p l a c e r f r o n t .

With methylene

190 c h l o r i d e s o l u t i o n s no d i s p l a c e m e n t t o o k p l a c e e i t h e r and t h e components were e l u t e d by t h e s o l v e n t p r o p e r a b o v e t h e d i s p l a c e r f r o n t .

On t h e o t h e r hand

d i s p l a c e m e n t o c c u r r e d w i t h c h l o r o f o r m s o l u t i o n s of t h e d i s p l a c e r a t a l l concentrations investigated.

T h u s , c h l o r o f o r m was c h o s e n a s t h e c a r r i e r f o r t h e

s e p a r a t i o n i n s i l i c a g e l column w i t h d i e t h y l e t h y l e n e d i a m i n e a s t h e d i s p l a c e r a s shown i n F i g . 16.

Displacer.

I n t h e d e s i g n of d i s p l a c e m e n t development t h e s e l e c t i o n of a s u i t a -

b l e d i s p l a c e r may r e p r e s e n t t h e g r e a t e s t o b s t a c l e a t p r e s e n t b e c a u s e o f t h e p a u c i t y of e x p e r i e n c e i n t h i s r e g a r d . requirements:

The d i s p l a c e r must meet s e v e r a l s t r i n g e n t

( i ) it has t o have g r e a t e r a f f i n i t y t o t h e s t a t i o n a r y phase t h a n

any of t h e f e e d components, i.e.

i t s a d s o r p t i o n i s o t h e r m must o v e r l i e t h o s e of

t h e components; ( i i ) i t should be r e a d i l y s o l u b l e i n t h e c a r r i e r s o l v e n t ;

(iii)

i t should n o t i n t e r a c t w i t h t h e f e e d components, f o r example, v i a complex forma-

t i o n , b e s i d e s d i s p l a c i n g them from t h e s o r b e n t s u r f a c e ; ( i v ) i t should b e e a s i l y removable i n t h e c o u r s e of column r e g e n e r a t i o n .

Futhermore t h e g e n e r a l r e q u i r e -

ments of low t o x i c i t y and v i s c o s i t y a l s o a p p l y t o t h e d i s p l a c e r a s w e l l .

It i s

a l s o d e s i r a b l e t h a t t h e d i s p l a c e r c a n b e e a s i l y s e p a r a t e d f r o m t h e l a s t component of t h e d i s p l a c e m e n t t r a i n i f t h e r e i s e f f l u e n t f r a c t i o n which c o n t a i n s both. With s i l i c a g e l columns secondary and t e r t i a r y amines such a s d i e t h y l e t h y l e n e d i a m i n e o r t r i e t h a n o l a m i n e can s e r v e a s a p p r o p r i a t e d i s p l a c e r s u n l e s s t h e f e e d m i x t u r e c o n t a i n s a c i d i c substances.

As a g e n e r a l r u l e , t h e employment

of a d i s p l a c e r s u b s t a n c e c a r r y i n g e l e c t r o s t a t i c c h a r g e o p p o s i t e i n s i g n t o t h o s e p r e s e n t i n any of t h e f e e d components have t o be avoided because t h e new s p e c i e s formed by e l e c t r o s t a t i c i n t e r a c t i o n s w i l l impede s e p a r a t i o n due t o t h e r e a c t i o n zone t h u s o b t a i n e d .

With a l k y l - s i l i c a columns, a l k y l o r a l k y l - a r y l q u a r t e r n a r y

ammonium s a I t s such a s t r i m e t h y l o c t y lammon ium , t r ime t h y lbenzy lammonium o r t e t r a b u t y l a m m o n i u m s a l t s a r e v e r y e f f e c t i v e d i s p l a c e r s when no n e g a t i v e l y charged components a r e p r e s e n t i n t h e feed. N e u t r a l d i s p l a c e r s a r e o f t e n needed i n r e v e r s e d phase chromatography which i s f r e q u e n t l y used f o r t h e s e p a r a t i o n of l e s s p o l a r s p e c i e s having i o n i c func-

tions.

Some of t h e d i s p l a c e r s which have been used under such c o n d i t i o n s a r e

n-propanol, n-butanol, phenol, 2 - b u t o x y e t h a n o l ,

2-(2-butoxyethoxy)ethanol

and

dipropyleneglycolmonomethylether. E l u t i o n e x p e r i m e n t s w i t h a column i d e n t i c a l i n r e t e n t i o n p r o p e r t i e s t o t h a t used f o r d i s p l a c e m e n t development can be v e r y h e l p f u l i'n s c r e e n i n g f o r a s u i t a ble displacer.

P r i o r t o d i s p l a c e m e n t e x p e r i m e n t t h e r e t e n t i o n f a c t o r s of a l l

components i n c l u d i n g t h e d i s p l a c e r a r e measured and a s c e r t a i n e d t h a t t h e d i s p l a c e r i s indeed t h e most r e t a i n e d e l u i t e .

The r e l a t i v e r e t e n t i o n s o f n e i g h -

b o r i n g peaks a r e t h e s e p a r a t i o n f a c t o r s which d e t e r m i n e t h e d i f f i c u l t y of s e p a r -

191 a t i o n , i. e . t h e l e n g t h of c o l u m n r e q u i r e d f o r d e v e l o p m e n t o f t h e d i s p l a c e m e n t train.

T hus , t h e e l u t i o n ch r o m ato g r am o f t h e s p e c i e s i n v o l v e d p r o v i d e s n o t o n l y

i n f o r m a t i o n on t h e q u a n t i t a t i v e c o m p o s i t i o n of t h e f e e d m i x t u r e b u t a l s o a means t o e s t i m a t e t h e s u i t a b i l i t y of t h e d i s p l a c e r and t h e d i f f i c u l t y o f s e p a r a t i o n by displacement development. Tine c o n c e n t r a t i o n o f t h e d i s p l a c e r i s a n o t h e r i m p o r t a n t d e s i g n p a r a m e t e r . The v e l o c i t y o f t h e d i s p l a c e r f r o n t t h r o u g h t h e co l u mn i n c r e a s e s w i t h t h e d i s p l a c e r c o n c e n t r a t i o n a c c o r d i n g t o eq. t i o n r e s u l t s i n f a s t e r separation.

1, t h e r e f o r e ,

higher d i s p l a c e r concentra-

F u r t h e r m o r e , a s s e e n i n Fi g . 1, t h e s l o p e of

t h e o p e r a t i n g l i n e d e c r e a s e s and a s a r e s u l t , t h e c o n c e n t r a t i o n of t h e p r o d u c t s i n t h e e f f l u e n t i s h i g h e r s o t h a t t h e v o l u m e o c c u p i e d by a c o m p o n e n t b e c o m e s smaller.

A s u n d e r o t h e r w i s e i d e n t i c a l o p e r a t i n g c o n d i t i o n s t h e v o l u me o f t h e

o v e r l a p p i n g r e g i o n of a d j a c e n t zones i s i n v a r i a n t , t h e e f f i c i e n c y of d i s p l a c e ment c h r o m a t o g r a p h y i n t e r m s of t h e f r a c t i o n of a p r o d u c t r e c o v e r e d i n p u r e f o r m d e p e n d s o n t h e v o l u m e o c c u p i e d by t h e z o n e o f p u r e p r o d u c t r e l a t i v e t o t h e v o l u m e o f t h e i n t e r m i x e d r e g i o n a s i l l u s t r a t e d i n F i g 8. T h e c o n c e n t r a t i o n o f t h e d i s p l a c e r can a l s o be adjusted t o o p t i m i z e separation.

A 1 ,111,

I 0

al c 0

a %

0

-

0 0

L

m C 0

c

0

+

? 0 C)

0 C

0

for CD

Concentrotion in Mobile

Phose

F i g . 8. E f f e c t o f t h e amount o f component i i n t h e f e e d on i t s r e c o v e r y i n pure f o r m . I n A , B and C r e c o v e r i e s a r e 0 , 5 0 a n d 90%, r e s p e c t i v e l y . F r o m R e f . 6.

Fig. 9.

O p e r a t i o n a l range o f d i s p l a c e r c o n c e n t r a t i o n .

From R e f . 8.

The o p e r a t i o n a l c o n c e n t r a t i o n of t h e d i s p l a c e r i s a l s o c o n s t r a i n e d b y t h e

192

s o l u b i l i t y of t h e d i s p l a c e r and t h e p r o d u c t s i n t h e c a r r i e r , a s mentioned above, and t h e r e i s a n upper l i m i t f o r t h e a l l o w a b l e d i s p l a c e r c o n c e n t r a t i o n a s i l l u s t r a t e d i n Fig.

9.

The lower l i m i t i s g i v e n by t h e r e q u i r e m e n t t h a t t h e opera-

t i n g l i n e i n t e r s e c t s t h e i s o t h e r m of each f e e d component t o be d i s p l a c e d .

As

shown i n Fig. 9 t h e l o w e s t c o n c e n t r a t i o n of t h e d i s p l a c e r i s d e t e r m i n e d by t h e chord t o i t s i s o t h e r m which s t i l l i n t e r s e c t s t h e i s o t h e r m of t h e l e a s t weakly a d s o r b i n g component.

Optimization of operating condition.

Displacement chromatography f a l l s s h o r t of

s i m p l e measures of column e f f i c i e n c y and r e s o l u t i o n germane t o l i n e a r e l u t i o n chromatography a l t h o u g h s e v e r a l a t t e m p t s (49,56) have been made t o e x p r e s s t h e e f f i c i e n c y o f t h e c h r o m a t o g r a p h i c s y s t e m . In d i s p l a c e m e n t c h r o m a t o g r a p h y no f u r t h e r s e p a r a t i o n o c c u r s a f t e r r e a c h i n g i s o t a c h i c c o n d i t i o n s and t h e l e n g t h of column r e q u i r e d f o r t h e f u l l development of t h e d i s p l a c e m e n t t r a i n depends on t h e p r o p e r t i e s of t h e column and t h e f e e d components a s w e l l a s on t h e amount of feed.

Under i d e a l c o n d i t i o n s t h e amount of s t a t i o n a r y phase, i.e.

t h e l e n g t h of

a column of f i x e d d i a m e t e r , needed t o r e a c h i s o t a c h i c c o n d i t i o n s i s p r o p o r t i o n a l t o t h e amount of feed. For t h i s r e a s o n i t i s convenient t o d e f i n e t h e " s t a t i o n a r y phase e f f e c t i v e -

ness" by t h e m a s s of p r o d u c t r e c o v e r e d i n p u r e f o r m p e r u n i t m a s s o f c o l u m n packing.

A s t h e s e l e c t i o n of t h e d i s p l a c e r g r e a t l y a f f e c t s t h e s e p a r a t i o n we

can d e f i n e t h e " d i s p l a c e r e f f e c t i v e n e s s " a s t h e r e c o v e r e d mass of p u r e product d i v i d e d by t h e amount of d i s p l a c e r r e q u i r e d f o r a g i v e n column and f e e d t o r e a c h isotachic conditions.

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 and e x p e r i m e n t a l r e s u l t s sug-

g e s t t h a t i f t h e column l e n g t h i s v a r i a b l e h i g h e r d i s p l a c e r c o n c e n t r a t i o n y i e l d s more e f f i c i e n t u s e of t h e s t a t i o n a r y p h a s e and r e s u l t s i n l e s s e r amount o f displacer overall.

Of c o u r s e , t h e r e a r e c e r t a i n c o n s t r a i n t s o n t h e maximum

d i s p l a c e r c o n c e n t r a t i o n due t o t h e s o l u b i l i t y l i m i t s of t h e d i s p l a c e r and f e e d components as d i s c u s s e d above. I n p r e p a r a t i v e work o n e o f t h e f u n d a m e n t a l m e a s u r e s o f t h e r e s u l t s o f

d i s p l a c e m e n t chromatography i s t h e r e c o v e r y g i v e n by t h e p e r c e n t a g e of a component under c o n s i d e r a t i o n which i s c o n t a i n e d i n s u c c e s s i v e e f f l u e n t f r a c t i o n s a t a g i v e n p u r i t y , say, 99.9%.

On t h e o t h e r hand, t h e e f f i c i e n c y of t h e p r o c e s s

c a n b e e x p r e s s e d by t h e t h r o u g h p u t , i.e. r a t e o f p r o d u c t i o n , w h i c h i s g i v e n by t h e amount of recovered p r o d u c t s d i v i d e d by t h e d u r a t i o n of t h e chromatographic run c o n v e n i e n t l y measured by t h e r e t e n t i o n t i m e of t h e d i s p l a c e r f r o n t . When t h e l e n g t h of t h e c o l u m n i s f i x e d t h e r e i s f r e q u e n t l y some o p t i m u m v a l u e o f t h e o p e r a t i o n a l v a r i a b l e s w h e r e t h e t h r o u g h p u t i s maximum a s i l l u s t r a t e d i n F i g s . 1 0 t o 12.

The f e e d c o n t a i n e d t w o d e g r a d a t i o n p r o d u c t s o f a n

a n t i c a n c e r drug (53) which were t e n t a t i v e l y i d e n t i f i e d a s 1-methylfuran-1,2,3,4tetrahydroxyoxane (A) and 1-methylfuran-4-oxymethy 1 - l 1 2 , 3 , 4 - t r i h y d r o x y o x a l a n e

193

(B).

T he s e p a r a t i o n w a s p e r f o r m e d w i t h t h r e e d i s p l a c e r s :

dipropyleneglycol-

!

m o n o m e t h y l e t h e r , tripropyleneglycolmonomethylether and 2 - b u t o x y e t h a n o l .

5 -

.*/'\a 4 a \

3 -

2

m.

2 -

1 -

0'

0'

Normalized breakthrough volume of the displacer

25

50

1 -

75

Feed (me)

F i g . 1 0 . T h r o u g h p u t y e r s u s d i s p l a c e r b r e e k t h r o u g h v o l u m e . C o l u m n , 2 5 0 x 4.6 mm, 5-pm Zorbax 0 0 s ; c a r r i a r , w a t e r ; f l o w r a t e , 0.1 m l / m i n ; temp., 22' C; f e e d , 3 1 mg o f e a c h c o m p o n e n t . D i s p l a c e r s g i v e n i n t h e t e x t w e r e u s e d i n w e t e r a t c o n c e n t r a t i o n s f r o m 10 t o 40 mg/ml. From R e f . 53. Fig. 11. Throughput versus column loading. C o n d i t i o n s 8s i n F i g . 1 0 , t h e d i s p l a c e r was 10 mg/ml d i p r o p y l e n e g l y c o l m o n o m e t h y l e t h e r i n w a t e r . From R e f . 53.

The d i s p l a c e r s and t h e i r c o n c e n t r a t i o n w e r e v a r i e d i n o r d e r t o o b t a i n d i f f e r e n t v e l o c i t i e s of t h e d i s p l a c e r f r o n t a t a f i x e d f l o w r a t e and t h e t h r o u g h p u t o f b o t h p r o d u c t s was e v a l u a t e d .

The r e s u l t s a r e shown i n Fi g .

10 by

p l o t s o f t h e t h r o u g h p u t a g a i n s t t h e n o r m a l i z e d b r e a k t h r o u g h v o l u me of t h e d i s p l a c e r whic h is g i v e n by t h e a d j u s t e d r e t e n t i o n v o l u me o f t h e d i s p l a c e r f r o n t d i v i d e d by t h e v o i d v o l u m e o f t h e c o l u m n .

I t i s n o t e d t h e s h a p e of t h e o p e r -

a t i n g l i n e i s p r o p o r t i o n a l t o t h e a d j u s t e d r e t e n t i o n v o l u me o f t h e d i s p l a c e r , c o n s e q u e n t l y t h e same o p e r a t i n g l i n e c a n b e o b t a i n e d w i t h a n y of t h e d i s p l a c e r s a t t h e a p p r o p r i a t e c o n c e n t r a t i o n a s d e t e r m i n e d by t h e i r a d s o r p t i o n i s o t h e r m s . When t h e b r e a k t h r o u g h v o l u m e i s s m a l l , t h e z o n e w i d t h s o f t h e p r o d u c t s a r e n a r r o w and zone o v e r l a p r e s u l t s i n r e l a t i v e l y low r e c o v e r y and c o n c o m m i t a n t l y t h e t h r o u g h p u t i s low.

When t h e b r e a k t h r o u g h v o l u me i s l a r g e , t h e r e c o v e r y i s

h i g h b u t t h e t i m e of s e p a r a t i o n i s a l s o h i g h s o t h a t t h e t h r o u g h p u t may b e low again.

A s s h o w n i n F i g . 10 t h e r e i s a n o p t i m u m b r e a k t h r o u g h v o l u m e , i . e . a n

'optimum o p e r a t i o n l i n e , t h a t g i v e s t h e maximum t h r o u g h p u t u n d e r o t h e r w i s e f i x e d operating conditions.

194 S i m i l a r l y t h e r e i s a n o p t i m u m amount o f f e e d a s s e e n f r o m F i g . 11.

I t is

because w i t h s m a l l amounts of f e e d t h e zone w i d t h s a r e s m a l l and r e c o v e r y i s low b e c a u s e of zone o v e r l a p .

On t h e o t h e r h a n d , w i t h a l a r g e a m o u n t o f f e e d t h e

f i x e d c o l u m n l e n g t h d o e s n o t s u f f i c e t o r e a c h i s o t a c h i c c o n d i t i o n s and t h e incomplete s e p a r a t i o n r e s u l t s i n low recovery.

I n b o t h c a s e s t h e throughput i s

reduced and h a s a maximum a t an i n t e r m e d i a t e amount of f e e d a t which t h e f u l l y d i s p l a c e m e n t t r a i n o c c u p i e s t h e column. 16

1::

1

g r a p h i c system i s s t r o n g l y a f f e c t e d

P

by t h e f l o w v e l o c i t y .

The magnitude

of a x i a l d i s p e r s i o n i n c r e a s e s w i t h t h e f l o w v e l o c i t y and a s a r e s u l t d e p a r t u r e from i d e a l chromatography

r\,,,

e n g e n d e r s i n c r e a s i n g zone o v e r l a p .

On t h e o t h e r hand, t h e t i m e r e q u i r e d

,/

for the separation decreases with

the flow rate.

As

t h e value of

t h r o o g h p u t encompasses b o t h t h e r e -

0

d e g r e e o f t h e s e p a r a t i o n and t h e time

of

separation,

plots

of

throughput a g a i n s t t h e f l o w r a t e go Fig. 1 2 . Throughput e r s s flow rate. s h o w n in Fig. C o n d i t i o n s 8 s i n F i n- . 1;. . t:e f e e d was 31 t h r o u g h a maximum mg o f each component. From Ref. 53. 12. SELECTED APPLICATIONS The s e p a r a t i o n of a s i m p l e t w o c o m p o n e n t m i x t u r e of d i h y d r o x y b e n z e n e by d i s p l a c e m e n t chromatography w i t h two r e v e r s e d phase a n a l y t i c a l columns i n s e r i e s

i s i l l u s t r a t e d i n F i g . 13.

C a t e c h o l and r e s o r c i n o l a r e r e l a t i v e l y p o l a r and

r e a d i l y d i s p l a c e d f r o m t h e o c t a d e c y l - s i l i c a s u r f a c e by n - p r o p a n o l .

The h i g h

column load a s w e l l a s h i g h p r o d u c t c o n c e n t r a t i o n and p u r i t y i n t h e e f f l u e n t , shown i n Fig. 13, d e m o n s t r a t e t h a t t e c h n i q u e o f f e r s an e f f i c i e n a l t e r n a t i v e t o e l u t i o n chromatography. P e p t i d e s were among t h e f i r s t s u b s t a n c e s t o be s e p a r a t e d by d i s p l a c e m e n t chromatography on c h a r c o a l column (2).

Recent work h a s d e m o n s t r a t e d t h a t micro-

p a r t i c u l a t e a l k y l - s i l i c a p h a s e s a r e eminently s u i t a b l e f o r peptide separation n o t o n l y i n t h e e l u t i o n b u t a l s o i n t h e d i s p l a c e m e n t mode (8).

F i g u r e 14

i l l u s t r a t e s t h e s e p a r a t i o n of t h r e e d i p e p t i d e s under d i f f e r e n t c o n d i t i o n s w i t h r e s p e c t t o t h e l e n g t h o f t h e c o l u m n and t h e a m o u n t o f f e e d .

The pH of t h e

b u f f e r e d c a r r i e r was 2.0 s o t h a t t h e d i s s o c i a t i o n of t h e c a r b o x y l i c group i n t h e p e p t i d e s was l a r g e l y s u p r e s s e d and tetrabutylammonium s a l t could be used a s t h e

195 displacer with r e l a t i v e l y small t a i l i n g of t h e l a s t component i n t o t h e d i s p l a c e r zone.

The c h r o m a t o g r a m s i n A

and B a r e r e s u l t s of e x p e r i m e n t s i n which

t h e amount of

l e n g t h of Thus,

feed

per

t h e c o l u m n was t h e

unit same.

both chromatograms appear t o

r e p r e s e n t t h e same s t a g e of d i s p l a c e ment development which i s n o t c o m p l e t e a s m a n i f e s t e d by t h e n o n - r e c t a n g u l a r shape of t h e di-L-valine

zone.

Since

t h i s component is c o m p l e t e l y s e p a r a t e d from t h e o t h e r s and f u r t h e r development would lower i t s c o n c e n t r a t i o n t h e sepa r a t i o n s depicted i n Fig. 14 a r e act u a l l y m o r e e f f i c i e n t t h a n t h e y would

VOLUME [ m l ]

be under i s o t a c h i c c o n d i t i o n s . Fig. 13. D i s p l a c e r c h r o m a t o g r a m o f r e e o r c i n o l and c e t e c h o l . Column, 500 x 4.6 m m ; 5-pm o c t a d e c y l - S p h e r i s o r b ; C a r r i e r . w a t e r : d i s o l a c e r . 0.8 M n-propanol . . i n w a t e r flow ; r i t e , 0.15 ml/min; temp., 2 5 0 c * T h e displacer i s s h o w n b y the shaded r e g i o n . From Ref. 6.

Compar-

i s o n of t h e t w o c h r o m a t o g r a m s p r o v e s t h e p o i n t a l r e a d y made above t h a t wider zones o b t a i n e d w i t h l a r g e r amount of f e e d a l l o w b e t t e r r e c o v e r y t h a n narrowe r ones due t o s m a l l e r f e e d a t t h e same s t a g e of development when zone o v e r l a p

i s about t h e same. The s e p a r a t i o n of a m i x t u r e of t h r e e a d e n y l i c a c i d s and a d e n o s i n e o r o c t a decyl-silica

by d i s p l a c e m e n t development i s i l l u s t r a t e d i n Fig.

15.

n-Butanol

was chosen a s a n e u t r a l d i s p l a c e r because t h e f e e d m i x t u r e c o n t a i n e d b o t h a c i d i c and b a s i c c o m p o n e n t s .

F u r t h e r m o r e b u t a n o l i s v o l a t i l e hence c a n be r e a d i l y

removed from t h e p r o d u c t i f n e c e s s a r y .

B u t a n o l c o n c e n t r a t i o n was l i m i t e d t o

0.28 M by t h e s o l u b i l i t y of a d e n o s i n e which p r e c i p i t a t e d i n t h e column a t h i g h e r d i s p l a c e r c o n c e n t r a t i o n whereas t h e n u c l e o t i d e s t h e m s e l v e s were d i s p l a c e d by.0.5 M n-butanol w i t h o u t any untoward e f f e c t s .

The c o m p o n e n t s o f c o m m e r c i a l p o l y m y x i n B were s e p a r a t e d on o c t y l - s i l i c a column by d i s p l a c e m e n t chromatography and t h e r e s u l t s a r e shown on t h e chromatog r a m d e p i c t e d i n F i g . 16.

The d i s p l a c e r was o c t y l d o d e c y l d i m e t h y l a m m o n i u m -

c h l o r i d e d i s s o l v e d i n t h e c a r r i e r which was w a t e r c o n t a i n i n g 10% a c e t o n i t r i l e . A t t e m p t s t o u s e n-butanol,

phenol o r N,N-dimethylcyclohexylamine

as displacers

f a i l e d because t h e a n t i b i o t i c s e x h i b i t r e l a t i v e l y s t r o n g r e t e n t i o n on a l k y l s i l i c a s t a t i o n a r y p h a s e s due t o combined hydrophobic and s i l a n o p h i l i c i n t e r a c tions.

T h e i r s e p a r a t i o n by d i s p l a c e m e n t chromatography on s i l i c a - g e l o r phenyl-

s i l i c a was l e s s e f f i c i e n t t h a n t h a t i l l u s t r a t e d i n Fig. 16.

196 Corticosterones w e r e s e p a r a t e d by d i s placement chromatography

1

on s i l i c a g e l column. The r e s u l t s o b t a i n e d

? 50-

with chloroform a s the

Q-

c a r r i e r and d i e t h y l -

n-

ethylenediamine a s t h e

m10

-

0

.I$ 0

displacer a t different c o n c e n t r a t i o n s a r e shown 0

8

9 V d n rm .!

i n F i g 17.

The f e e d

components were ReichF i g . 14. Separation o f three dipeptides. A; c o l u m n , 5 - ~ m S p a r i s o r b O O S , 250 X 4.6 m m ; c a r r i e r , 50 mM phosphate b u f f e r ; pH, 2.0; d i s p l e c a r , 150 mM tetrebutylammonium bromide i n the c a r r i e r ; f l o w f e e d , 10 mg o f Lr a t a , 0.86 m l / m i n ; temp, 3 0 ° C ; valyl-L-valine, 15 mg o f g l y c y l - 1 - l e u c i n e and 15 mg 6, o f L-laucyl-L-valina i n 0.5 m l o f c a r r i a r . c o l u m n , 5-pm S p h a r i s o r b O D s , 500 x 4.6 m m ; f l o w r a t e , 0.72 ml/min; feed, 20 mg o f L - v a l y l - L - v a l i n a ; 30 mg o f g l y c y l - L - l a u c i n e and 30 mg o f L-leucylval i n e i n 1.0 m l o f c a r r i a r ; o t h a r c o n d i t i o n s 8 s i n A. From R e f . 8.

stein's

substances Q, S

and H and t h e i r c h e m i c a l structures a r e given in Fig. 7 which showes t h e e m p l o y m e n t o f TLC i n scouting for a suitable carrier.

F r a c t i o n s of

t h e column e f f l u e n t i n t h e s e p a r a t i o n were c o l lected

and

the

zone

b o u n d a r i e s o f t h e comp o n e n t s could be conveniently

e s t a b l i s h e d by

u s i n g TLC a n a l y s i s o f the individual fractions

on s i l i c a g e l . Recent

work

has

demonstrated t h a t d i s placement development i s n o t r e s t r i c t e d t o column chromatography but can VOLUME ( m l l

Fig. 15. Displacement chromatogram o f 5'-AMP, 3'-AMP, 2'-AMP and a d e n o s i n e . Column,S-pm S u p e l c o s i 1 Lc-18 "250 X 4.6 m m l + [ I 5 0 X 4.6 m m l l ; c a r r i e r , 1 0 m M a c e t a t e b u f f e r , pH 5.0; d i s p l a c e r , 0.28 M n - b u t a n o l i n t h e c a r r i e r ; f l o w r a t e , 0.1 ml/min; temp. 22' C; feed, 1 5 mg o f 5'-AMP, 6 mg o f 3'-AMP, 2 4 mg o f 2'-AMP a n d 1 5 mg o f a d e n o s i n e i n 1.5 m l o f c a r r i e r . From R e f . 8.

be a p p l i e d t o p l a n a r chromatograpy a s w e l l

(57).

A particularly

i n t e r e s t i n g example i s t h e s e p a r a t i o n of phenylethylamine derivat i v e s and t h e i r metabol i t e s by c a r r i e r d i s -

197 p l a c e m e n t i n TLC o n s i l i c a g e l (58).

I t was found t h a t t h e components of t h e

d y e s t u f f Sudan Black a r e s u i t a b l e s p a c e r s f o r t h e components of t h e sample t o be analyzed.

A f t e r development of t h e chromatogram t h e sample components occupy

s p a c e s between t h e v e r y t h i n c o l o r e d l i n e s of t h e s p a c e r s .

10

I0

COLUMN E F F L U E N T

[ ml]

30

20 FRACTION NUMBER

F i g . 16. D i s p l a c e m e n t chromatogram o f p o l y m y x i n 8. Column, 5-gm L i c h r o s o r b RP8; c a r r i e r , w a t e r c o n t a i n i n g 1 0 % [ v / v l a c e t o n i t r i Le; d i s p l a c e r , 50 m M o c t y l dodecyldimethyammonium c h l o r i d e i n t h e c a r r i e r . The shaded zone r e p r e s e n t s t h e From Ref. 55. displacer. F i g . 17. D i s p l a c e m e n t chromatogram o f c o r t i c o s t e r o i d e s . Column, 2 x [25D x 4.6 m m l , P a r t i s i 1 PX 5-525 5-pm s i l i c a g e l ; c a r r i e r , c h l o r o f o r m ; d i s p l a c e r , (A1 2.5 [B) 5.0 and [ C l 10% d i e t h y l e t h y l e n e d i a m i n e i n c h l o r o f o r m ; f l o w r a t e , 0.1 ml/min; temp., 22' C ; f e e d , 6 0 mg o f each c o m p o n e n t . S y m b o l s a s i n F i g . 7. F r o m R e f . 7.

I n a n y c a s e t h e f u n d a m e n t a l s of d i s p l a c e m e n t c h r o m a t o g r a p h y c a n b e conveniently demonstrated,

w i t h o u t u s i n g an e l a b o r a t e HPLC s y s t e m , by t h i n l a y e r

d i s p l a c e m e n t chromatography (57).

For t h e n o v i c e i t can s e r v e a s an e d u c a t i o n a l

t o o l t o g e t a c q u a i n t e d w i t h t h e e f f e c t of 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 .

For

c h r o m a t o g r a p h e r s who c a r r y o u t d r s p l a c e m e n t chromatograpy w i t h HPLC i n s t r u m e n t s , TLC can s e r v e a s an e x p e d i e n t t o o l f o r e s t a b l i s h i n g a p p r o p r i a t e o p e r a t i n g condi-

t i o n s and . f o r a n a l y z i n g e f f l u e n t f r a c t i o n s . Displacement chromatography was used i n c o n j u n c t i o n w i t h a r e c i r c u l a t i n g enzyme r e a c t o r w i t h immobilized r i b o n u c l e a s e T , t o s e p a r a t e t h e components of t h e r e a c t i o n m i x t u r e i n which guanosine-2':3'-cyclic d i n e , U, r e a c t t o f o r m g u a n y l y l (3'->

5')

u r i d i n e , GpU.

p h o s p h a t e , G>P, and u r i With l a r g e e x c e s s of U

a b o u t h a l f o f G > P i s c o n v e r t e d i n t o GpU and some o f i t h y d r o l y z e s t o y i e l d

198 guanosine-3’-phosphate,

Gp. I n t h e s y s t e m d e s c r i b e d h e r e t h e p r o d u c t of t h e

r e a c t i o n i s s e p a r a t e d by d i s p l a c e m e n t chromatography from t h e u n r e a c t e d r e a g e n t s which a r e r e c y c l e d i n t o t h e r e a c t o r .

The a d v a n t a g e of u s i n g d i s p l a c e m e n t chro-

matography i n combination w i t h t h e r e a c t o r r e s t s w i t h t h e n e g l i g i b l e d i l u t i o n d u r i n g t h e c h r o m a t o g r a p h i c p r o c e s s so t h a t d i r e c t r e c y c l i n g o f t h e u n r e a c t e d reagents i s facilitated.

The f o r m a t i o n o f GpU by e n z y m a t i c c o n d e n s a t i o n was

used t o e x p l o r e t h e p o t e n t i a l of such an i n s t r u m e n t a s a s y n t h e t i z e r .

The

f l o w s h e e t of t h e a p p a r a t u s w i t h o u t f e a t u r i n g d i r e c t r e c y c l i n g of t h e u n r e a c t e d r e a g e n t s b a c k t o t h e r e a c t o r i s d e p i c t e d i n F i g . 18.

The t e m p e r a t u r e s o f t h e

r e a c t o r and t h e chromatographic column were c o n t r o l l e d i n d e p e n d e n t l y w i t h t h e r m o s t a t t e d w a t e r baths.

The tandem arrangement of t h e r e c y c l i n g enzyme r e a c t o r

and t h e d i s p l a c e m e n t chromatograph a l l o w s f u l l y automated o p e r a t i o n .

F i g . 18. F l o w s h e e t o f t h e s y n t h a t i r e r c o n s i s t i n g o f a r e c i r c u l a t i n g enzyme r a a c t o r and a displacement chromatograph. From Ref. 54. F i g . 19. D i s p l a c e m e n t c h r o m a t o g r a m s o f t h e r e a c t i o n m i x t u r e f r o m t h e enzyme c a t a l y z e d f o r m a t i o n o f GpU. Twa colums were usad i n s e r i e s f o r t h e s e p a r a t i o n . Column I,150 x 4.6 m m , 5- p m S u p e l c o s i 1 LC-18; Column 11, 250 x 4.6 m m , 5-pin Z o r b a x C18; c a r r i e r ; 5 0 m M p h o s p h a t e b u f f e r , pH 3.0; d i s p l e c e r , 0.25 M nI n B the bulk o f uridina b u t a n o l i n c a r r i e r ; f l o w r a t e , 0.1 ml/min; temp. 2’ C. was s e p a r a t a d by f r o n t a l chromatography i n Column Iand w i t h d r a w n b e f o r e e n t e r i n g Column 11. From R e f . 54.

I n t h e chromatographic s e p a r a t i o n of t h e r e a c t i o n m i x t u r e which was c a r r i e d o u t w i t h two a l k y l - s i l i c a columns i n s e r i e s , f i r s t t h e l a r g e e x c e s s of u r i d i n e , t h e l e a s t r e t a i n e d components of t h e m i x t u r e , was s e p a r a t e d by f r o n t a l chromato-

199

g r a p h y , w i t h d r a w n a t t h e end of t h e f i r s t column and r e t u r n e d t o t h e r e a c t o r . The r e s t of t h e f e e d was s e p a r a t e d by d i s p l a c e m e n t d e v e l o p m e n t w i t h n - b u t a n o l b e c a u s e t h e f e e d c o m p o n e n t s w e r e a m p h o l y t e s and a d i s p l a c e r h a v i n g no i o n i c c h a r g e s had t o b e s e l e c t e d .

The co lu m n was k e p t a t low t e m p e r a t u r e i n o r d e r t o

m i n i m i z e h y d r o l y t i c d e g r a d a t i o n o f t h e s t a r t i n g r e a g e n t G>P a n d t h e p r o d u c t , GpU. T y p i c a l r e s u l t s o f t h e d i s p l a c e m e n t c h r o m a t o g r a p h i c s e p a r a t i o n a r e shown i n Fig. 19.

I t i s s e e n i n B t h a t most o f u r i d i n e was r e c o v e r e d a t h i g h c o n c e n t r a -

t i o n s a t t h e end o f t h e r e l a t i v e l y s h o r t f i r s t column a f t e r b e i n g s e p a r a t e d by f r o n t a l c hroma tog r ap h y .

On t h e o t h e r h an d , i n A u r i d i n e was n o t skimmed o f f b u t

l e t p a s s t h r o u g h b o t h c o l u m n s and a s a r e s u l t i t emerged a s a w i d e band h a v i n g s i g n i f i c a n t l y lower concentration.

The o t h e r co mp o n en t s w e r e r e c o v e r e d a t t h e

e nd o f t h e s e c o n d c o l u m n a f t e r d i s p l a c e m e n t d e v e l o p m e n t w a s c o m p l e t e d .

Th e

p r o d u c t GpU was c o l l e c t e d , w h e r e a s t h e r e a c t a n t s U and G>P w er e r e t u r n e d t o t h e reactor.

T h e s i d e p r o d u c t Gp w a s a l s o r e t u r n e d a n d l e t a c c u m u l a t e f o r l a t e r

removal from t h e mixture.

In s u c h a p p l i c a t i o n s d i s p l a c e m e n t d e v e l o p m e n t o f f e r s s i g n i f i c a n t a d v a n t a g e s o v e r e l u t i o n chromatography due t o t h e r e l a t i v e l y h i g h c o n c e n t r a t i o n s i n t h e e f f l u e n t stream.

By u s i n g a p a r a l l e l a r r a n g e m e n t o f t w o s e t s o f c o l u m n s , o n e i s

s e p a r a t i n g w h i l e t h e o t h e r is being r e g e n e r a t e d , t h e c a p a c i t y of t h e s y n t h e t i z e r can be doubled without d i f f i c u l t i e s .

The u s e of a s i m i l a r r e a c t o r

cum

chromato-

graph u n i t i s c u r r e n t l y explored i n our laboratory f o r enzymatic peptide synthesis.

0UTL.OOK

A f t e r e s t a b l i s h i n g i t s e l f a s t h e most v e r s a t i l e a n a l y t i c a l t o o l , high performance l i q u i d chromatography i s j u s t about t o g a i n prominence a s a preparat i v e and even an i n d u s t r i a l s c a l e s e p a r a t i o n p r o c e s s m a i n l y d u e t o demands e ng e nde re d by t h e e m e r g i n g b i o t e c h n o l o g y .

Whereas t h e e l u t i o n mode of c h r o m a t o -

g r a p h y i s e m i n e n t l y s u i t a b l e f o r a n a l y t i c a l a p p l i c a t i o n s it h a s d i s t i n c t d i s a d v a n t a g e s i n p r e p a r a t i v e work.

Poor u t i l i z a t i o n of t h e column, l a r g e s o l v e n t

c o n s u m p t i o n and h i g h c o s t of p r o d u c t r e c o v e r y f r o m t h e e f f l u e n t due t o t h e d i l u t i o n i n h e r e n t t o t h e e l u t i o n p r o c e s s d i m i n i s h t h e a p p e a l o f u s i n g t h i s mode of c h r o m a t o g r a p h y f o r l a r g e s c a l e s e p a r a t i o n s . R e c e n t l y o u r i n t e r e s t t u r n e d t o d i s p l a c e m e n t d e v e l o p m e n t , t h e o t h e r mode o f chromatography f o r multicomponent s e p a r a t i o n s .

For a number of r e a s o n s d i s -

p l a c e m e n t c h r o m a t o g r a p h y a f t e r a p r o m i s i n g s t a r t 40 y e a r s a g o was r a t h e r d o r m a n t o v e r t h e p a s t two d e c a d e s u n t i l a d v a n c e s i n l i q u i d c h r o m a t o g r a p h y h a v e p r o mp t ed an examination of i t s p o t e n t i a l .

P r o g r e s s i n t h e t h e o r y of non-l i near

chromato-

g r a p h y h a s a l s o c o n t r i b u t e d t o t h e u n d e r s t a n d i n g o f t h e p h y s i c o - c h e m i c a l phenomena u n d e r l y i n g d i s p l a c e m e n t d ev elo p m en t.

200 So f a r t h e a p p l i c a t i o n s o f h i g h p e r f o r m a n c e d i s p l a c e m e n t chromatography

have been d e m o n s t r a t e d by s e m i - p r e p a r a t i v e s e p a r a t i o n s of low and i n t e r m e d i a t e m o l e c u l a r weight compounds and by u s i n g t h e column and i n s t r u m e n t a t i o n g e n e r a l l y employed i n HPLC.

The b a s i c p r i n c i p l e s f o r t h e d e s i g n of t h e chromatographic

system and f o r s e l e c t i n g optimum o p e r a t i n g c o n d i t i o n s have been e s t a b l i s h e d . A t p r e s e n t , however, wide u s e of d i s p l a c e m e n t development i s g r e a t l y hampered by l a c k of s u f f i c i e n t l y broad e x p e r i e n c e and d a t a b a s e , t h e burden o f and r e l u c t a n cy t o a c c e p t i n g n o n - l i n e a r c h r o m a t o g r a p h y a n d by t h e d i f f i c u l t i e s a s s o c i a t e d w i t h a d a p t i n g a new way of c a r r y i n g o u t chromatographic s e p a r a t i o n s . A t t e m p t s i n o u r l a b o r a t o r y t o s e p a r a t e b i o p o l y m e r s by d i s p l a c e m e n t chromato g r a p h y w i t h c o l u m n s t r a d i t i o n a l l y u s e d i n HPLC f o r c h r o m a t o g r a p h y o f s m a l l m o l e c u l e s were n o t y e t s u c c e s s f u l .

Recent developments i n t h e d e s i g n of h i g h

performance s t a t i o n a r y p h a s e s f o r t h e s e p a r a t i o n of p r o t e i n s and n u c l e i c a c i d by hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n chromatography, however, l e n d new opp o r t u n i t i e s f o r d i s p l a c e m e n t chromatography of b i o p o l y m e r s a s well.

T h i s ap-

proach o f f e r s p a r t i c u l a r l y p r o m i s i n g a p p l i c a t i o n s i n v a r i o u s a r e a s of b i o t e c h nology. The knowledge of t h e a d s o r p t i o n ' i s o t h e r m s of f e e d components and p o t e n t i a l d i s p l a c e r s on t h e s t a t i o n a r y phase of i n t e r e s t g r e a t l y f a c i l i t a t e s method and p r o c e s s development i n d i s p l a c e m e n t chromatography.

Recent employment of h i g h

performance f r o n t a l chromatography f o r r a p i d i s o t h e r m measurements w i t h m i n u t e amounts of s o l u t e s o f f e r s a c o n v e n i e n t method f o r g a t h e r i n g i s o t h e r m d a t a .

Of

c o u r s e t h e a v a i l a b i l i t y of such q u a n t i t a t i v e i n f o r m a t i o n i s of physico-chemical s i g n i f i c a n c e a s f a r a s a d s o r p t i o n from l i q u i d s and t h e i n t e r a c t i o n of b i o l o g i c a l s u b s t a n c e s w i t h s u r f a c e s a r e concerned. I n t h e a n a l y t i c a l f i e l d d i s p l a c e m e n t chromatography can b e used a s a h i g h l y e f f i c i e n t means of sample c o n c e n t r a t i o n .

Our e x p e r i e n c e i n u s i n g t h i s approach

w i t h narrow b o r e packed columns h a s been v e r y p r o m i s i n g .

The e m p l o y m e n t o f

d i s p l a c e m e n t chromatography w i t h narrow b o r e columns e i t h e r i n tandem o p e r a t i o n of t h e l i q u i d chromatograph w i t h t h e mass s p e c t r o m e t e r o r i n m i c r o p r e p a r a t i v e work may a l s o o f f e r c e r t a i n a d v a n t a g e s o v e r t h e u s e of e l u t i o n chromatography. I t i s expected t h a t scaling-up

t h e chromatographic p r o c e s s i n t h e d i s p l a c e -

ment mode w i l l r e q u i r e more c a r e f u l e n g i n e e r i n g t h a n t h a t employed p r e s e n t l y i n e l u t i o n chromatography.

I t i s l i k e l y t h a t some of t h e o l d e r i d e a s such a s t h e

a t t e n u a t i o n of t h e c o l u m n d i a m e t e r a t t h e o u t l e t end t o r e d u c e t h e v o l u m e o f zone i n t e r m i x i n g h a v e t o b e i n c o r p o r a t e d i n t o t h e d e s i g n o f a l a r g e s c a l e d i s p l a c e m e n t chromatograph i n o r d e r t o e x p l o i t t h e f u l l p o t e n t i a l of t h e process.

Other problems a s s o c i a t e d w i t h sample i n t r o d u c t i o n o r a x i a l d i s p e r s i o n

a r i s i n g from c h a n n e l i n g a r e expected t o be common f o r a l l t y p e s of p r e p a r a t i v e s c a l e chromatographs.

I n any c a s e , t h e a v a i l a b i l i t y of s u i t a b l e column m a t e r i -

a l s w i l l b e a key f a c t o r i n t h e t r a n s f e r of chromatographic s e p a r a t i o n p r o c e s s

201 from t h e l a b o r a t o r y t o i n d u s t r i a l p l a n t s . A p a r t i c u l a r f e a t u r e o f d i s p l a c e m e n t c h r o m a t o g r a p h y i s t h e much l o w e r d i l u t i o n o f t h e f e e d c o m p o n e n t s i n t h e c o u r s e of t h e s e p a r a t i o n p r o c e s s t h a n t h a t o c c u r s i n e l u t i o n chromatography.

T h i s makes p a r t i c u l a r l y a t t r a c t i v e t h e

c o m b i n a t i o n o f a c h e m i c a l r e a c t o r w i t h a d i s p l a c e m e n t chromatograph when t h e u n r e a c t e d components of t h e r e a c t i o n m i x t u r e have t o be s e p a r a t e d from t h e p r o d u c t and r e c y c l e d t o t h e r e a c t o r . The h i s t o r y o f c h r o m a t o g r a p h y h a s s e v e r a l p r e c e d e n t s t h a t a p a r t i c u l a r t e c h n i q u e reached prominence only a f t e r a long dormant p e r i o d . nowned i s perhaps A.J.P.

The . b e s t r e -

Martin’s r e v e r s e d phase chromatography (59) t h a t , a f t e r

20 y e a r s of b e i n g c o n s i d e r e d a n odd and v e r y s p e c i a l method, h a s become t h e most w i d e l y used and g e n e r a l l y a p p l i c a b l e branch of HPLC.

Should p r e p a r a t i v e l i q u i d

chromatography l i v e up t o t h e e x p e c t a t i o n s and become a s e p a r a t i o n p r o c e s s of broad i n d u s t r i a l s i g n i f i c a n c e it might j u s t be due t o t h e u s e of a n o t h e r approach, s t i l l o f f b e a t today, d i s p l a c e m e n t chromatography.

ACKNOWLEDGEMENTS

The a u t h o r g r a t e f u l l y acknowledges s u p p o r t of t h i s work by g r a n t s No. 21948 and GM 20993 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e and N a t i o n a l I n s t i t u t e f o r G e n e r a l M e d i c a l S c i e n c e s , U.S.

P u b l i c H e a l t h and Human s e r v i c e s a n d by t h e

N a t i o n a l Foundation f o r Cancer Research.

A . T i s e l i u s , Arkiv Kemi M i n e r a l . Geol., A . T i s e l i u s , i n A . T i s e l i u s (Ed.),The A.J.P.Martin

and A.Synge,

R.Consden, A.H.Gordon A.T.James

and A.J.P.Martin,

Cs.Horvith,

35 (1941) 1358. Biochem. J . , 38 (1944) 224.

Biochem. J . (London), 50 (1952) 679.

Cs.Horvith, A.Nahum and J.H.Frenz, H.Kalisz and Cs.Horvith,

(1943) 1.

Svedberg,Almqvist,Uppsala,l944,p370.

Biochem.J.,

and A.J.P.Martin,

16A ( 1 8 )

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J. Chromatogr., 239 (1982) 423.

J.Frenz and Z . E l R a s s i , J . Chromatogr., 255 (1983) 273.

F.H.Spedding,

J.E.Powel1,

E.I.Fulmer and T.A.Butler,

J . Am.

Chem. SOC.,

7 2 (1950) 2354. 10

F.H.Spedding,

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and H.J.Svec,

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Chem. SOC., 7 7 (1955) 6125.

11 C.N.Davidson, C.K.Mann and R.E.Sheline,J.Am.Chem.Soc., 12

2.Hagiwara and Y.Takakura,

13

S . F u j i n e , K.Saito and K.Shiba, Sep. S c i . Tech.,

14

S.Claesson, Rec. Trav. Chim.,T65 (1946) 571.

8 3 (1961) 2389.

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15 C.S.G.Phillips, Disc. Faraday SOC., 7 (1949) 241. and C.S.G.Phillips, in A.Goldup (Ed.),Gas Chromatography, 16 C.G.Scott Butterworths, (London), 1964. J.A.Harris, K.F.Scott, M.J.Walker and C.S.G.Phillips, J. 17 C.M.A.Badger, Chromatogr., 126 (1976) 11. 18 J.P.Horrocks, J.A.Jarris, C.S.G.Phi1lips and K.F.Scott, J. Chromatogr., 197 (1980) 109. 19 C.L.Mantel1, Adsorption, 2nd Ed., McGraw-Hill, New York (1951). 20 D.P.Broughton, in Kirk-Othmer Encyclopedia of Chemical Technology, Wiley, New York, 1978,p 563. 21 S. Claesson, Ann. N.Y. Acad. Sci., 49 (1948) 183. 22 L.Hagdah1, Acta Chem. Scand., 2 (1948) 573. 23 L.Hagdah1 and R.T.Holman, J. Am. Chem. SOC., 72 (1950) 701. 24 L.Hagdah1, R.J.P.William6 and A.Tiselius, Arkiv Kemi, 4 (1952) 193. 25 J.Porath, Acta Chem. Scand.,6 (1952) 1237. 26 J.Porath, Acta Chem. Scand., 8 (1954) 1813. 27 C.C.Shepard and A.Tiselius, Disc. Faraday SOC., 7 (1949) 275. 28 C.H.Li, A.Tiselius, K.O.Pederson, L.Hagdah1 and H.Carstensen, J. Biol. Chem., 190 (1951) 317. 29 S.M.Patridge, Disc. Faraday SOC., 7 (1949) 296. 30 S.M.Patridge and R.C.Brimley, Biochem. J., 48 (1951) 313. 31 S.M.Patridge and R.C.Brimley, Biochem. J., 49 (1951) 153. 32 S.M.Patridge, Chem. Ind., (1950) 383. 33 M.Page and M.Belles-Isles, Canad. J. Biochem., 56 (1978) 853. 34 J.P.Emond and M.Page, J. Chromatogr., 200 (1980) 57. 35 E.A.Peterson, Anal. Biochem., 90 (1978) 767. 36 E.A.Peterson and A.R.Torres, Anal. Biochem., 130 (1983) 271. 37 E.A.Peterson and A.R.Torres, Methods in Enzymology, 104 (1984) 113. 38 F.Helfferich, Ind. Eng. Chem. Fund., 6 (1967) 362. Multicomponent Chromatography- Theory of 39 F.Helfferich and G.Klein, Interference, Marcel Dekker, New York (1970). 40 F.Helfferich and D.B.James, J. Chromatogr., 46 (1970) 1. 41 D.DeVault, J. Am. Chem. SOC., 65 (1943) 532. 42 J.Weiss, J. Chem. SOC. (London), (1943) 297. 43 E.Glueckauf, Proc. Roy. SOC. (London),A, 186 (1946) 35. 44 E.Glueckauf and J.I.Coate.9, J. Chem. SOC. (London), (1947) 1315. 45 L.G.Sillen, Arkiv Kemi, 2 (1950) 477. 46 S.Claesson, Arkiv. Kem. Mineral., Geol., 24A (16) (1947) 1. 47 H.K.Rhee, R.Aris and N.R.Amundson, Phil. Trans. Roy. Soc.(London), (1970) 419.

48 H.K.Rhee and N.R.Amundson, Am. Inst. Chem. Eng. J., 28 (1982) 423.

267

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49 J.Frenz and Cs.Horvith, Am. Inst. Chem. Eng. J., in press. 50 J.Frenz and Cs.Horvith,in Cs. Horvith (Ed.), HPLC- Advances and

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tives, Vol.IV, Academic Press, Orlando, in press. 51

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52 J.Frenz, J. Jacobson and Cs .Aorvith, in preparation. 53 J.Frenz, Ph.van der Schrieck and Cs.Horv;th, J. Chromatogr., submitted. 54 Z.El Rassi and Cs.Horvith, J. Chromatogr., 266 (1983) 319. 55 H.Kalisz and Cs.Horvith, J. Chromatogr., 215 (1981) 295. 56 G.E.Veress,Cs.Horvith and E.Pungor,in H.Kalisz (Ed.) New Approaches in Liquid Chromatography, AkadGmiai Kiad6,Budapest 1984, pp 45-56. 57 H.Kalisz and Cs.Horvith, in H.Kalisz (Ed.) ,New Approaches in Liquid Chromatography, AkadGmiai Kiad6, Budapest,1984, pp 57-61. 58 H.Kalisz, J. High Resolut. Chromatogr. Chrom. Corn., 6 (1983) 49. 59 G.A.Howard and A.J.P.Martin, Biochem. J., 46 (1950) 532.

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205

RETENTION I N LIQUID/SOLID CHROMATOGRAPHY

ERVIN

SZ.

KOV~TS

L a b o r a t o i r e de Chimie-technique de 1 ' E c o l e P o l y t e c h n i q u e FCderale de Lausanne,

1015 Lausanne ( S w i t z e r l a n d ) .

SWARY R e t e n t i o n d a t a i n l i q u i d / s o l i d chromatography a r e recorded on t h e b a s i s o f e q u a t i o n s which were developed f o r l i q u i d / l i q u i d and g a s / l i q u i d chromatography. It i s shown t h a t these e q u a t i o n s a r e inadapted.

It i s a l s o shown t h a t r e t e n t i o n

i n l i q u i d / s o l i d chromatography can be g i v e n i n terms o f Gibbs' d e s c r i p t i o n o f t h e a d s o r p t i o n process. As i n systems w i t h r e v e r s i b l e a d s o r p t i o n e q u i l i b r i u m , t h e i n t r o d u c t i o n o f a c o n v e n t i o n i s necessary.

In the l i g h t o f t h i s retention

equation, t h e e n e r g e t i c s o f t h e a d s o r p t i o n process i s discussed as w e l l as d i f f e r e n t methods o f t h e d e t e r m i n a t i o n o f t h e hold-up volume.

INTRODUCTION

De V a u l t was t h e f i r s t t o g i v e an e x a c t s o l u t i o n o f r e t e n t i o n i n chromatography, c o n s i d e r e d t o proceed t h r o u g h i n f i n i t e s i m a l e q u i l i b r i u m steps ( r e f . 1). I n t h e mathematical s o l u t i o n , matrix.

r e t e n t i o n volumes appear as e i g e n v a l u e s o f a

The r e s u l t s were a p p l i e d b y Helfferich and K l e i n f o r t h e d i s c u s s i o n o f

chromatography ( r e f .

2).

R e c e n t l y i t was shown t h a t t h e model o f de V a u l t be-

comes g e n e r a l l y a p p l i c a b l e b y i n t r o d u c i n g a new s i m p l e thermodymanic f u n c t i o n the i s o c r a t i c capacity,

?iK ( r e f . 3 ) . This new f u n c t i o n i s t h e m a t e r i a l con-

t e n t o f an open system ( t h e column) i n e q u i l i b r i u m w i t h an i n f i n i t e r e s e r v o i r o f a f l u i d m i x t u r e ( s e e F i g . 1). The s p e c i f i c s o l u t i o n f o r a n a l y t i c a l chromatography under i s o t h e r m a l and i s o c r a t i c c o n d i t i o n s i s g i v e n b y eqn. 1

where vR,i

i s t h e r e t e n t i o n volume o f an i n f i n i t e s i m a l sample o f i (component

206

-

I n f i n i t e reservoir with a f l u i d m i x t u r e o f the composition: s;" = x ou,AYXp,B(=l-XA) 0 A

x

= xu

where x

, ~ * X u , ~ y X p , ~ ~

su

+

0

/

I s o c r a t i c c a p a c i t y o f t h e open system (column)

K"

= no

no K,B

-

n

K,A'

K

A

n

K

- 'K,A'

where n

K ,SU

K,B'

n

K,SU

+O

F i g . 1 The i s o c r a t i c c a p a c i t y o f t h e column, ;n i n equilibrium with a binary m i x t u r e o f A and B o f t h e composition, x i and t h e i s o c r a t i c c a p a c i t y n, i n t h e presence o f a t h i r d component, su ( s o l u t e ) o f i n f i n i t e s i m a l c o n c e n t r a t i o n , xsu. o f the eluent, e l u e n t , nK,j

A,B

... o r

solute,

su),

v;

i s t h e mean molar volume o f t h e

i s t h e column c a p a c i t y o f component,

i, and p*

i s the composition

o f t h e m o b i l e phase. The i d e a l i z e d chromatographic column i s c o n s i d e r e d t o have a u n i f o r m c r o s s s e c t i o n a t any d i s t a n c e from t h e i n l e t , f i l l e d w i t h a quasi continuum o f a porous powder. E q u i l i b r i u m i s i n s t a n t a n e o u s i n any c r o s s s e c t i o n o f t h e column and t h e r e i s no a x i a l d i f f u s i o n . Therefore, t h e p e r t u r b a t i o n o f t h e e l u e n t b y an i n f i n i t e s i m a l s i g n a l a t t h e column i n l e t w i l l appear a t t h e column o u t l e t r e t a i n e d , b u t n o t deformed.

RETENTION I N M S / L I Q U I D AND LIQUID/LIQUID CHROMATOGRAPHY I n o r d e r t o demonstrate t h e use o f eqn. 1 i t w i l l now b e a p p l i e d t o g a s / l i q u i d chromatography w i t h a m o b i l e phase composed o f a m i x t u r e o f two i d e a l gases,

S and I. Component S i s s o l u b l e i n t h e s t a t i o n a r y l i q u i d , whereas compo-

nent I i s i n s o l u b l e . V,

= w,/d,

The volume o f t h e s t a t i o n a r y phase can be c a l c u l a t e d as

b y supposing t h a t t h e s t a t i o n a r y l i q u i d f i l m on t h e i n e r t sup-

p o r t has t h e same d e n s i t y , given by

d,,

as t h e b u l k .

The column c a p a c i t i e s are t h e n

207

where c i s t h e c o n c e n t r a t i o n ,

[mol

1-11, t h e s u b s c r i p t s p and

5

refer t o the

m o b i l e and s t a t i o n a r y phases r e s p e c t i v e l y and t h e o t h e r symbols a r e as b e f o r e . Having t h e necessary e x p r e s s i o n s a t hand (eqns. 2 and 4) a p p l i c a t i o n o f eqn. 1 g i v e s t h e same e x p r e s s i o n f o r t h e r e t e n t i o n volume f o r t h e p e r t u r b a t i o n o f t h e e l u e n t c o m p o s i t i o n b y i n j e c t i o n s o f v e r y small amounts of S o r I. I n j e c t i o n o f e i t h e r o f t h e gases provokes a concentration peak pressed w i t h x,s

where V,

. Its

r e t e n t i o n volume,

ex-

as t h e independent v a r i a b l e , i s g i v e n i n eqn. 5

i's t h e hold-up volune (dead volume) o f t h e column. T h i s same r e s u l t

was found by Valentin and Guiochon ( r e f . 4).

I n t h e s p e c i a l case where t h e r e i s

o n l y an i n s o l u b l e c a r r i e r and t h e s o l u b l e gas i s i n j e c t e d as s o l u t e ( S

+

su),

eqn. 5 s i m p l i f i e s t o

where

KSu

i s the partition coefficient o f the solute at

infinite dilution

Eqn.6 i s t h e c l a s s i a l r e l a t i o n s h i p o f Martin and Synge f o r r e t e n t i o n volume i n

1 i q u i d / l i q u i d chromatography ( r e f . 5)

, calculated

today as " C r a i g ' s c o u n t e r c u r r e n t b a t t e r y " machine" ( r e f . 6).

w i t h t h e a i d o f a model known

or the "droplet

counter c u r r e n t

I n t h e i r model t h e chromatographic column was compared w i t h

a h y p o t h e t i c a l b a t t e r y o f s e p a r a t i n g c e l l s g i v i n g t h e same chromatogram as t h e chromatographic column i n q u e s t i o n . The number o f t h e c e l l s o f t h e h y p o t h e t i c a l b a t t e r y was a measure o f t h e peak w i d t h and t h e r e b y t h e e f f i c i e n c y o f t h e c h r o matographic column. T h i s d e s c r i p t i o n i n i t i a l l y c r e a t e d some c o n f u s i o n about t h e meaning o f a c e l l , which was c a l l e d a p l a t e , s u g g e s t i n g t h e same r o l e p l a y e d b y a p a r t i t i o n i n g c e l l as t h a t b y a s e p a r a t i n g p l a t e i n d i s t i l l a t i o n . Furthermore, s i g n a l d e f o r m a t i o n i n t h i s model i s due t o t h e f i n i t e volume o f t h e s e p a r a t i n g c e l l s and n o t t o d i f f u s i o n and t o k i n e t i c s o f e q u i l i b r i u m a t t a i n m e n t . Nevertheless, t h e c e l l model gave t h e r i g h t answer f o r t h e r e l a t i o n s h i p of t h e r e t e n t i o n volume w i t h t h e p a r t i t i o n c o e f f i c i e n t i n l i q u i d / l i q u i d and gas/ l i q u i d chromatography ( r e f . 7 ) . It was t h e r e f o r e proposed t o use t h e net-reten-

t i o n volune.

f o r the characterization o f retention.

The hold-up volume,

Vp,

c o u l d be de-

t e r m i n e d as t h e r e t e n t i o n volume o f a n o n - r e t a i n e d substance, a substance i n s o -

208

l u b l e i n t h e s t a t i o n a r y phase. A r e t e n t i o n volume s m a l l e r t h a n t h e hold-up v o lume was n o t p o s s i b l e .

In gas chromatography t h e n e t - r e t e n t i o n volume i s r e l a -

t e d t o Henry's coefficient b y

where

nu and wu a r e t h e number o f moles and t h e mass o f t h e s t a t i o n a r y li-

q u i d i n t h e column r e s p e c t i v e l y , constant,

Tc i s t h e column temperature,

hsu i s Henry's c o e f f i c i e n t

R i s t h e gas

and gsu i s H e n r y ' s m o l a l c o e f f i c i e n t

o f the solute a t i n f i n i t e d i l u t i o n i n t h e actual solvent.

Eqn. 9 i s d e r i v e d

from eqn. 8. It g i v e s t h e r e l a t i o n s h i p o f t h e r e t e n t i o n volume w i t h t h e

diffe-

rence o f t h e standard chemical potential of the substance between t h e i d e a l d i l u t e s o l u t i o n and t h e gas s t a t e as e i t h e r h ~ p i u( r e l a t e d t o hsu) o r 9 A p i u ( r e l a t e d t o g s u ) . The gas phase i s c o n s i d e r e d t o be a m i x t u r e o f t h e i d e a l c a r r i e r and t h e substance vapor as i d e a l gas. RTclnVN,su = RTcln(n uRTc ) - h ~ p Z u= RTcln(wuRTc/lOOO)

- 9~pru

This simple r e l a t i o n s h i p between t h e l o g a r i t h m o f t h e n e t r e t e n t i o n volume and t h e standard chemical p o t e n t i a l d i f f e r e n c e has been v e r y u s e f u l f o r e s t a b l i shing l i n e a r f r e e energy r e l a t i o n s h i p s f o r t h e p r e d i c t i o n o f chromatographic data. It a l s o helped t h e u n d e r s t a n d i n g o f i n t e r a c t i o n f o r c e s . S i m i l a r r e l a t i o n s h i p s can be d e r i v e d , mutatis mutandis, f o r l i q u i d / l i q u i d chromatography.

RETENTION IN LIQUID/SOLID CHROMATOGRAPHY The success o f l i q u i d / l i q u i d chromatography had a s e r i o u s impact on t h e development o f chromatographic techniques. This impact was even a m p l i f i e d by t h e d i s c o v e r y o f g a s / l i q u i d chromatography b y Martin and James ( r e f .

7).

It gave

t h e impetus and l e a d i n g ideas f o r t h e achievement o f h i g h e r performance i n l i q u i d / s o l i d chromatography. The s u c c e s s f u l a p p l i c a t i o n o f t h e column t e c h n o l o g y o f g a s / l i q u i d chromatography suggested t h e seducing i d e a t o a l s o a p p l y t h e succ e s s f u l r e s u l t s o f t h e model o f Martin and Synge t o l i q u i d / s o l i d chromatography b y f o r m a l analogy, and t o a p p l y eqns. 7, 8 and 9 w i t h o u t a d a p t a t i o n t o t h e desc r i p t i o n o f r e t e n t i o n . Therefore, s e v e r a l papers d i s c u s s t h e meaning o f t h e vo-

lune o f the stationary phase and t h e r e l a t e d problem o f t h e hold-up volune. Before d i s c u s s i n g these essays,

l e t us f i r s t a p p l y eqn. 1 f o r t h e r e t e n t i o n

volume i n l i q u i d / s o l i d chromatography. The molar i s o c r a t i c c a p a c i t y o f a column f i l l e d w i t h an adsorbent i s g i v e n by

209

and =

nK,tOt

where

S

Vp/CX/V$&

+

rtot/cx

i s t h e s u r f a c e area o f t h e adsorbent, T i i s t h e s u r f a c e c o n c e n t r a t i o n

of t h e i t h component and a l l o t h e r symbols a r e as b e f o r e . I n a system where adsorption i s r e v e r s i b l e , surface concentration i s not a defined q u a n t i t y without introducing

an a d d i t i o n a l e q u a t i o n d e f i n i n g

concerning t h e

adsorption

equilibrium.

By

a convention (Convention X =CX) using the

particular

convention

Nothing t h a t t h e sum o f t h e s u r f a c e c o n c e n t r a t i o n s [pmol w 2 ] i s equal t o zero (is Adsorbed i n terms o f number o f moles, n: nNA);

a d s o r p t i o n i s d e s c r i b e d i n terms o f reduced surface concentrations, ri/,,NA PAC-symbol:

n$n)

/S).

Using eqns. 10 and 11, a p p l i c a t i o n o f eqn.

(IU-

1 gives f o r

t h e r e t e n t i o n volume:

if t h e nNA-convention i s a p p l i e d . It i s seen t h a t t h e h o l d - u p volume, Vp/nNA i s defined

i n connection w i t h the given convention.

similar

A

d e r i v e d i f a d s o r p t i o n i s expressed i n an unusual u n i t , Y [ p l m-'1

equation

, is

and u s i n g an

unusual convention, vNA (Nothing i s Adsorbed i n terms o f volume, v ) . This g i v e s

where, g, i s f o r t h e volume f r a c t i o n . For chromatography w i t h a binary eluent

composed o f A and B eqn. 14 g i v e s

eqn. 15 f o r t h e r e t e n t i o n volume o f a c o n c e n t r a t i o n p e r t u r b a t i o n ( i n j e c t i o n o f a small amount o f A o r 6 ) t o g i v e a c o n c e n t r a t i o n peak, cc,

Thus,

t h i s r e t e n t i o n volume i s r e l a t e d t o t h e d e r i v a t i v e o f t h e a d s o r p t i o n

i s o t h e r m o f component A o f t h e e l u e n t , ' Y A / ~ N A . ( T h i s r e l a t i o n s h i p i s an approx i m a t i o n , i t i s s t r i c t l y v a l i d o n l y f o r p e r f e c t l i q u i d m i x t u r e s ) . The r e t e n t i o n volume o f a l a b e l l e d component o f t h e e l u e n t , A* o r B*,

i s directly related to

210 t h e a d s o r p t i o n isotherm:

F i n a l l y , t h e r e t e n t i o n volume o f a s o l u t e i s g i v e n b y

These r e l a t i o n s h i p s a l s o d e f i n e t h e a c t u a l h o l d - u p volume b y g i v i n g t h e e x p e r i mental method o f i t s d e t e r m i n a t i o n . Considering t h a t

YA/,NA

+

Y ! B / ~ N A = 0,

the

sum o f eqn. 16 g i v e s a f t e r rearrangement:

Eqn.

18 was f i r s t d e r i v e d by Knox on t h e b a s i s o f a d i f f e r e n t argumentation

( r e f . 8).

( S i m i l a r r e l a t i o n s h i p s a r e r e a d i l y d e r i v e d from eqn.

t h e method o f c a l c u l a t i o n o f V,,/nNA

13 g i v i n g a l s o

(ref. 3)).

As a t e s t o f these r e l a t i o n s h i p s l e t us examine e x p e r i m e n t a l s p e c i f i c r e t e n t i o n volumes o f l a b e l l e d ( d e u t e r a t e d ) a c e t o n i t r i l e ,

l a b e l l e d water ( H D O ) ,

and

t h a t o f t h e c o n c e n t r a t i o n peak, p l o t t e d i n F i g . 2 as a f u n c t i o n o f e l u e n t comp o s i t i o n , w i t h tetradecyldimethylsiloxy m o d i f i e d s i l i c a as adsorbent ( r e f . 9 ) . R e t e n t i o n volumes were c o r r e c t e d w i t h t h e hold-up volume from eqn. 18 t o g i v e n e t - r e t e n t i o n volumes.

The surface specific retention volune was t h e n

calcu-

l a t e d as

P o i n t s on t h e reduced excess a d s o r p t i o n isotherm,

Y A / ~ N A, c o u l d be c a l c u l a t e d

w i t h t h e a i d o f t h e r e l a t i o n s h i p s summarized i n eqns. 19 from t h e s p e c i f i c r e t e n t i o n volumes o f A* and B* ( n o t e t h a t Y B / ~ N A=

- Y!A/~NA) p l o t t e d i n Fig. 3 as a f u n c t i o n o f e l u e n t c o m p o s i t i o n . The i s o t h e r m i s o f t h e S-type. S i m i l a r isotherms were observed by Schay and Nagy a t n o n - p o l a r

measurements ( r e f .

interfaces i n static

10). The r e g r e s s i o n f u n c t i o n o f t h e a d s o r p t i o n isotherm,

shown i n F i g . 3 p e r m i t s us i n t u r n t o p r e d i c t t h e s p e c i f i c r e t e n t i o n volumes o f A* and B* and t h a t o f t h e c o n c e n t r a t i o n peak. The t r a c e o f t h e s e f u n c t i o n s i s shown i n F i g . 2. The agreement i s e x c e l l e n t .

211

F i g . 2. Surface s p e c i f i c r e t e n t i o n volume o f d e u t e r a t e d a c e t o n i t r i l e and HDO, and that o f t h e c o n c e n t r a t i o n peak in a c e t o n i t r i l e / H 2 0 as a f u n c t i o n o f t h e c o m p o s i t i o n o f t h e e l u e n t ( r e f . 9 ) . The volume f r a c t i o n , 0, was c a l c u l a t e d w i t h partial molar volumes. Temperature: 2 P C ; s t a t i o n a r y phase: L i c h r o s o r b - S I 1 0 0 covered with tetradecyldimethylsiloxy substituents. Curves c a l c u l a t e d w i t h eqns. 15 and 1 7 f r o m t h e a d s o r p t i o n i s o t h e r m shown i n F i g . 3.

0 Y

.o

-.2

'H20 H,O/vNA

F i g . 3. A sor t i o n isotherms, \y ~~0 V~~ , from ace ,n i r i l e / H 2 0 m i x t u r e s a( t h e of s u r f ace t e t r adecyl d i m e t h y l s i 1o x y covered s i l i c o n d i o x i d e . F u l l symbols and c u r v e f o r Tc = 20.0 O C ; open symbols and Points dashed l i n e f o r Tc = 4O.O0C. c a l c u l a t e d w i t h eqn. 19 ( r e f . 9).

-.4

-.6 0

A f i r s t c o n c l u s i o n from t h i s d i s c u s s i o n i s t h a t t h e hold-up volume i n l i q u i d / s o l i d chromatography has t o b e c o n s i d e r e d as a correctilrg volune f o r t h e c a l c u l a t i o n o f t h e n e t - r e t e n t i o n volume f o l l o w i n g eqn. 7, and as such i t cannot be i d e n t i f i e d with any physical volune inside the colunn. It was s t a t e d t h a t t h e hold-up volume, c a l c u l a t e d w i t h t h e vNA c o n v e n t i o n does correspond t o

212

t h e volume o f t h e m o b i l e phase i n t h e column. S t r i c t l y speaking,

t h i s i s not

t r u e . F i r s t l y , experiments o f Ash and Findenegg show t h a t t h e d e n s i t y o f a p u r e l i q u i d near an i n t e r f a c e i s d i f f e r e n t from t h a t i n t h e b u l k ( r e f . 11). Obviousl y , t h e l i q u i d has a d i f f e r e n t s t r u c t u r e i n t h e neighbourhood o f a s o l i d as

i l l u s t r a t e d i n F i g . 4.

Secondly,

i n a b i n a r y m i x t u r e t h e p a r t i a l molar volume

1i q u i d

solid

a

b

Fig. 4. I l l u s t r a t i o n o f t h e m o l e c u l a r o r d e r i n p u r e l i q u i d s near a l i q u i d / s o l i d i n t e r f a c e : a: t h e s t r u c t u r e o f t h e l i q u i d near t h e i n t e r f a c e i s t h e same as i n t h e b u l k ; b: t h e l i q u i d i s ordered near t h e i n t e r f a c e , t h e r e f o r e i t has a h i g h e r d e n s i t y ( c f . r e f . 11).

o f an adsorbed component i s c e r t a i n l y d i f f e r e n t from t h a t i n t h e b u l k , because t h e p a r t i a l molar volume i s a f u n c t i o n o f c o m p o s i t i o n and t h e c o m p o s i t i o n near t h e i n t e r f a c e i s d i f f e r e n t from t h a t i n t h e b u l k . These a r e m i n o r e f f e c t s i n b i n a r y systems used as e l u e n t s i n l i q u i d / s o l i d chromatography w i t h a non-polar solid,

and a l t h o u g h w a t e r -

ideality,

organic modifier mixtures deviate s e r i o u s l y from

t h i s volume can be equated w i t h a v e r y good a p p r o x i m a t i o n t o t h e

t o t a l volume o f t h e m o b i l e phase i n t h e column.

V,,/NA

,

I n experiments t h e volume,

i s almost independent o f t h e n a t u r e o f t h e m o b i l e phase ( r e f . 9).

o t h e r words,

In

b y t h e c o n v e n t i o n vNA t h e Gibbs d i v i d i n g p l a n e i s s i t u a t e d v e r y

near t o t h e r e a l , p h y s i c a l d i v i d i n g p l a n e between t h e s o l i d and t h e l i q u i d . A second c o n c l u s i o n i s t h a t a stationary phase cannot be i d e n t i f i e d

. Nume-

r o u s a t t e m p t s have been made t o c r e a t e an i m a g i n a r y s t a t i o n a r y phase b y propos i n g monolayer o r b i l a y e r a d s o r p t i o n models (see F i g . 5 ) .

In r e a l systems t h e

s i t u a t i o n i s more c o m p l i c a t e d as shown by Somorjai i n t h e s t u d y o f " f r o z e n " ads o r p t i o n e q u i l i b r i a i n l i q u i d metal m i x t u r e s ( r e f .

12). Even i f , i n c e r t a i n

systems, a monolayer o r b i l a y e r a d s o r p t i o n may be a v e r y good a p p r o x i m a t i o n f o r

I

0

1

1, -

a

1

213

-

0

1

FA

....

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

....... ................... ................... ................... .................. ................... ................... ................... 9

solid

a

C

F i g . 5. C o n c e n t r a t i o n p r o f i l e s near a l i q u i d / s o l i d i n t e r f a c e : a: monomolecular a d s o r p t i o n ; b: b i m o l e c u l a r a d s o r p t i o n ; c : a more complex s i t u a t i o n ( c f . r e f . 12). the real situation,

t h e whole adsorbed m a t e r i a l

brium w i t h t h e b u l k : t h e where

one of

i s always i n dynamic e q u i l i -

surface phase i s not autonomous. ( A c t u a l l y , t h e case

t h e components

is really

immobilized

a t t h e surface

i s not

i n t e r e s t i n g from t h e v i e w p o i n t o f t h e chromatographic process. T h i s case has t o be considered as chromatography a t t h i s newly formed l i q u i d / s o l i d i n t e r f a c e ) . A t h i r d c o n c l u s i o n i s t h a t t h e net r e t e n t i o n volune o f a solute can be negat i v e r e g a r d l e s s o f which c o n v e n t i o n i s used f o r t h e d e t e r m i n a t i o n o f t h e h o l d up volume. The s o l u t e can have a n e g a t i v e reduced a d s o r p t i o n i f i t i s l e s s adsorbed then e i t h e r o f t h e components o f t h e e l u e n t s ( c f . eqn. 17). The l a s t c o n c l u s i o n concerns t h e e n e r g e t i c s o f t h e r e t e n t i o n .

In l i q u i d /

s o l i d chromatography t h e r e t e n t i o n volume o f a s o l u t e i s d i r e c t l y r e l a t e d t o t h e specific Helmholtz f r e e energy o f t h e i n t e r f a c e ( r e f . 1ink

13). The necessary

s g i v e n b y Gibbs' a d s o r p t i o n e q u a t i o n ( c o n s t a n t t e m p e r a t u r e ) :

dy =

where y i s t h e s p e c i f i c f r e e energy o f t h e i n t e r f a c e ( i n t e r f a c i a l t e n s i o n ) , psu

i s t h e chemical p o t e n t i a l o f t h e s o l u t e and rs,/ANA

(IUPAC

t h e r e l a t i v e excess surface concentration o f t h e s o l u t e ,

symbol:

n$,A)/s)

r e l a t i v e t o A,

A is Not Adsorbed a t t h e i n t e r f a c e (ANA). The t h e c o n v e n t i o n t h a t component c o r r e s p o n d i n g hold-up volume i s g i v e n b y

is with

214

as,

b y convention,

component

A i s not

adsorbed.

The c o r r e s p o n d i n g s u r f a c e

s p e c i f i c r e t e n t i o n volume i s g i v e n b y ( c f . eqn. 13):

Combination o f eqns.

20 and 22 gives,

after

t h e necessary t r a n s f o r m a t i o n s

( r e f . 13):

F o l l o w i n g eqn. 23 t h e r e t e n t i o n volume o f a s o l u t e i s d i r e c t l y r e l a t e d t o t h e

decrease o f the specific f r e e energy o f the i n t e r f a c e

,

a d s o r p t i o n and not t o the chemical potential o f the solute

y,

caused b y s o l u t e

. Therefore,

eqn. 23

i s o f l i t t l e use i n t h e understanding and p r e d i c t i o n o f chromatographic d a t a .

CONCLUSIONS L e t us r e t u r n now t o t h e d i s c u s s i o n o f t h e current description o f renten-

t i o n i n 1 i q u i d / s o l i d chromatography. The most p o p u l a r method o f p r e s e n t a t i o n o f such d a t a i s t o c a l c u l a t e t h e k'-value o f a s o l u t e d e f i n e d b y

Furthermore, i t i s b e l i e v e d t h a t eqn. 23 i s a p p l i c a b l e RTcln k,;

=

C

-

~g~~ t

25 1

where t h e c o n s t a n t , C, i s a r b i t r a r y because (as i t i s s t a t e d ) t h e volume o f t h e s t a t i o n a r y phase i s n o t d e f i n e d .

On t h e b a s i s o f t h e f o r e g o i n g d i s c u s s i o n , eqn. 24 i s meaningless ( o r h s a t r i v i a l meaning) and eqn. 25 i s wrong. Even b y a d m i t t i n g , f o r t h e sake o f d i s cussion, t h a t t h e k ' - v a l u e i n s i m i l a r columns,

i s p r a c t i c a l f o r t h e comparison o f d a t a determined

i t i s seen t h a t t h i s c h a r a c t e r i s t i c v a l u e measures t h e

r e t e n t i o n i n t h e u n i t s o f t h e hold-up volume. F i r s t l y , t h e hold-up volume i s small,

i t can be determined w i t h a p r e c i s i o n o f o n l y f 3%. Secondly,

current

215 apinions h i g h l y d i f f e r

as t o t h e method o f

i t s determination

meaning. L e t us s h o r t l y r e v i e w t h e p r o p o s a l s ( r e f s .

and about

its

14 and 1 5 ) .

I n j e c t one o f t h e marked components (A*) o f t h e e l u e n t and accept V,

= VR,A*

( r e f . 1 6 ) . This h o l d - u p volume i s e q u i v a l e n t t o t h a t d e r i v e d w i t h t h e A i s n o t adsorbed c o n v e n t i o n (ANA; see eqn. 21) I n j e c t b o t h marked components and c a l c u l a t e V,

w i t h eqn. 8

(see discussion

t h e r e ) ( r e f . 8). I n j e c t b o t h marked components and always accept t h e s m a l l e r o f t h e r e t e n t i o n volumes, VR,A* lieved that

VR,B*

or

,

as t h e h o l d - u p volume.

r e t e n t i o n volumes

smaller

than

V,

I n t h i s p r o p o s a l i t i s beare n o t possible

(as

in

l i q u i d / l i q u i d and g a s / l i q u i d chromatography) ( r e f . 17). The column i s f i l l e d w i t h two p u r e l i q u i d s s u c c e s s i v e l y and weighed ( r e f . 1 8 ) . The h o l d - u p volume i s c a l c u l a t e d as

where Aw i s t h e weight d i f f e r e n c e o f t h e column f i l l e d w i t h t h e two l i q u i d s (Aw = wc,1-wc,2) (Ad = d l d2).

-

and Ad i s t h e d i f f e r e n c e ' o f t h e d e n s i t y o f t h e two l i q u i d s T h i s experiment i s e s s e n t i a l l y t h e same as t h a t o f Ash and

Findenegg ( r e f . 11) b u t i t i s supposed t h a t t h e r e a r e no d e n s i t y changes i n the

liquids

near

the

interface.

If

hold-up volume i s about t h e same as V,N,/ A

this

is

approximately

true,

this

(see eqn. 1 8 ) .

The l o g a r i t h m of t h e r e t e n t i o n volumes o f a homologous s e r i e s a r e " l i n e a r i zed" as t h e f u n c t i o n o f

z, t h e carbon number, t o g i v e

Vp

(ref.19).

This

procedure i s d e r i v e d by f o r m a l analogy w i t h gas chromatography. Note t h a t i n l i q u i d / s o l i d chromatography n e t r e t e n t i o n can be n e g a t i v e

and t h a t

the

l o g a r i t h m o f a n e g a t i v e number has no sense. I n j e c t a "chosen s o l u t e " , y, b e l i e v e d t o be n o n - r e t a i n e d and accept V R , ~ as hold-up volume ( r e f . 20). From t h e v i e w p o i n t o f Gibbs' d e s c r i p t i o n o f adsorption,

t h i s proposal

i n t r o d u c e s t h e c o n v e n t i o n yNA,

i.e.

y

i s not

adsorbed a t any e l u e n t c o m p o s i t i o n . Hold-up volumes determined f o l l o w i n g these p r o p o s a l s d i f f e r b y a t l e a s t as much as f 20%. Consequently,

r e t e n t i o n d a t a g i v e n i n u n i t s o f t h e hold-up volumes

w i l l v a r y a t l e a s t b y t h e same o r d e r o f magnitude. As a c o n c l u s i o n , t h e h o l d - u p volume i s n o t a s u i t a b l e u n i t f o r r e c o r d i n g r e t e n t i o n d a t a even i f i t s method o f d e t e r m i n a t i o n c o u l d be agreed upon.

If hold-up

volume i s used as a correction i n s t e a d o f a u n i t

i n order t o

c a l c u l a t e n e t - r e t e n t i o n volumes, t h e e r r o r o f t h e c o r r e c t i o n appears as a s m a l l r e l a t i v e e r r o r i n t h e d e t e r m i n a t i o n o f h i g h e r r e t e n t i o n volumes.

In t h e l i g h t

216

o f t h i s proposal,

t h e a b s o l u t e v a l u e o f t h e hold-up volume i s of

l e s s impor-

tance. From t h e n e t - r e t e n t i o n volumes s u r f a c e s p e c i f i c r e t e n t i o n volumes can be c a l c u l a t e d . With c a r e f u l work a p r e c i s i o n o f f 2% i s easy t o a t t a i n i f a r e l i a b l e method i s a v a i l a b l e f o r t h e d e t e r m i n a t i o n o f t h e s u r f a c e area o f t h e adsorbent i n t h e column.

Recent r e s u l t s suggest,

t h a t t h e s u r f a c e area o f

the

c h e m i c a l l y bonded l a y e r i s n e a r l y t h e same as t h a t o f t h e u n d e r l y i n g s i l i c o n d i o x i d e ( r e f s . 21 and 22). (See analogy i n t h e p r o p o s a l o f M a r t i n f o r t h e e s t i m a t i o n o f t h e s u r f a c e area o f a duplex f i l m on an i n e r t s u p p o r t ( r e f . 23)). In conclusion,

i t i s proposed t o r e c o r d r e t e n t i o n i n l i q u i d / s o l i d chromato-

graphy as s u r f a c e s p e c i f i c r e t e n t i o n volume. A c t u a l l y , t h e analogous v a l u e , t h e weight s p e c i f i c r e t e n t i o n volume,

played an i m p o r t a n t r o l e i n t h e development

o f t h e o r e t i c a l aspects o f r e t e n t i o n i n gas chromatography.

vs In the calculation,

=

3S

[kl

rn-23

s u r f a c e area can be t a k e n as t h e s u r f a c e

area o f

the

s i l i c o n d i o x i d e i n t h e column. It i s recommended t o use V P / , ~ ~ f o r t h e hold-up volume determined e i t h e r b y t h e weighing method o r b y t h e measurement g i v e n i n eqn. 18.

ACKNOWLEDGEMENTS T h i s paper r e p o r t s on t h e p r o j e c t supported b y t h e Fonds National Suisse de l a Recherche E i e n t i f i q u e Eng 1 is h

.

. We

thank Mrs. L i s a B e l v i t o f o r c o r r e c t i o n o f t h e

REFERENCES

1 D. de Vault, J. h e r . Chem. SOC., 62 (1940) 1583. 2 F. H e l f f e r i c h and G. K l e i n , i n J.C. Giddings and R.A. 3 4 5

6 7 8 9 10

Keller (Editors), "Mu1 ticomponment Chromatography - Theory o f I n t e r f e r e n c e " , Marcel Dekker , New York, 1970. F. Riedo and E.sz. Kovdts, J. Chromatogr., 239 (1982) 1. P. V a l e n t i n and G. Guiochon, J. Chromatogr. Sci., 14 (1976) 56 and 132; see a l s o J.F.K. Huber and R.G. G e r r i t s e , J. Chromatogr., 58 (1971) 137. A.J.P. M a r t i n and R.L.M. Synge, Biochem. J., 35 (1941) 1358. See e.g. K. Hostettman, i n J.C., Giddings, E. Grushka, J. Cazes and Ph.R. Brown ( E d i t o r s ) , "Advances i n Chromatography", Marcel Dekker Vol 21 (1982). A.T. James and A.J.P. M a r t i n , Biochem. J., 50 (1952) 679. J.H. Knox and E.sz. Kovdts, d i s c u s s i o n c o n t r i b u t i o n , Faraday Symposium No. 15, B r i g h t o n , 1980, Royal S o c i e t y of Chemistry, London, 1980, p. 171. N.L. Ha, J. Ungvarai and E.sz. Kovdts, Anal. Chem.,54 (1982) 2410. See e.g. G. Schay and L. Gy. Nagy, " A d s o r p t i o n a t L i q u i d / S o l i d and L i q u i d / Gas I n t e r f a c e s " ( i n Hungarian), i n " A k6mia djabb eredm6nyei"; 6. Csdkvdry ( E d i t o r ) , A d a d h i a i Kiadb, Budapest, 1974, Vol. 18 p.7.

217 11. S.G. Ash and G.H. Findenegg, Spec. D i s c u s s i o n Faraday SOC., 1 (1970) 105; see a l s o L . L o r i n g and G.H. Findenegg, (1. C o l l o i d I n t e r f a c e S c i . , 84 (1981) 355. 12. G.A. Somorjai, " Chemistry i n Two Dimensions: Surfaces", C o r n e l l Univ. Press It h a c a and London. 1981 , p. 100-1 75. 13. F. Riedo and E.sz. Kovats, J . Chromatogr., 186 (1979) 47. 14. See e.9. G.E. Berendsen, P.J. Shoemakers, L. de Galan, G. Vigh, Z. Varga-Puhony and J. Inczedy, J . L i q u i d Chromatogr., 3 (1980) 1669. 15. A.M. K r s t u l o v i c , H. C o l i n and G. Guiochon, Anal. Chem., 54 (1987) 2482. 16. R.M. McCormick and B.L. Karqer, $1. Chromatogr., 199 (1980) 259; see a l s o f r o m t h e same a u t h o r s , Anal. Chem., 57 (1980) 2249. 17. W.R. Velander, J.-F. E r a r d and Cs. Horvath, J . Chromatoqr., 282 (1983) 211. 18. Cs. H o r v a t h and H.-J. L i n , J . Chromatoqr., 118 (1975) 401; see a l s o E.H. S l a a t s , J . C . Kraak, W.J.T. Brugman and H. Poppe, 2 . Chromatogr., 149 (1978) 255. 19. See e.g. J.K. Haken, M.S. Wainwriqht and R.J. Smith, J . Chromatogr., 133 (1977) 1 . 20. See e.g. M.J.M. W e l l s and C . R . C l a r k , A n a l . Chem., 53 (1981) 1341; B.L. Karger, J.R. Gant, A . H a r t k o o f and P.H. Weiner, J. Chromatogr., 128 (1976) 65. 21. J . Gobet and E.sz. Kovats, A d s o r p t i o n S c i . Technol., 1 (1984) 111. 22. J . Gobet and E.sz. Kovats, A d s o r p t i o n S c i . Technol., i n p r e s s . 23. R.L. M a r t i n , Anal. Chem., 35 (1963) 116.

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219

CHROMATOGRAPHY FOR THE EVALUATION OF THE ATMOSPHERIC ENVIRONMENT

A. L i b e r t i and P. C i c c i o l i I s t i t u t o Inquinamento A t m o s f e r i c o d e l C.N.R.Via S a l a r i a Km 29,300

1.1

10

-C.P.

-

Area d e l l a R i c e r c a d i Roma

Monterotondo S t a z i o n e (Roma) ITALY

INTRODUCTION I t was customary u n t i l few y e a r s ago t o r e f e r t o t h e atmospheric environment

as t h e dark c o n t i n e n t i n o r d e r t o o u t l i n e t h e u n c e r t a i n t y r e l a t e d t o t h e e v a l u a t i o n o f t h e v a r i o u s s p e c i e s which m i g h t be p r e s e n t i n t h e a i r as a consequence of n a t u r a l as w e l l a n t h r o p o g e n i c a c t i v i t y .

The wide a p p l i c a t i o n o f chromatogra-

phy and, s p e c i f i c a l l y gas chromatography has c l a r i f i e d a v a r i e t y o f atmospheric systems and w i t h t h e i n t r o d u c t i o n o f gas chromatographic-mass s p e c t r o m e t r i c (GC-MS) techniques i t became p o s s i b l e t o i d e n t i f y a number o f compounds p r e s e n t i n t r a c e s i n t h e atmosphere, t o b e g i n t o i n v e s t i g a t e t h e i r r e a c t i o n p r o d u c t s and t o u n v e i l t h e mechanism o f s e v e r a l r e a c t i o n s o c c u r r i n g i n t h e t r o p o s p h e r e . I t i s w e l l e s t a b l i s h e d t h a t even s o - c a l l e d u n r e a c t i v e compounds may e x e r t a d e f i n i t e chemical a c t i o n and t a k e p a r t i n complex r e a c t i o n s i n t h e atmosphere. U n t i l 30 y e a r s ago most chemical s p e c i e s i m m i t t e d i n t o t h e environment were n o t c o n s i d e r e d bothersome u n l e s s t h e y had o b j e c t i o n a b l e p r o p e r t i e s ; however today t h e r e i s g r e a t awareness o f t h e r o l e t h a t most species may p l a y and h i g h l y spec i a l i z e d t e c h n i q u e s a r e needed. I n o r d e r t o c l a s s i f y a i r i n terms o f v a r i e t y o f species which m i g h t be i m m i t t e d o r formed as a consequence o f t h e v a r i o d s e m i t t e r sources and r e a c t i o n s which can occur, t h e f o l l o w i n g i n d i c e s have been i n t r o d u c e d by e n v i r o n m e n t a l i s t s evaluate

to

a i r quality: s u l f u r oxides

SOX

nitrogen oxides

NOX

HC

hydrocarbons

co

carbon monoxide

03-peroxides

ozone and p e r o x i d e s (photochemical o x i d a n t s )

P a r t i c u l a t e Matter U s u a l l y r e f e r e n c e i s made t o t h e s e terms

-

which w i l l be examined and discus-

sed - t o show t h e achievements r e a l i z e d by chromatography.

After looking a t

these i n d i c e s two o b s e r v a t i o n s must be made; t h e f i r s t one i s t h a t w i t h t h e excep t i o n o f carbon monoxide, each i n d e x i s r e p r e s e n t e d by more t h a n one s p e c i e s and, specifically organic not

,

t h e terms

species w i t h

molecularly

HC and p a r t i c u l a t e m a t t e r r e s p e c t i v e l y mean

an a p p r e c i a b l e

dispersed i n the

vapour t e n s i o n and

a i r , which m i g h t be

any chemical

any species

p r e s e n t as an a e r o s o l .

220

F u r t h e r , i t must be s t r e s s e d t h a t most s p e c i e s p r e s e n t i n t h e a i r e i t h e r i n gas o r i n a condensed phase a r e always a t t h e t r a c e l e v e l i n t h e range o f ppm

-

p p t c o n c e n t r a t i o n so t h a t an enrichment process i s o f t e n r e q u i r e d and c l e a n - u p procedures have t o be adopted t o o b t a i n meaningful r e s u l t s f r o m c o l l e c t e d samples. T h i s r e v i e w w i l l deal w i t h t h e development o f chromatographic m e t h o d s a p p l i e d t o t h e e v a l u a t i o n o f t h e i n d i c e s above r e p o r t e d and o f species which m i g h t be r e l a t e d t o them; sampling w i l l be discussed f o r each s e c t i o n . T h i s i s o f fundamental importance, p a r t i c u l a r l y when an enrichment process i s r e q u i r e d t o o b t a i n a detectable quantity. 2.1

SULFUR OXIDES AND SULFUR COMPOUNDS S u l f u r d i o x i d e i s t h e main atmospheric p o l l u t a n t and i t s v a l u e i s u s u a l l y

taken as a general i n d i c a t o r o f t h e a i r q u a l i t y o f a c e r t a i n area. Anthropogenic emissions o f SO2 come f r o m combustion o f f o s s i l f u e l s ( o i l and carbon), which g e n e r a l l y c o n t a i n a p p r e c i a b l e q u a n t i t i e s o f i n o r g a n i c s u l f i d e s and s u l f u r - c o n t a i n i n g o r g a n i c compounds. A s u b s t a n t i a l amount o f SO2 i s a l s o n a t u r a l l y e m i t t e d d u r i n g v o l c a n i c e r u p t i o n s . The d e l e t e r i o u s e f f e c t o f atmospheric SO2,

such as

damage t o v e g e t a t i o n , d e t e r i o r a t i o n o f t e x t i l e s and c o r r o s i o n o f metal s,are we1 1 documented ( 1 ) . The o t h e r s u l f u r o x i d e

i m n i t t e d i n t h e atmosphere i s S O 3 . I t i s

p r e s e n t i n sniall amounts i n some i n d u s t r i a l emissions and i s formed i n

t h e atmosphe-

r e by c o n v e r s i o n o f SO2 i n t h e presence o f o x i d a n t s o r p a r t i c u l a t e m a t t e r ( 2 ) . Since water vapour c o n v e r t s r a p i d l y SO3 i n t o s u l f u r i c a c i d , t h e a n a l y s i s o f f r e e SO3 i n t h e atmosphere i s n o t m a t t e r o f s p e c i f i c concern.

S u l f u r i c a c i d e m i t t e d f r o m i n d u s t r i a l processes o r formed by o x i d a t i o n o f SO2 i s an i m p o r t a n t p o l l u t a n t as i t i s t o x i c t o man and p l a n t s . Other s u l f u r gases p r e s e n t i n t h e atmosphere

a r e hydrogen s u l f i d e , c a r b o n y l

s u l f i d e , carbon d i s u l f i d e and d i m e t h y l s u l f i d e . Hydrogen s u l f i d e i s n a t u r a l l y e m i t t e d f r o m geothermal sources o r anaerobic d e g r a d a t i o n o f s u l f a t e s , s u l f i d e s and s u l f u r - c o n t a i n i n g o r g a n i c compounds. The source o f carbonyl s u l f i d e i s n o t y e t f u l l y understood, whereas carbon d i s u l f i d e , which i s n a t u r a l l y produced by some p l a n t s , i s a l s o e m i t t e d f r o m manychemical and i n d u s t r i a l processes. Large amount of d i m e t h y l s u l f i d e a r e produced by p l a n c t o n i c organisms.

A common f e a t u r e o f most s u l f u r gases i s t h e i r h i g h o l f a c t o r y l e v e l which can produce an o f f e n s i v e odor even 2.2

a t ppbv l e v e l s .

Chromatographic Methods f o r S u l f u r Oxides and reduced s u l f u r compounds Although chromatography i s n o t t h e o n l y a n a l y t i c a l t e c h n i q u e a v a i l a b l e f o r

t h e d e t e r m i n a t i o n o f SO2 and s u l f u r gases i n a i r , i t s use i s o f t e n p r e f e r r e d as i t y i e l d s a c c u r a t e r e s u l t s and i n some cases a comprehensive p i c t u r e o f these

compounds i n an a i r sample.

221 I t i s well-known t h a t a flame p h o t o m e t r i c d e t e c t o r (FPD) s p e c i f i c a l l y measu-

r e s t h e chemiluminescent e m i s s i o n energy o f s u l f u r c o n t a i n i n g molecules, which a r e p a r t l y c o n v e r t e d t o a c t i v a t e d S*2 s p e c i e s . The e m i s s i o n i n t e n s i t y i s p r o p o r t i o n a l t o t h e square o f t h e f l a m e s u l f u r atom c o n c e n t r a t i o n . Most commercial apparatus use FPD w i t h a d i r e c t i n j e c t i o n system t o measure SO2 b u t d i f f i c u l t y i s experimented i n t h e GC a n a l y s i s o f SO2 and o t h e r s u l f u r gases as t h e i r h i g h r e a c t i v i t y can cause a d s o r p t i o n l o s s e s o f t h e sample and an i r r e p r o d u c i b l e t r a n s f e r t o t h e GC system. Stevens e t a l . ( 3 ) have overcome many l i m i t a t i o n s by d e v e l o p i n g an a c c u r a t e sampling procedure: a known volume o f a i r , sampled i n t o a l o o p by a membrane pump, i s i n j e c t e d by means o f a PTFE c o a t e d v a l v e and separ a t e d on a 36 f t column packed w i t h Chromosorb

T

( o r PTFE), coated w i t h 12% PPE

and 0.5% H3P04. To p r e v e n t a d s o r p t i o n l o s s e s , a l l gas l i n e s and columns were made b y PTFE. B e t t e r l i n e a r i t y and s h o r t e r a n a l y s i s t i m e have been r e p o r t e d by B r u n e r e t a l . (4, 5, 6 ) on 1.4 m columns packed w i t h hydrogen t r e a t e d Carbopack

B coated

w i t h 0.5% H3P04 and 0.3% D e x i l o r , a l t e r n a t i v e l y , w i t h 1.0% H3P04 and 1.5% XE60. Using a f l o w r a t e o f ca. 120 mL/min H2S, SO2 and methylmercaptan were c o m p l e t e l y separated and e l u t e d i n l e s s than 1 m i n u t e on b o t h columns. The minimum amounts o f H2S, SO2 and methyl mercaptan which can be d e t e c t e d by t h i s method a r e i n t h e range o f 5-10 ppbv. L i n e a r p l o t s o f t h e l o g a r i t h m o f peak h e i g h t v s . l o g a r i t h m

o f c o n c e n t r a t i o n were o b t a i n e d w i t h t h e FPD by u s i n g a dual v a l v e i n j e c t i o n system which p e r m i t s an e f f e c t i v e l o o p p u r g i n g between two analyses. The column coated w i t h X E 60 was a l s o used f o r t h e s e p a r a t i o n o f SF6, COSY methylmercaptan, e t h y l mercaptan, d i m e t h y l s u l f i d e and carbon d i s u l f i d e i n l e s s t h a n 10 m i n u t e s . An example o f t h e chromatographic a n a l y s i s o f s u l f u r compounds i n a i r o b t a i n e d by t h i s method i s shown i n F i g u r e l a and b.

b)

s 0, (20PPb)

-

t(min) 0

1

a

I 0

I

2

I

I

1

4

6

8

t (mid

1

10

Figure 1 . Separation of s u l f u r gases on Carbopack B columns ( f o r t h e type of liquid coatings see the t e x t ) ; a ) monitoring of s u l f u r compounds in a i r b ) separation of H2S ( 1 ) , s F 6 ( 2 ) , c o s (3),S02 ( a ) , C H ~ S H( 5 ) , C ~ H ~ (S6 H ), CH3SCH3 ( 7 ) and CS2 ( 8 ) .

222

More r e c e n t l y o t h e r s t a t i o n a r y phases such as Porapak QS (7), 25% TCEP on S h i m a l i t e (8) and d e a c t i v a t e d s i l i c a (9) have been used f o r t h e s e p a r a t i o n o f s u l f u r gases. The p o s s i b i l i t i e s a f f o r t e d

by v a r i o u s column packings have been surveyed

by Thompson and S t a b i s l a v l j e v i c ( l o ) , who a l s o i n v e s t i g a t e d and e v a l u a t e d l i m i t s o f GC-MS systems. While d e t e c t i o n l i m i t s o f t h e o r d e r o f 10 ppbv a r e s u f f i c i e n t f o r t h e d e t e r m i n a t i o n o f SO2 i n many atmospheric samples, background l e v e l s o f o t h e r s u l f u r gases can be much l o w e r and t h u s , more s e n s i t i v e methods a r e r e q u i r e d f o r t h i s a n a l y s i s . The use o f Helium i o n i z a t i o n ( l l ) , e l e c t r o n c a p t u r e ( 1 2 ) and c h e m i l u minescent ( 1 3 ) d e t e c t i o n have been proposed t o improve t h e s e n s i t i v i t y of t h e GC system. Helium i o n i z a t i o n d e t e c t i o n , w o r k i n g i n t h e s a t u r a t i o n r e g i o n , was

found s u i t a b l e o n l y f o r SO2 w i t h a d e t e c t i o n l i m i t o f t h e same o r d e r of magnitude o f t h a t observed w i t h t h e FPD. E l e c t r o n c a p t u r e d e t e c t i o n (ECD) was found t o be e x t r e m e l y s e n s i t i v e (10-30 p p t v l e v e l s ) b u t i t s u s e i s l i m i t e d t o t h e d e t e r m i n a t i o n o f COS and CS2. Chemiluminescent d e t e c t i o n induced by ozone was found s p e c i f i c f o r reduced s u l f u r compounds ( e s p e c i a l l y H2S and d i m e t h y l s u l f i d e ) b u t t h e s e n s i t i v i t y was n o t s u f f i c i e n t f o r GC purposes.

A more s u c c e s s f u l approach i s t h e p r e c o n c e n t r a t i o n o f t h e sample by c r y o g e n i c f r e e z e t r a p p i n g o r s e l e c t i v e a d s o r p t i o n p r i o r t o t h e a n a l y s i s . The use o f gold-coated g l a s s beads f o r t h e s e l e c t i v e enrichment o f hydrogen s u l f i d e , dimet h y 1 s u l f i d e and o t h e r o r g a n o s u l f u r compounds was d e s c r i b e d by Braman e t a l . ( 1 4 ) . A f t e r a d s o r p t i o n , t h e c o n c e n t r a t e was desorbed a t 500-600°C,

cryofocused

on an empty t u b e k e p t a t t h e temperature o f l i q u i d n i t r o g e n and t h a n analyzed by a GC, equipped w i t h a FPD. D e t e c t i o n l i m i t s i n t h e p p t v ranges were r e p o r t e d f o r H2S and d i m e t h y l s u l f i d e . The method proposed by Concawe’s S p e c i a l Task F o r c e on Odours ( 1 5 ) c o n s i s t s i n t h e p r e c o n c e n t r a t i o n o f 100 m l o f a i r i n t o a P o r a s i l D t r a p c o o l e d a t -70°C w i t h a d r y ice-acetone m i x t u r e . The e n r i c h e d sample i s i n j e c t e d i n t o t h e column by h e a t i n g t h e t r a p t o 40°C. H2S, SO2, CH3SH, CS2 and d i m e t h y l s u l f i d e can be separated on a 1 metre column packed w i t h P o r a s i l 0, coated w i t h 5% PPE and 0.2% H3P04. D e t e c t i o n l i m i t s o f 1-2 ppbv can be o b t a i n e d w i t h t h i s method.

A c r y o g e n i c f r e e z e - o u t t r a p p i n g t e c h n i q u e f o r t h e sampling o f s u l f u r compounds f r o m H2S t o d i m e t h y l d i s u l f i d e has been d e s c r i b e d by F a r w e l l e t a l . ( 1 6 ) . The sample was analyzed on a 30 m c a p i l l a r y column c o a t e d w i t h OV 101 o r SE 54 by programming t h e oven temperature from -70 t o 100°C. Sub-ppbv l e v e l s o f s u l f u r gases were d e t e c t e d i n a i r samples. The t e c h n i q u e was, however, u n s u i t a b l e f o r t h e d e t e r m i n a t i o n of SO2, because o f t h e u n s a t i f a c t o r y e l u t i o n o f t h i s compound f r o m t h e column. More r e c e n t l y a new t e c h n i q u e f o r sampling and f o r e n r i c h i n g components i n t r a c e s has been developed which i n v o l v e s use o f denuders o r d i f f u s i o n t u b e s t r i p -

223

pers (17).

A denuder i s a t u b u l a r d e v i c e i n which a i r i s made t o f l o w under

l a m i n a r c o n d i t i o n s . The tube w a l l s a r e coated w i t h a r e a c t i v e l a y e r f o r t h e spec i e s which must be m o n i t o r e d so t h a t t h e molecules o f t h e t e s t e d g a s can d i f f u se t o t h e t u b e w a l l s where a r e i r r e v e r s i b l y adsorbed. The p a r t i c l e s l a r g e r t h a n 0.01 ,urn proceed u n a f f e c t e d because t h e i r d i f f u s i o n c o e f f i c i e n t s a r e s e v e r a l o r d e r s o f magnitude l o w e r t h a n t h o s e o f gaseous s p e c i e s . I n a d d i t i o n t o an e f f e c t i ve g a s - p a r t i c l e s e p a r a t i o n , a denuder o f f e r s t h e p o s s i b i l i t y t o sample s e l e c t i v e l y r e a c t i v e gases by d e t e r m i n i n g d i r e c t l y t h e sorbed species. F l o w l i m i t a t i o n s which r e q u i r e a l o n g sampling t i m e have been overcome by t h e use o f r e c e n t l y developed a n n u l a r denuders. They c o n s i s t s o f two c o a x i a l tubes, where t h e a i r t o be sampled f l o w s l a m i n a r l y t h r o u g h t h e a n n u l a r space. On t h e s u r f a c e o f t h e t u bes a r e coated l a y e r s o f s o l u t i o n s , which may a c t as i r r e v e r s i b l e s i n k s f o r t h e species which have t o be m o n i t o r e d so t h a t a s p e c i f i c f r a c t i o n a t i o n o f gases w i t h a d i f f e r e n t r e a c t i v i t y occurs ( 1 8 ) . The denuders a r e v e r y e f f e c t i v e f o r t h e sampling o f atmospheric s u l f u r compounds. SO2 can be sampled s p e c i f i c a l l y , w i t h o u t i n t e r f e r e n c e s and a r t i f a c t s , due t o t h e presence o f o t h e r gaseous components and p a r t i c u l a t e d m a t t e r . F i g u r e

2 shows a t r i p l e - s t a g e denuder f o r sampling o f S O p , NH3, HNOQ and HC1 as w e l l as p a r t i c u l a t e d m a t t e r f r e e f r o m mutual i n t e r f e r e n c e s . tiN03 and HC1 a r e adsorbed on ammonia i s t r a p p e d

t h e w a l l s o f t h e denuder c o a t e d w i t h a sodium f l u o r i d e ,

by a d s o r p t i o n on a s h o r t e r denuder coated w i t h o x a l i c a c i d , and SO2 and NO2 by a n o t h e r sodium carbonate coated tube. A f t e r a known volume o f a i r i s made t o f l o w though t h e denuders, t h e c o a t i n g s a r e e x t r a c t e d w i t h w a t e r and SO4-, NO3and C1- a r e analyzed b y I o n Chromatography. D e t e c t i o n l i t i i i t s as l o w as a few p p t v c a n e a s i l y be determined w i t h t h i s procedure.

4

air in

20 cm

u -P

15

-

20

4

/

Pump

F i g u r e 2. Assembly o f a n n u l a r d i f f u s i o n t u b e s (denuders) f o r t h e sampling o f s u l f u r and n i t r o g e n compounds i n t h e atmosphere.

224

The use of diffusion tubular denuders makes a l s o possible t h e d i r e c t d e t e r mination of aerosolic ammonium s u l f a t e s and s u l f u r i c acid by FPD. The ammonium s u l f a t e s and s u l f u r i c acid aerosols can be detected together by placing a Ph O2 denuder before the detector ( 1 9 ) , whereas speciation of p a r t i c u l a t e s u l f u r compounds can be performed by a subtraction technique using two denuders f o r SO2 connected in s e r i e s by a g l a s s l i n e which can be heated a t temperature ranging between 125-135°C (20, 2 1 ) . While aerosolic s u l f a t e s flow unaffected through the heated l i n e , s u l f u r i c acid aerosols a r e converted i n t o gaseous SO2 and removed by the gas stream by t h e second denuder. The decrease in t h e FPD signal measured, when t h e connecting l i n e i s heated u p , i s r e l a t e d t o t h e content of sulfur i c acid in t h e atmosphere. 3.1

NITROGEN OXIDES, NITRIC ACID AND NITROGEN CONTAINING COMPOUNDS The content of nitrogen oxides usually indicated a s NO, r e f e r s t o concentration of n i t r i c oxide ( N O ) and nitrogen dioxide ( N O 2 ) , the l a t t e r being of spec i f i c i n t e r e s t f o r i t s t o x i c i t y ; among t h e other nitrogen oxides F120 -which occurs in appreciable concentrations-has a s l i g h t environmental impact whereas N203 i s present in much smaller concentrations a s i t a c t s a s intermediate i n the formation of n i t r i c acid ( 2 2 ) . Although, on a global s c a l e , the major portion of NOx i s n a t u r a l l y produced by bacterial degradation a s NO, the concentration measured in urban atmospheres i s one t o two orders of magnitude higher than t h a t measured in rural a r e a s . T h u s problems associated with NOx r e f l e c t the importance of anthropogenic sources over natural sources. The former a r e associated with t h e use of motor vehicles, f o s s i l fuel combustion, and t h e manufacture of some chemicals. NO, e x e r t a strong influence on many p o l l u t a n t s gas cycles a s they a r e precursors in the formation of photochemical oxidants, c o n t r i b u t e t o atmospheric a c i d i t y and take p a r t in t h e formation of p a r t i c u l a t e matter. N i t r i c acid - harmful and toxic t o man and plants - i s primarly formed by oxidation of NO2 in the presence of OH r a d i c a l s produced by photochemical reactions: i t can be emitted by i n d u s t r i a l and chemical processes. Ammonia a n d , t o a l e s s e r e x t e n t , amines a r e o t h e r nitrogen-containing compounds which a r e released in t o the atmosphere. Both can be produced by anthropogenic and natural sources. Deleterious e f f e c t s of ammonia a r e primarlyassociated with i t s r o l e in t h e formation of p a r t i c u l a t e matter. Chromatographic Methods f o r NO,, HN03, NH3 and nitrogen containing compounds Prior t o the introduction of methods based on chemiluminescence d e t e c t i o n , several attempts have been made t o develop GC methods capable t o give a d i r e c t measure of NO and NO2 i n the atmosphere. As these e f f o r t s f a i l e d because of t h e 3.2

225

i n t r i n s i c d i f f i c u l t y o f f i n d i n g packing m a t e r i a l s s u f f i c i e n t l y i n e r t t o permit t h e e l u t i o n of such r e a c t i v e p o l l u t a n t s a t t h e ppbv l e v e l s , t h i s approach has been no l o n g e r pursued. Today, t h e i n t e r e s t i n chromatographic methods f o r NOx i s r e s t r i c t e d t o NO2 as i t s d e t e c t i o n by chemiluminescent methods i s s u b j e c t t o i n t e r f e r e n c e s f r o m p e r o x y a c e t y l n i t r a t e and n i t r i c a c i d whereas s p e c t r o s c o p i c methods u t i l i z e d f o r i t s s p e c i f i c m o n i t o r i n g a r e expensive and d i f f i c u l t t o be used i n t h e f i e l d . Adequate s e n s i t i v i t y has been achieved by making use o f s u i t a b l e e n r i c h i n g dev i c e s whereas chromatographic problems have been p a r t l y s o l v e d by c o n v e r t i n g

NO2 i n t o a l e s s r e a c t i v e compound easy t o be analyzed. T r a p p i n g o f NO2 on T r i e t h a n o l a m i n e c o a t e d p l a t e s f o l l o w e d by e x t r a c t i o n o f t h e r e a c t i o n p r o d u c t s and t h e i r c o n v e r s i o n t o d e r i v a t i v e s p r i o r t o t h e GC a n a l y -

s i s has been r e p o r t e d Aoyama e t a l . ( 2 3 ) . D e t e c t i o n and i d e n t i f i c a t i o n o f t h e n i t r o s o d e r i v a t i v e s has been c a r r i e d o u t by GC-MS w i t h a thermal energy a n a l y z e r . The sampling system i s s i m p l e as t h e p l a t e a c t s as a p a s s i v e sampler, b u t long exposure

and expensive apparatus i s r e q u i r e d f o r a n a l y s i s .

A novel approach f o r t h e sampling o f NO2 by making use o f c o m m e r c i a l l y a v a i l a b l e Thermosorb/F c a r t r i d g e s f i l l e d w i t h F l o r i s i l has been d e s c r i b e d b y L i p a r i ( 2 4 ) . The s o r b e n t m a t e r i a l i s coated w i t h d i p h e n y l a m i n e which r e a c t s spec i f i c a l l y w i t h NO2. The p r o d u c t s a r e e l u t e d f r o m t h e c a r t r i d g e , analyzed byHPLC w i t h U.V.

d e t e c t i o n and r e l a t e d t o t h e amount o f NO2 p r e s e n t i n t h e a i r . No i n -

t e r f e r e n c e was e v i d e n t i a t e d by NO, 03, HN03 and SO2 w h i l e l i t t l e i n t e r f e r e n c e was caused by t h e presence o f P e r o x y a c e t y l n i t r a t e . D e t e c t i o n l i m i t s a r e l o w as 0.1 ppbv can be o b t a i n e d by sampling 2 m3 o f a i r . Annular denuders shown i n F i g u r e 2 p r o v i d e d a s i m p l e r and more r e l i a b l e t e c h n i q u e f o r t h e sampling o f NO2 and NH3 ( 2 5 ) . By e x t r a c t i n g t h e c o a t i n g mater i a l (Potassium I o d i d e ) i t i s p o s s i b l e t o d e t e r m i n e NO2 as NO3

by I o n L i q u i d

Chromatography. A l s o c y l i n d r i c a l denuders coated w i t h t u n g s t i c a c i d were found s u i t a b l e f o r t h e sampling o f n i t r i c a c i d and ammonia from t h e a i r (26, 2 7 ) . A f t e r sample c o l l e c t i o n t h e denuder i s s e t i n t o a h e a t e r and t h e gases a r e r e l e a s e d a t a p r o grammed t e m p e r a t u r e under a stream o f He-02. While PIH3 i s c o n v e r t e d i n t o NO dur i n g t h e d e s o r p t i o n process, Ht4O3, which i s r e l e a s e d as NO2,

i s reduced by c a t a -

l i t i c c o n v e r t e r p r i o r t o d e t e c t i o n , which i s c a r r i e d o u t by means o f a c h e m i l u minescent a n a l y z e r . D e t e r m i n a t i o n of ammonia and l o w b o i l i n g amines i n a i r b y gas chromatography has been d e s c r i b e d by K a s h i h i r a e t a l . ( 2 8 ) . These compounds c o n c e n t r a t e d on por o u s polymers a r e separated b y a packed column and d e t e c t e d a f t e r p y r o l y s i s and o x i d a t i o n by a m o d i f i e d chemiluminescent n i t r o g e n d e t e c t o r . A method f o r t h e d e t e r m i n a t i o n of l o w b o i l i n g a l i p h a t i c amines has been de-

s c r i b e d by Kuwata e t a l . ( 2 9 ) . P r e c o n c e n t r a t i o n was c a r r i e d o u t on SEP-PAK (c18)

226

c a r t r i d g e s k e p t a t room temperature. The vapours were e x t r a c t e d w i t h w a t e r

-

me-

t h a n o l s o l u t i o n s and small a l i q u o t s i n j e c t e d i n t o t h e column. A s p h e r i c a l l y shaped polymer a1 k a l i n i z e d w i t h potassium h y d r o x i d e was used f o r t h e s e p a r a t i o n o f C1-C3

amines. C o e x i s t i n g o r g a n i c components c o l l e c t e d on t h e t r a p d i d n o t i n -

t e r f e r e w i t h t h i s d e t e r m i n a t i o n because d e t e c t i o n o f a1 i p h a t i c amines was c a r r i e d o u t w i t h an a l k a l i f l a m e d e t e c t o r . W i t r o s o amines were e n r i c h e d on Thermosorb/N c a r t r i d g e s m a i n t a i n e d a t room temperature ( 3 0 ) . A f t e r e x t r a c t i o n , a p o r t i u n o f t h e s o l u t i o n was f u r t h e r i n j e c t e d i n t o a Tenax GC t r a p and t h e excess s o l v e n t vented w i t h Helium. A d u a l t r a p p i n g system was used t o t r a n s f e r t h e f r a c t i o n o f i n t e r e s t i n t o t h e a n a l y t i c a l column. GC-MS and chemiluminescence d e t e c t i o n a l l o w e d t h e p o s i t i v e i d e n t i f i c a t i o n o f nitrosoamines. 4.1

HYDROCARBONS Hydrocarbons, (HC), a r e i m p o r t a n t p r i m a r y p o l l u t a n t s r e l e a s e d i n t o theatmo-

sphere by anthropogenic as w e l l as by n a t u r a l sources. However, t h e t e r m HCalso i n c l u d e s carbon compounds which may have heteroatoms, such as oxygen, s u l f u r , n i t r o g e n and halogens i n t h e i r m o l e c u l e . Anthropogenic emissions come f r o m v a r i o u s a c t i v i t i e s o f mans, which can be c l a s s i f i e d as m o b i l e o r s t a t i o n a r y sources. The former a r e r e l a t e d t o t r a n s p o r t a t i o n a c t i v i t i e s (motor v e h i c l e s , a i r c r a f t , r a i l r o a d s , ships etc.),

whereas t h e l a t t e r a r e a s s o c i a t e d w i t h f o s s i l

f u e l combustion (carbon and o i l ) , and t h e use o f f u e l and s o l v e n t s i n a v a r i e t y o f i n d u s t r i a l processes. N a t u r a l HC a r e namely produced by b a c t e r i a l d e g r a d a t i o n o f o r g a n i c m a t t e r and b i o g e n i c e m i s s i o n f r o m p l a n t s . As t h e v o l a t i l i t y o f HC i s r e l a t e d a p p r o x i m a t e l y t o carbon number, i t i s u n l i k e l y t h a t compounds w i t h a carbon number g r e a t e r t h a n 16 can r e a c h appreciab l e c o n c e n t r a t i o n s i n t h e gaseous phase. Sampling and d e t e r m i n a t i o n o f h i g h e r m o l e c u l a r w e i g h t components, which a r e u s u a l l y p r e s e n t i n t h e atmosphere as aer o s o l s o r s o l i d p a r t i c l e s , w i l l be discussed i n t h e s e c t i o n on p a r t i c u l a t e matter. H C may a f f e c t t h e a i r q u a l i t y i n d i f f e r e n t ways: t h e y may be r e l e a s e d i n such amounts t o b u i l d up harmful c o n c e n t r a t i o n s i n t o theatmosphere o r may t a k e p a r t t o chemical r e a c t i o n s g i v i n g r i s e t o p r o d u c t s which may, i n t u r n , cause det r i m e n t a l e f f e c t s on t h e environment. Photochemical smog and d e p l e t i o n o f t h e s t r a t o s p h e r i c ozone l a y e r a r e examples o f t h e e f f e c t s t o which HC e m i s s i o n l a r gely contribute. The wide range o f p h y s i c a l , chemical and b i o l o g i c a l p r o p e r t i e s o f HC makes d i f f i c u l t t o develop an a n a l y t i c a l procedure which can r e p r e s e n t t h e i r e n v i r o n mental impact. One general approach i s t o measure t h e T o t a l Hydrocarbons Content (THC), t h i s v a l u e being r e l a t e d somehow t o t h e t o t a l o r g a n i c carbon mass concent r a t i o n p r e s e n t i n t h e atmosphere.

221

4.2

D e t e r m i n a t i o n of t h e T o t a l Hydrocarbons Content (THC) i n t h e Atmosphere The e a s i e s t way t o e v a l u a t e t h e THC i s based on t h e d i r e c t measure o f HC by

means o f a f l a m e i o n i z a t i o n d e t e c t o r ( F I D ) . The a i r sampled by a sealed PTFE coated diaphram pump

-

t h r o u g h a f i l t e r t o remove t h e p a r t i c u l a t e m a t t e r , f l o w s

t o t h e d e t e c t o r ( 3 2 ) . I t s s i g n a l p r o v i d e s an i n t e g r a l measure o f HC and t h e r e sponse i s expressed as o r g a n i c carbon c o n t e n t e q u i v a l e n t t o a c e r t a i n compound taken as r e f e r e n c e ( u s u a l l y CH4). A l t e r n a t i v e l y , t h e d e t e r m i n a t i o n can be c a r r i e d o u t s e m i c o n t i n u o u s l y by c o n n e c t i n g a sampling l o o p t o t h e FID w i t h a t i m e c o n t r o ll e d switching valve, The d e t e r m i n a t i o n o f THC i s , however, o f l i t t l e p r a c t i c a l i n t e r e s t as methane, which i s a n a t u r a l a i r component, c o n t r i b u t e s t o a l a r g e e x t e n t t o t h e THC. The d e t e r m i n a t i o n o f Nonmethane T o t a l Hydrocarbons Content ( NMTHC) has been t h u s proposed as a more r e p r e s e n t a t i v e i n d e x . I t s e v a l u a t i o n can be accomplished by s e v e r a l methods. One procedure c o n s i s t s i n t h e c o n t i n u o u s measure o f t h e THC and semicontinuous d e t e r m i n a t i o n o f methane which i s f r a c t i o n a t e d f r o m o t h e r l o w b o i l i n g HC ( s u c h as ethane, e t h y l e n e and a c e t y l e n e ) on a GC column. N M H C i s c a l c u l a t e by s u b t r a c t i n g t h e c o n c e n t r a t i o n s o f THC and methane o b t a i n e d u s i n g i n d i p e n d e n t measurements (33, 3 4 ) . The same d e t e r m i n a t i o n can be performed semic o n t i n u o u s l y by u s i n g t h e column f o r s e p a r a t i n g methane f r o m t h e r e s t o f HC. The gas chromatograph i s designed i n such a way t h a t , a f t e r t h e e l u t i o n and det e c t i o n o f methane, t h e f l o w r a t e o f t h e c a r r i e r gas i s r e v e r s e d and HC w i t h carbon numbers g r e a t e r than two a r e b a c k f l u s h e d . A f t e r d e s o r p t i o n , t h e compounds a r e d i v e r t e d t o t h e FID and d e t e c t e d t o g e t h e r as a GC peak. By e x p l o i t i n g t h e same p r i n c i p l e , some i n s t r u m e n t s have been m o d i f i e d t o p e r m i t t h e d e t e r m i n a t i o n o f CO and, i f necessary C02, i n t h e same r u n (35, 3 7 ) . An a d d i t i o n a l column i s used f o r s e p a r a t i n g CH4 from CO and C02. The l a s t two compounds a r e d e t e c t e d by the

FID a f t e r r e d u c t i o n on a N i k e l Raney c a t a l y s t k e p t a t 350°C. I n o r d e r t o o b t a i n a s i g n a l response p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f

t o t a l o r g a n i c carbon, t h i s method has been f u r t h e r m o d i f i e d by adding an o x i d a t i o n s t e p (Copper o x i d e a t 700°C) t o c o n v e r t NYTHC t o C02 p r i o r t o t h e f i n a l r e d u c t i o n t o methane (38). V a r i o u s c o m b i n a t i o n o f columns packed w i t h porous p o l y mers (Porapak Q), m o l e c u l a r s i e v e s and s o l i d supports, c o a t e d w i t h l i q u i d phases ( t y p i c a l l y 10% methyl s y l i c o n e on S u p e l c o p o r t o r s i m i l a r a b s o r b e n t s ) , have been used f o r t h e s e p a r a t i o n o f CH4, CO and C02. Since t h e equipments f o r NMTHC a r e somewhat complex and a r t i f a c t s may a r i s e from decomposition o r i r r e v e r s i b l e a d s o r p t i o n o f HC i n t h e column, procedures n o t i n v o l v i n g GC s e p a r a t i o n o f t h e sample have been proposed. One c o n s i s t s i n t h e c o n t i n u o s measure o f NM HC by P h o t o i o n i z a t i o n d e t e c t i o n (PID) ( 3 9 ) . A l t h o u g h t h e d a t a c o l l e c t e d w i t h t h i s method c o r r e l a t e w e l l w i t h t h o s e measured by a NMTHC gas chromatograph equipped w i t h t h e FID, c o n s i d e r a t i o n s on t h e i o n i z a t i o n p o t e n t i a l o f d i f f e r e n t HC cause some doubts r e g a r d i n g accuracy o f t h i s procedure.

228

Another method i n v o l v e s c r y o g e n i c c o n c e n t r a t i o n o f NI1 HC f o l l o w e d by d e t e c t i o n w i t h t h e FID (40, 4 1 ) . A l t h o u g h t h i s approach p r o v i d e s a simple, y e t accur a t e measure o f NM HC down t o p p b v l e v e l s , i t r e q u i r e s manual o p e r a t i o n and c a r e f u l sample c o l l e c t i o n . A l t h o u g h t h e NM THC i s a v e r y u s e f u l i n d e x t o e v a l u a t e t h e q u a l i t y o f t h e atmosphere, i t p r o v i d e s o n l y a g e n e r a l i n f o r m a t i o n so t h a t s p e c i a t i o n o f HC i s o f t e n required. 4.3

S p e c i a t i o n o f Atmospheric Hydrocarbons The s p e c i a t i o n o f HC i n t h e a i r can be c a r r i e d o u t f o r d i f f e r e n t aims ( d e -

t e r m i n a t i o n o f a i r q u a l i t y , e v a l u a t i o n o f photochemical c o n v e r s i o n , measure o f d e p o s i t i o n v e l o c i t y e t c . ) and, a c c o r d i n g l y , t h e a n a l y t i c a l method must f u l f i l l t h e s p e c i f i c need. Two p r i m a r y procedures may be f o l l o w e d . One i n v o l v e s t h e f r a c t i o n a t i o n o r s p e c i f i c d e t e c t i o n o f HC a c c o r d i n g t o t h e i r r e a c t i v i t y and t h e o t h e r t h e i r d e t e r m i n a t i o n on t h e b a s i s o f t h e i r v o l a t i l i t y , i . e . carbon number. 4.3.1 D e t e r m i n a t i o n o f hydrocarbons a c c o r d i n g t o t h e i r r e a c t i v i t y The s e p a r a t i o n o f HC i n t o c l a s s e s has been r e s t r i c t e d t o t h e f r a c t i o n a t i o n of alkanes, alkenes and a r o m a t i c s , t h e n a i n i n t e r e s t b e i n g r e l a t e d t o t h e l a s t two c l a s s e s , which e x h i b i t h i g h photochemical r e a c t i v i t y . The f r a c t i o n a t i o n has been c a r r i e d o u t on v a r i o u s columns o r column c o m b i n a t i o n s c a p a b l e o f r e t a i n i n g one c l a s s o f compounds w i t h r e s p e c t t o t h e o t h e r s . Column packed w i t h potassium carbonate, magnesium p e r c h l o r a t e , c o n c e n t r a t e d s u l f u r i c a c i d ( 4 2 ) sodium i o d i d e ( 4 3 ) , m e r c u r y and p a l l a d i u m s a l t s ( 4 4 ) , supported on s u i t a b l e a d s o r b e n t s have been d e s c r i b e d i n t h e t e c h n i c a l l i t e r a t u r e . D e t e c t i o n has been c a r r i e d o u t by F I D u s i n g b o t h group a n a l y s i s (45, 46) and s u b t r a c t i v e t e c h n i q u e s ( 4 7 ) . The b e s t r e s u l t s have been r e p o r t e d by Saltzmann e t a l . ( 4 7 ) , who m o d i f i e d a THC a n a l y z e r by i n s e r t i n g a chromium t r i o x i d e - s u l f u r i c a c i d column between t h e sampler and t h e d e t e c t o r t o remove o l e f i n s and h i g h e r a r o m a t i c compounds sel e c t i v e l y f r o m t h e sample. By a l t e r n a t i v e l y r e c o r d i n g t h e s i g n a l c o r r e s p o n d i n g t o t h e THC and t h e one o b t a i n e d by d i v e r t i n g t h e sample t o t h e column, i t has been p o s s i b l e t o d e t e r m i n e p h o t o c h e m i c a l l y r e a c t i v e HC b y a s u b t r a c t i v e t e c h n i que. I n g e n e r a l , however, t h e o p e r a t i n g c o n d i t i o n s o f t h e s e methods a r e c r i t i c a l and v e r y o f t e n n o t s u f f i c i e n t l y s e n s i t i v e . One way t o a l l e v i a t e t h e s e l i m i t a t i o n s i s t o i n c r e a s e t h e s e l e c t i v i t y o f t h e d e t e c t o r so t h a t t h e use o f separat i o n columns can be avoided. Becher e t a l . ( 4 8 ) m o d i f i e d a chemiluminescence det e c t o r f o r t h e s e l e c t i v e measurement o f o l e f i n s by a d d i n g ozone t o t h e a i r samp l e ; t h e r e s u l t i n g s i g n a l was found t o c o r r e l a t e w e l l w i t h t h e c o n c e n t r a t i o n o f e t h y l e n e measured by gas chromatography. However, t h e dependance o f t h e d e t e c t o r response on t h e degree o f i n s a t u r a t i o n and m o l e c u l a r w e i g h t o f t h e o l e f i n s made, an a c c u r a t e e s t i m a t i o n o f t h e t o t a l o l e f i n

c o n t e n t by

means

of this

method

229

difficult. 4.3.2 D e t e r m i n a t i o n of Hydrocarbons a c c o r d i n g t o carbon number A n a l y s i s o f tIC i n a i r i s more f r e q u e n t l y r e f e r r e d as t h e d e t e r m i n a t i o n o f components c a r r i e d o u t a c c o r d i n g t o t h e i r v o l a t i l i t y . The sampling t e c h n i q u e as w e l l as t h e a n a l y t i c a l column i s o p t i m i z e d i n o r d e r t o d e t e r m i n e s p e c i e s w i t h i n a w e l l - d e f i n e d range o f carbon atoms; u s u a l l y two c l a s s e s a r e c o n s i d e r e d : C2-C5 and c6-c16. O c c a s i o n a l l y t h e d e t e r m i n a t i o n o f h i g h m o l e c u l a r w e i g h t components has been extended t o i n c l u d e compounds w i t h carbon number u p t o C20. A f t e r t h e f r a c t i o n a t i o n o f t h e a i r sample t h e compounds a r e i d e n t i f i e d by e l u t i o n o r d e r w i t h s p e c i f i c d e t e c t o r s and q u a n t i f i e d

i n d i v i d u a l l y e i t h e r by

e x t e r n a l o r i n t e r n a l s t a n d a r d s . T h i s approach i s more c o m p l i c a t e d t h a n t h o s e above described, b u t i t does p r o v i d e d e t a i l e d i n f o r m a t i o n on t h e e m i s s i o n r a t e s and r e a c t i v i t y o f e v e r y s i n g l e s p e c i e s . As sampling i s t h e most c r i t i c a l step, i t i s customary t o c l a s s i f y t h e n e thods f o r t h e d e t e r m i n a t i o n o f HC a c c o r d i n g t o t h e c o l l e c t i o n techniqueemployed. The more common methods adopted f o r d e t e r m i n i n g C2-C5 HC a r e l i s t e d

i n Table I .

They i n c l u d e d i r e c t a n a l y s i s o f t h e a i r sample c o l l e c t e d " i n s i t u " o r " g r a b samp l i n g " and p r e c o n c e n t r a t i o n o f HC by c r y o g e n i c t e c h n i q u e s . An a u t o m a t i c gas chromatograph e n a b l i n g d i r e c t a n a l y s i s o f v o l a t i l e components has been d e s c r i b e d by J e l t e s e t a1

.

( 5 2 ) . The compounds p r e s e n t i n 8 m l o f

a i r were f r a c t i o n a t e d on an alumina column. A d e h y d r a t i n g a g e n t was used t o r e move w a t e r which c o u l d i n t e r f e r e w i t h d e t e c t i o n o f t h e l o w e r components and change t h e chromatographic column. S i l i c a ( 6 8 ) o r a c t i v a t e d alumina c o a t e d w i t h 178 B - B ' o x y d i p r o p i o n i t r i l e ( 7 5 ) has a l s o been used f o r t h e r a p i d s e p a r a t i o n o f 17 l i g h t HC. S i n c e t h e d e t e c t i o n l i m i t s which can be achieved b y d i r e c t i n j e c t i o n o f t h e sample range f r o m 100 ppbvto few ppmv, t h i s method i s o f l i m i t e d a p p l i c a tions, s u i t a b l e o n l y i n p o l l u t e d areas. P r e c o n c e n t r a t i o n o f t h e sample i s t h e procedure w i d e l y used t o a c h i e v e t h e s e n s i t i v i t y requested f o r t r a c e a n a l y s i s and i t i s a mandatory s t e p i f t h e h i g h e f f i c i e n c y o f c a p i l l a r y columns i s t o be f u l l y e x p l o i t e d . B e i e r and Bruckmann ( 5 5 ) e n r i c h e d 8 m l o f a i r sample on a m i c r o b o r e t r a p p r i o r t o a n a l y s i s on a 70 m c a p i l l a r y column coated w i t h aluminium o x i d e . The c o n c e n t r a t e was t r a n s f e r r e d a t t h e column i n l e t by warming t h e t r a p w i t h h o t water.

A more e x t e n s i v e c r y o g e n i c t r a p p i n g (200 m l ) has been d e s c r i b e d by Shaw e t a l . ( 5 8 ) . The t r a p was submerged i n 1 i q u i d oxygen and i t s c o n t e n t f r a c t i o n a t e d on a 65 m SCOT column coated w i t h OV 101 p l u s 50% I g e p a l CO-880 on s i l i c a . Cox e t a l . ( 5 6 ) have s l i g h t l y m o d i f i e d t h i s procedure b y u s i n g t r a p s f i l l e d w i t h g l a s s beads. The s e p a r a t i o n was c a r r i e d o u t on a 60 m SE 30 wide-bore, t h i n - f i l m fused s i l i c a c a p i l l a r y column. D e t e c t i o n o f t h e v a r i o u s components was performed

230 TABLE I and P r e c o n c e n t r a t i o n methods f o r C 2 - C 5 HC i n A i r

=ling Method

Ref.

I ) Direct injection

49-54

Type o f Sample Urban heavy p o l 1u t e d

2 ) Cryogenic p r e c o n c e t r a__ tion Cryogenic f o c u s i n g

Urban

55

Cryogenic f r e e z e t r a p p i n g

56-60

Cryogenic g r a d i e n t

Urban, Rura1,Forestal

61

Urban

3 ) E n c e n t r a t i o n on s o r b e n t m a t e r i a l s Carbosieve C

T(-78

"C

62-64

Urban, R u r a l

Carbosieve C t Porous S i l i c a T=-38"C 65 Carbopack A

T=-T60"C

34,66,67,102

Urban, Suburban

Porous S i l i c a T= l i q u i d O2

68-71

Urban, Rural

A c t i v e Charcoal T= ambient

66,72,73

Urban, Suburban

Chromosorb 102

74

Urban

75-77

Urban

T=-80"C

GC Supports

TABLE I 1 Sampling and P r e c o n c e n t r a t i o n methods f o r HC w i t h Carbon Number g r e a t e r t h a n 5 Method

Ref

Type o f Sample

1) D i r e c t i n j e c t i o n

78

Urban,Suburban

2) Cryogenic p r e c o n c e n t r a t i o n Cryogenic f o c u s i n g ( V< 0.5 1 )

56,58,79

Cryogenic f r e e z e t r a p p i n g

59,80

Urban, R u r a l ,Fcresta 1 Urban, R u r a l

3 ) .__---__ P r e c o n c e n t r a t i o n on s o _______ rbent m a t e r i a l s A c t i v e Charcoal (L.E.) 74,81-85 Tenax GC

(T.D.)

64,86-100 2

Urban,?ural,Forestal Urb n,Suburban,Rural,Forest

GCBS ( s . s . a . = 80-110 m / g ) (T.D.)

67,72,73,101-105

(T. D., L .E. )

86,90,91,106-108

Porapaks

Urban,Suburban,Forestal Urban

Chromosorbs c e n t u r y s e r i e s (T.D.)

109-113

Urban

Other p o l y m e r i c sorbents (T.D.)

103

Urban

C h e m i c a l l y bonded phases (L.E.)

114-115

GC

15,62,63,68-70 77,116-118

Supports (T.D.)

4) Co-condensation w i t h l i q u i d s ~

L.E.= L i q u i d E x t r a c t i o n T.D.=

119 Thermal D e s o r p t i o n

Urban Urban,Rural,Suburban Urban

1

231 by a FID p l a c e d p a r a l l e l t o a P I D . H i g h r e s o l u t i o n combined w i t h m u l t i p l e d e t e c t i o n p r o v i d e d a h i g h degree of i d e n t i f i c a t i o n o f o r g a n i c s i n environmental a i r . By u s i n g potassium c a r b o n a t e t o p r e v e n t condensation o f water, Westberg e t

a l . ( 5 7 ) were a b l e t o extend t h e f r e e z e - o u t t r a p p i n g t e c h n i q u e t o volume l a r g e r than 1 1 . The c o n c e n t r a t e was f r a c t i o n a t e d on a 6 m micropacked column f i l l e d w i t h Durapack n - o c t a n e / P o r a s i l C p a r t i c l e s a l l o w i n g t h e s e p a r a t i o n o f more t h a n

24 components i n l e s s t h a n 30 m i n u t e s . The chromatographic s e p a r a t i o n was improved b y programming t h e temperature o f t h e column f r o m -70 t o 65°C. E x c e l l e n t chromatograms of samples c o l l e c t e d i n urban areas have been r e p o r t e d .

A s o p h i s t i c a t e d c r y o g e n i c g r a d i e n t t e c h n i q u e was used by Jonsson e t a l . ( 6 1 ) f o r t h e enrichment o f p o l a r components. The sample was t r a n s f e r r e d i n t o a espec i a l l y designed GC by g e n t l e h e a t d e s o r p t i o n . The s e p a r a t i o n o f oxygenated compounds from t h e r e s t of t h e c o n c e n t r a t e was c a r r i e d o u t by two-dimensional chromatography. The f r a c t i o n o f i n t e r e s t was r e f o c u s e d on a f u s e d s i l i c a c o l d t r a p p r i o r t o t h e i n j e c t i o n i n t o a f u s e d s i l i c a c a p i l l a r y column. P o s i t i v e i d e n t i f i c a t i o n o f s e v e r a l components was c a r r i e d o u t by GC-MS. Cryogenic f r e e z e t r a p p i n g methods f o r t h e enrichment o f a i r samples l a r g e r t h a n 10 1 have been r e p o r t e d by s e v e r a l a u t h o r s (59, 6 0 ) . Although, t h i s method m i g h t appear, i n p r i n c i p l e , t h e b e s t one t o o b t a i n e x t r e m e l y h i g h s e n s i t i v i t i e s ( p p t v l e v e l s ) , i t has a number o f p r a c t i c a l l i m i t a t i o n s . The main one a r i s e s from t h e use o f d e s i c c a n t s necessary t o remove w a t e r f r o m t h e sample as t h e y can a l s o s e l e c t i v e l y adsorb t h e 1 i g h t p o l a r components. P r e c o n c e n t r a t i o n on s o l i d adsorbents m a i n t a i n e d a t low temperatures has been a l s o proposed as a s u i t a b l e a l t e r n a t i v e t o c o l l e c t q u a n t i t a t i v e l y l i g h t HC i n air. K a i s e r ( 6 2 ) d e s c r i b e d an o r i g i n a l c r y o g r a d i e n t t e c h n i q u e t o be used i n comb i n a t i o n w i t h Carbon m o l e c u l a r s i e v e s (Carbosieve)

adsorbents. The c o n c e n t r a t e

was t r a n s f e r r e d i n t o t h e a n a l y z e r by g e n e r a t i n g a thermal g r a d i e n t w i t h i n t h e a d s o r p t i o n t u b e . Rapid d e t e r m i n a t i o n s o f e t h y l e n e , ethane and a c e t y l e n e a t ppbv l e v e l s were c a r r i e d o u t on s h o r t columns packed w i t h t h e same a d s o r b e n t used f o r sample p r e c o n c e n t r a t i o n . By combining t h e a d s o r p t i o n p r o p e r t i e s o f porous s i l i c a and Carbosieve C,

Rudolph e t a l . ( 6 5 ) were a b l e t o e n r i c h 2 1 o f a i r c o l l e c t e d by " g r a b sampling" on evacuated c o n t a i n e r s . The vapours were desorbed a t 250°C and t r a n s f e r r e d a t t h e column i n l e t which was m a i n t a i n e d a t -90°C.

The compounds were c o m p l e t e l y

separated on a 7 m narrow b o r e column packed w i t h u n t r e a t e d porous s i l i c a (Spher o s i l X 0B 075) by programming t h e t e m p e r a t u r e o f t h e oven f r o m -90" t o 70°C. T h i s method was s u i t a b l e f o r t h e d e t e r m i n a t i o n o f HC a t p p t v l e v e l s . Bruner e t a l . ( 6 6 ) e x p l o i t e d t h e p o s s i b i l i t i e s o f g r a p h i t i z e d carbon b l a c k s (GCB) t o e v a l u a t e l i g h t components i n atmospheric samples. 9ne t o two l i t e r s o f approximately a i r were c o l l e c t e d i n t o a t r a p f i l l e d w i t h Carbopack A k e p t a t

232

-160°C t o r e t a i n HC q u a n t i t a t i v e l y . A f t e r thermal d e s o r p t i o n a t 250"C,

t h e con-

c e n t r a t e was f r a c t i o n a t e d on a 3 m column packed w i t h Carbopack B c o a t e d w i t h 2.6% o f Carbowax 20

M. Due t o t h e hydrophobic n a t u r e o f b o t h t h e t r a p p i n g and

packing m a t e r i a l , no d i s t u r b a n c e o f water was observed d u r i n g t h e e l u t i o n o f t h e l o w e r components. S i l i c a g e l t r a p s i n c o m b i n a t i o n w i t h a n a l y t i c a l columns packed w i t h t h e same m a t e r i a l were p r e f e r r e d by Lonnemann ( 7 0 ) t o e l u c i d a t e t h e c o m p o s i t i o n o f C2-C5

i n many u r b a n and suburban l o c a t i o n s .

The use o f Chromosorb 102 has been d e s c r i b e d by Louw e t a l . ( 7 4 ) who a n a l y zed 500 m l o f a i r samples c o l l e c t e d by " g r a b sampling". Gaseous components were recovered by b a c k f l u s h i n g t h e c a r t r i d g e a t 160°C a f t e r c o u p l i n g i t i n t o t h e GC system a t a p n e u m a t i c a l l y operated s w i t c h v a l v e s i t u a t e d j u s t upstream t h e anal y t i c a l column. A 30 m SCOT column o p e r a t i n g under programmed t e m p e r a t u r e f r o m -10 t o 85°C p r o v i d e d s u f f i c i e n t s e l e c t i v i t y and s e n s i t i v i t y f o r t r a c e a n a l y s i s . The p o s i t i v e i d e n t i f i c a t i o n o f s e v e r a l components was made p o s s i b l e by s t a n d a r d a d d i t i o n and s e l e c t i v e s u b t r a c t i o n t e c h n i q u e s combined w i t h i n f r a r e d and mass spectrometry. Some p u b l i c a t i o n s d e s c r i b e t h e use o f s o l i d s u p p o r t s coated w i t h l i q u i d phases f o r t h e enrichment o f v o l a t i l e HC (66, 7 7 ) . A m a j o r drawback a s s o c i a t e d w i t h most o f t h e s e procedures i s caused by w a t e r which, on d e s o r p t i o n , a f f e c t s t h e chromatographic d e t e r m i n a t i o n o f some components. To o b t a i n s a t i s f a c t o r y r e s u l t s , i t i s mandatory t o remove w a t e r f r o m t h e a i r stream by means o f dehydrat i n g agents which, as s a i d b e f o r e , have o b j e c t i o n a b l e p r o p e r t i e s . Although sample enrichment on c a r t r i d g e s f i l l e d w i t h a c t i v e c h a r c o a l s o f d i f f e r e n t s u r f a c e areas (72, 73) p r o v i d e d e f f i c i e n t t r a p p i n g o f l i g h t HC a t temperatures,where condensation o f w a t e r i s p r e v e n t e d , i t

gave poor r e c o v e r y o r de-

c o m p o s i t i o n o f t h e r m a l l y u n s t a b l e compounds d u r i n g t h e d e s o r p t i o n process ( 6 6 ) . F a c t o r s a f f e c t i n g t h e sampling o f HC w i t h a carbon number g r e a t e r t h a n 5 a r e l e s s c r i t i c a l t h a n f o r t h e o t h e r c l a s s . The more w i d e l y used methods a r e lis t e d i n T a b l e 11. Although f r e e z e - o u t t r a p p i n g t e c h n i q u e s may be employed f o r t h e p r e c o n c e n t r a t i o n o f t h e sample, a d s o r p t i o n on t r a p s o p e r a t i n g a t room temper a t u r e i s , by f a r ,

p r e f e r r e d . I t i s s i m p l e and does n o t n e c e s s a r e l y r e q u i r e

h i g h l y e f f i c i e n t columns o r s e n s i t i v e d e t e c t o r s f o r t h e d e t e r m i n a t i o n o f t h e l e s s abundant components as volumes much l a r g e r t h a n 1 1 can be s a f e l y e n r i c h e d . Basically,

t h e vapours t r a p p e d on s o r b e n t m a t e r i a l s can be

recovered i n two ways; e x t r a c t i o n w i t h s o l v e n t s and thermal d e s o r p t i o n under a gas f l o w . The main advantage o f e x t r a c t i o n w i t h s o l v e n t s i s t h a t a r t i f a c t s a r i s i n g from p y r o l i s i s , p o l y m e r i z a t i o n , i s o m e r i z a t i o n o r i n c o m p l e t e r e c o v e r y o f some HC can be minimized. Some l i m i t a t i o n s a r i s e f r o m l o s s e s o c c u r r i n g d u r i n g t h e e x t r a c t i o n process and t h e c o n c e n t r a t i o n o f t h e recovered vapours. S i n c e chromatogra-

233

p h i c a n a l y s i s i s l i n i t e d t o s m a l l amounts o f l i q u i d samples, h i g h volumes o f a i r need t o be e n r i c h e d t o a c h i e v e a s a t i s f a c t o r y d e t e c t i o n l i m i t . Consequently, t h e s o r b e n t m a t e r i a l should be a b l e t o r e t a i n t h e HC so s t r o n g l y t h a t changes i n temperature, r e l a t i v e h u m i d i t y and HC c o n c e n t r a t i o n o c c u r r i n g d u r i n g t h e samp l i n g do n o t a f f e c t t h e a d s o r p t i o n p r o p e r t i e s o f t h e t r a p p i n g m a t e r i a l . I t i s r e c o g n i z e d t h a t a c t i v e c h a r c o a l s meet t h e s e r e q u i r e m e n t s t o a l a r g e e x t e n t as t h e y a r e hydrophobic m a t e r i a l s c h a r a c t e r i z e d by e x t r e m e l y h i g h a d s o r p t i o n capac i t i e s . The e x t r a c t i o n o f t h e c o n c e n t r a t e can be performed on a m i c r o - s o x h l e t apparatus o r by e l u t i o n t e c h n i q u e s . Grob and Grob ( 8 1 ) f u l l y e x p l o i t e d t h e advantage o f u s i n g c h a r c o a l t u b e s i n combination w i t h l i q u i d e x t r a c t i o n t e c h n i q u e s . The c o n c e n t r a t e was s e p a r a t e d on a h i g h r e s o l v i n g power c a p i l l a r y column (120 m) c o a t e d w i t h UCON HC 1500. To p r e v e n t l o s s e s o f t h e more v o l a t i l e components, an a l i q u o t o f t h e e x t r a c t was i n j e c t e d i n t o t h e a n a l y z e r w i t h t h e s p l i t - l e s s method. More t h a n 100 components, m o s t o f them i d e n t i f i e d by MS, were d e t e c t e d i n an atmospheric sample c o l l e c t e d i n Zurich. Based on t h e same p r i n c i p l e , a semiautomatic d e v i c e a l l o w i n g t h e c o l l e c t i o n o f two hours averaged samples ( c a . 50 1 ) on c h a r c o a l a d s o r p t i o n t u b e s has been developed by B u r g h a r d t and J e l t e s ( 8 3 ) . I n t h i s case t h e vapours were r e c o v e r e d by e l u t i o n w i t h known volumes o f carbon d i s u l f i d e . The method was s u i t a b l e f o r compounds r a n g i n g between 6 and 1 0 carbon atoms. Traps packed w i t h Porapak N were p r e f e r r e d by Van Tessel e t a l . (108) f o r t h e p r e c o n c e n t r a t i o n o f arenes and v o l a t i l e h a l o o r g a n i c s compounds i n u r b a n samp l e s . A f t e r t h e e x t r a c t i o n , t h e sample was analyzed on packed columns connected e i t h e r t o an ECD o r a

PID.

A p p l i c a t i o n o f c h e m i c a l l y bonded phases t o t h e sampling and f r a c t i o n a t i o n o f a i r p o l l u t a n t s has been d e s c r i b e d b y Aue and T e l i ( 1 1 4 ) . When t h e c o m p o s i t i o n o f d i l u t e d a i r samples has t o be e l u c i d a t e d o r s m a l l changes o f t h e HC

c o n c e n t r a t i o n need t o be d e t e c t e d , t h e use o f t h e method

j u s t d e s c r i b e d i s u n s a t i s f a c t o r y and p r e c o n c e n t r a t i o n on "1 i g h t a d s o r b e n t s " f o l l o w e d by thermal d e s o r p t i o n o f t h e c o n c e n t r a t e has t o be p r e f e r r e d . As t h e e n t i r e sample i s s u b m i t t e d t o t h e a n a l y s i s a g r e a t e r s e n s i t i v i t y i s d e f i n i t e l y achieved. The e n r i c h e d vapours can be r e c o v e r e d by d i f f e r e n t t e c h n i q u e s . H e a t i n g o f t h e adsorbent i n s t a t i c f l o w c o n d i t i o n s f o l l o w e d by i m p u l s i v e t r a n s f e r i n t o t h e a n a l y z e r , d e s o r p t i o n under programmed t e m p e r a t u r e c o n d i t i o n s , " t i m e d e l u t i o n " and b a c k f l u s h i n g o f t h e c o n c e n t r a t e a r e some o f t h e procedures employed t o ensure a q u a n t i t a t i v e r e c o v e r y o f t h e sample. I n many i n s t a n c e s , c r y o g e n i c f o c u s i n g o f t h e desorbed vapours i s necessary t o e l i m i n a t e band spreading occurr i n g a t t h e column i n l e t . Among t h e adsorbents s u i t a b l e f o r thermal d c s o r p t i o n , Tenax GC, porous p o l y mers and GCB

have been p r e f e r r e d as t h e y p r o v i d e e x c e l l e n t r e c o v e r y f o r HC

234

coupled w i t h s a t i s f a c t o r y a d s o r p t i o n c a p a c i t i e s . The main advantages i n u s i n g Tenax GC a r e t h e r e l a t i v e l y m i l d temperatures (250-275°C) r e q u e s t e d f o r t h e des o r p t i o n of vapours and t h e l o w s e l e c t i v i t y towards p o l a r compounds. A n a l y s i s o f atmospheric samples e n r i c h e d on Tenax GC c a r t r i d g e s have been r e p o r t e d b y B e r t s c h e t a l . ( 8 7 ) . A f t e r p r e c o n c e n t r a t i o n o f 200 1 o f a i r , t h e c a r t r i d g e was sealed, t a k e n i n t h e l a b o r a t o r y and i n s e r t e d i n t o a p r o p e r l y d e s i gned i n j e c t i o n p o r t f o r thermal d e s o r p t i o n . The vapours were c r y o f o c u s e d i n t o a narrow b o r e t r a p 2 m l o n g m a i n t a i n e d a t t h e t e m p e r a t u r e o f l i q u i d n i t r o g e n . A 100 m c a p i l l a r y column p r o v i d e d an e x c e l l e n t s e p a r a t i o n o f t h e v a r i o u s components. Several hundreds substances i n t h e C6-C16 100 i d e n t i f i e d by

r a n g e were d e t e c t e d and a l m o s t

MS.

The c o m p o s i t i o n o f HC i n d i f f e r e n t u r b a n environments has been e l u c i t a t e d b y P e l l i z z a r i e t a l . ( 8 9 ) d u r i n g a two y e a r s m o n i t o r i n g campaign. An a u t o m a t i c sampler equipped w i t h a m u l t i p o r t head a l l o w e d t h e s e q u e n t i a l c o l l e c t i o n o f HC

on Tenax GC c a r t r i d g e s . I n a t y p i c a l d e s o r p t i o n c y c l e , t h e c o n c e n t r a t e i s desorbed a t 270°C under a f l o w r a t e o f h e l i u m and t h e vapours t r a p p e d i n t o a l i q u i d n i t r o g e n c o o l e d c a p i l l a r y . By h e a t i n g t h e c a p i l l a r y a t 180°C t h e sample i s i n t r o d u c e d i n t o a SCOT column. The compounds ,emerging f r o m t h e column a r e a n a l y zed by MS. More t h a n 100 components i n t h e range C5-C12

were p o s i t i v e l y i d e n t i -

f i e d . The presence o f 20 halogenated HC was e v i d e n t i a t e d b y r u n n i n g t h e mass spectrometer i n S e l e c t e d Ion D e t e c t i o n (SID) mode. 40 oxygen, n i t r o g e n , s u l f u r and s i l i c o n c o n t a i n i n g o r g a n i c c o n s t i t u e n t s were d e t e c t e d i n a i r samples c o l l e c ted i n d i f f e r e n t locations. D i u r n a l v a r i a t i o n s o f monoterpenes i n t h e atmosphere o f a p i n e f o r e s t were measured by Yokouchi e t a l . ( 9 6 ) . Seven monoterpene HC were determined i n 1 1 a i r sample e n r i c h e d on Tenax GC t r a p s by d e t e c t i n g t h e e f f l u e n t o f t h e GC column b y MS o p e r a t i n g i n S i n g l e Ion M o n i t o r i n g . B i o g e n i c o l e f i n i c compounds c o l l e c t e d on t h e same adsorbent were f r a c t i o n a t e d by S e i l a ( 9 7 ) on a s h o r t DPN/Porasil C column. A d e t e c t i o n u n i t equipped w i t h a FID placed i n p a r a l l e l w i t h a chemiluminescent a n a l y z e r p r o v i d e d a h i g h degree of i d e n t i f i c a t i o n f o r t h e o l e f i n s e m i t t e d f r o m t r e e f o l i a g e . An e x t e n s i v e m o n i t o r i n g o f 25 s e l e c t e d contaminants p r e s e n t i n t h r e e d i f f e r e n t l o c a t i o n s has been r e p o r t e d by Harkov ( 1 0 0 ) . The vapours desorbed f r o m Tenax GC were, i n t h i s case, analyzed on a f u s e d s i l i c a column connected t o an ECD and a FID placed i n p a r a l l e l .

In s p i t e of t h e wide number o f a p p l i c a t i o n r e p o r t e d i n t h e l i t e r a t u r e , p r e c o n c e n t r a t i o n of atmospheric samples on Tenax GC t r a p s has been r e c e n t l y q u e s t i o ned b y P e l l i z z a r i (120, 121) who e v i d e n t i a t e d p r e v i o u s l y unrecognized source o f sampling a r t i f a c t s o c c u r r i n g when t h e c o n c e n t r a t i o n o f o x i d a n t s and i n o r g a n i c p o l l u t a n t s i n t h e atmosphere exceeds c e r t a i n v a l u e s . Among t h e v a r i o u s a1 t e r n a t i v e s a v a i l a b l e , GCE

adsorbents seem t o o f f e r r e a l advantage as t h e y a r e h i g h l y

235

i n e r t m a t e r i a l s , u n a f f e c t e d b y ozone (105) and o t h e r r e a c t i v e p o l l u t a n t s . A n a l y s i s o f atmospheric samples c o n c e n t r a t e d on GCB t r a p s has been f i r s t r e p o r t e d by Raymond and Guiochon ( 1 0 1 ) . The c o n c e n t r a t e was t r a n s f e r r e d i n t o t h e column head m a i n t a i n e d a t room temperature. More t h a n 70 components were separat e d on a 100

m c a p i l l a r y column by programming t h e t e m p e r a t u r e up t o 230°C. The m e r i t s o f Carbopack B ( a GCB h a v i n g a s p e c i f i c s u r f a c e area o f 80 m 2 / g )

f o r t h e enrichment o f C5-C12

n a t u r a l and a n t h r o p o g e n i c HC have been d i s c u s s e d

by C i c c i o l i e t a l . (102, 105). HC

c o n c e n t r a t e d on t h i s m a t e r i a l

, were

desorbed

by b a c k f l u s h i n g t h e t r a p a t 270°C. A s a t i s f a c t o r y s e p a r a t i o n o f t h e vapours was performed on a 2 m Carbopack C column c o a t e d w i t h 0.4% SP 1000. A m u l t i p l e d e t e c t i o n u n i t made o f a FIO, a FPO and ECD p l a c e d i n p a r a l l e l p e r m i t t e d t h e i d e n t i f i c a t i o n o f s e v e r a l s u l f u r and halogen c o n t a i n i n g compounds. By removing t h e FPD and c o n n e c t i n g t h e gas chromatograph t o a MS t h e i d e n t i f i c a t i o n o f more t h a n 40 components p r e s e n t i n t h e c o n c e n t r a t e o f

urban atmospheres was c a r r i e d o u t .

B a c k f l u s h o f Carbopack B t r a p s and c r y o g e n i c f o c u s i n g o f vapours p r i o r t o t h e GC-MS a n a l y s i s was, i n s t e a d , p r e f e r r e d f o r t h e m o n i t o r i n g o f n a t u r a l and anthropogenic HC i n f o r e s t a l areas d u r i n g photochemical smog episodes ( 1 0 4 ) . By o p e r a t i n g t h e mass spectrometer i n t h e SIO mode arene and monoterpene HC selectively quantified

were

a t pptv l e v e l s .

Porous polymers o f v a r i o u s t y p e and s o l i d s u p p o r t s c o a t e d w i t h l i q u i d phases have been a l s o t e s t e d f o r t h e c o l l e c t i o n o f HC

.

P e r r y e t a l . (109) developed a " t i m e d e l u t i o n " t e c h n i q u e f o r t h e r e c o v e r y o f a i r p o l l u t a n t s e n r i c h e d on Chromosorb 102 t r a p s . The c o n c e n t r a t e was separat e d on a 2 m column packed w i t h Chromosorb U AW DWCS coated w i t h Carbowax 20 m. Alkylbenzenes f r o m v e h i c u l a r emissions i n t r a f f i c roads were i d e n t i f i e d a t t h e 20 ng l e v e l .

A two s t a g e e x t r a c t i o n / i n j e c t i o n system s u i t a b l e f o r a i r samples preconcent r a t e d on Chromosorb 102 was developed by Dravniecks e t a l . ( 1 1 1 ) . Cryogenic f o c u s i n g o f t h e desorbed vapours was c a r r i e d o u t i n t h e i n j e c t i o n p o r t . A f t e r condensation, t h e i n j e c t o r was r a p i d l y heated and t h e compounds analyzed on a 30 m SCOT column c o a t e d w i t h Carbowax 20 M. The p o s s i b i l i t i e s and l i m i t a t i o n s o f Chromosorbs o f t h e c e n t u r y s e r i e s were c r i t i c a l l y d i s c u s s e d b y M i e u r e and O i e t r i c h ( 1 1 3 ) . Enrichment on t r a p s f i l l e d w i t h P o l y s o r b i m i d e adsorbent has been r e p o r t e d by I o f f e ( 1 0 3 ) . T h i s m a t e r i a l e x h i b i t s a d s o r p t i o n f e a t u r e s q u i t e s i m i l a r t o g r a p h i t i c carbons b u t does n o t have t h e same chemical s t a b i l i t y . A l t h o u g h t h e use o f s o l i d s u p p o r t s c o a t e d w i t h l i q u i d s t a t i o n a r y phases has been d e s c r i b e d by s e v e r a l authours, t h e o n l y a p p l i c a t i o n s o f a c e r t a i n r e l e v a n c e a r e t h o s e o b t a i n e d on t r a p s f i l l e d w i t h Gas Chrom Z coated w i t h 10% Carbowax and Chromosorb P c o a t e d w i t h 2% OV 101. The f o r m e r a d s o r b i n g m a t e r i a l was exten-

236

s i v e l y used by Lonnemann e t a l . ( 6 8 ) f o r t h e c r y o g e n i c enrichment o f C2-C8

HC

c o l l e c t e d by " g r a b sampling" i n s e v e r a l urban and suburban areas. A f t e r t h e r e moval o f condensed oxygen, t h e c o n c e n t r a t e was f r a c t i o n a t e d on a 100 m c a p i l l a r y column coated w i t h d i b u t y l m a l e a t e . T h i s method, used i n c o m b i n a t i o n w i t h

those developed by t h e same a u t h o r s f o r t h e a n a l y s i s o f C2-C5

and C6-C10HC

,

p e r m i t t e d t h e i d e n t i f i c a t i o n o f more t h a n 52 s p e c i e s i n s e v e r a l hundreds atmos p h e r i c samples coming f r o m d i f f e r e n t s i t e s . Chromosorb P coated w i t h a s i l i c o n o i l was recommended by Concawe's S p e c i a l Task Force on Odours ( 1 5 ) f o r t h e m o n i t o r i n g o f C5-C12 areas. The vapours, adsorbed a t -70°C,

HC i n r e l a t i v e l y p o l l u t e d

can be r e c o v e r e d by h e a t i n g t h e t r a p w i t h

h o t water. A s u i t a b l e f r a c t i o n a t i o n o f t h e v a r i o u s c o n s t i t u e n t s i s o b t a i n e d on a 10 m column packed w i t h t h e same m a t e r i a l used f o r c o n c e n t r a t i n g t h e a i r sample. The methods j u s t d e s c r i b e d g i v e an e x h a u s t i v e o v e r v i e w o f t h e d i f f e r e n t approaches which have been f o l l o w e d i n t h e a t t e m p t o f e l u c i d a t i n g t h e HC

composi-

t i o n i n t h e atmosphere. They a r e i n t e n d e d t o be o f g e n e r a l a p p l i c a t i o n i n t h e sense t h a t s e v e r a l compounds e x e r t i n g a d e f i n i t e a c t i o n on t h e environment can be s i m u l t a n e o u s l y evaluated. I f necessary, t h e s e methods can be t a i l o r e d , w i t h 1 i t t l e m o d i f i c a t i o n s , t o t h e m o n i t o r i n g o f c e r t a i n compounds o f s p e c i f i c i n t e r e s t . I t i s s u f f i c i e n t t o r e c a l l h e r e t h a t c r y o g e n i c t r a p f r e e z i n g (122) or a d s o r p t i o n on GCB (123, 124) and porous polymers (125) were found p a r t i c u l a r l y s u i t a b l e f o r t h e enrichment o f c h l o r o f l u o r o c a r b o n s suspected t o cause d e p l e t i o n o f s t r a t o s p h e r i c ozone, whereas Durapack n - o c t a n e / P o r a s i l C (122) and GCB (124, 125) columns p r e f e r r e d f o r t h e i r s e p a r a t i o n . Adequate s e n s i t i v i t y and s e l e c t i v i t y t o d e t e c t p p t v l e v e l s o f t h e s e c o n s t i t u e n t s i n a i r masses was p r o v i d e d by

t h e ECD (126) e s p e c i a l l y i f designed f o r c o u l o m e t r i c work (127, 128). GC-MS r u n n i n g i n S I D was, i n s t e a d , mandatory f o r t h e m o n i t o r i n g o f halocarbons i n r u r a l atmospheres o r i n t h e l o w e r t r o p o s p h e r e (129, 1 3 2 ) . I n s p i t e o f t h e n o t i c e a b l e d i f f e r e n c e i n t h e procedures f o l l o w e d and t h e occ u r r e n c e of p o s s i b l e sampling and a n a l y t i c a l a r t i f a c t s , t h e r e s u l t s shown by t h e v a r i o u s a u t h o r s a r e s u b s t a n t i a l l y i n f a i r agreement. The degree o f accuracy o f t h e e x i s t i n g d a t a d e f i n i t e l y improved t h e knowledge o f HC c y c l e i n t h e atmosphere and p e r m i t t e d t h e i d e n t i f i c a t i o n o f t h e i r p r i n c i p a l sources and s i n k s . I t has t o be noted, however, t h a t o n l y 70-80% o f t h e compounds measured i n an-

thropogenic e m i s s i o n sources have been a c t u a l l y f o u n d i n ambient a i r and o n l y a m i n o r number o f p r o d u c t s e m i t t e d f r o m v e g e t a t i o n have been i d e n t i f i e d and quant i t a t e d ( 1 3 3 ) . T h i s suggests t h a t e i t h e r t h e s e n s i t i v i t y o f t h e a n a l y t i c a l methods o r t h e accuracy o f t h e sampling procedure a r e s t i l l i n a d e q u a t e f o r a comp r e h e n s i v e knowledge o f t h e o r g a n i c s p e c i e s r e l e a s e d o r formed i n t h e atmosphere.

237

5.1

CARBON MONOXIDE Carbon monoxide i s t h e most abundant and ccmmonly o c c u r r i n g a i r p o l l u t a n t

as l a r g e amounts o f t h i s gas a r e produced a n t h r o p o g e n i c a l l y by i n c o m p l e t e combus t i o n o f f o s s i l fuels,

i n d u s t r i a l processes and b u r n i n g of waste m a t e r i a l . A m i -

n o r source i s n a t u r a l emission. It has t o be s t r e s s e d t h a t t h e impact o f s u r f a c e anthropogenic e m i s s i o n i s

n o t r e s t r i c t e d t o t h e boundary l a y e r as t u r b u l e n t m i x i n g can t r a n s p o r t carbon monoxide t o an a l t i t u d e as h i g h as s e v e r a l k i l o m e t e r s from t h e s u r f a c e . I n t h e upper atmosphere and under c e r t a i n c o n d i t i o n s i n t h e l o w troposphere, CO c a n be o x i d i z e d t o C02 by OH r a d i c a l s . Since t h e m a j o r reason f o r t h e atmospheric l e v e l o f CO c o n c e n t r a t i o n i s man's a c t i v i t y , t h i s v a l u e can be r e l a t e d t o t h e technol o g i c a l achievement which has been reached. 5.2.

Chromatographic d e t e r m i n a t i o n o f CO

A s u i t a b l e measure o f CO i n a i r , down t o 0.1 ppmvlevels, can be o b t a i n e d w i t h a gas chromatographic apparatus s i m i l a r t o t h a t d e s c r i b e d i n s e c t i o n 4.2. The a i r sample i s d i r e c t l y i n j e c t e d i n t o a column t o o b t a i n t h e s e p a r a t i o n o f CO from CH4 and LO2. A c a t a l y t i c c o n v e r t e r , p l a c e d a t t h e column o u t l e t , reduces

CO q u a n t i t a t i v e l y t o methane a l l o w i n g i t s d e t e c t i o n b y FID ( 1 3 4 ) . T h e system has a l i n e a r o u t p u t o f up t o 1,000 ppmv and p e r m i t s t h e m o n i t o r i n g o f CO i n p o l l u t e d areas as w e l l as i n r e l a t i v e l y c l e a n l o c a t i o n s .

P. l i n e a r response and a d e t e c t i o n l i m i t o f 0.5 ppm can a l s o be o b t a i n e d by d i r e c t i n j e c t i o n o f t h e sample i n t o a 2 m column packed w i t h m o l e c u l a r s i e v e s 5A connected t o a Helium I o n i z a t i o n d e t e c t o r (135 ) . T h i s method enables CO det e r m i n a t i o n e v e r y 15 m i n u t e s ;

t h e procedure i s a f f e c t e d b y water, which may

d e a c t i v a t e t h e column, and by t r a c e s o f gaseous components which a f f e c t t h e det e c t o r response. S i n c e t h e m a j o r l i m i t a t i o n i s due t o i n t e r f e r e n c e o f t h e n i t r o gen peak which o v e r l a p s w i t h t h e e l u t i o n o f CO, Marenco and Delaunay (136) modif i e d t h e gas chromatographic system b y i n s e r t i n g a s h o r t precolumn. The i n s t r u m e n t i s designed t o work as a two-dimensional gas chromatograph and p e r m i t s t h e d e t e c t i o n o f CO and Xenon ( w h i c h i s e l u t e d f i r s t ) w i t h o u t due i n t e r f e r e n c e o f n i t r o g e n . The system has been made f u l l y a u t o m a t i c b y c o n t r o l l i n g a l l operations ( i . e .

sampling, i n j e c t i o n i n t o t h e precolumn, h e a r t - c u t t i n g ,

f r a c t i o n a t i o n i n t o t h e main column, b a s e l i n e r e s e t , r e c o r d i n g o f CO and comparison w i t h c a l i b r a t i o n s t a n d a r d s ) by a programmable computer. D e t e c t i o n l i m i t o f

t h e a u t o m a t i c a n a l y z e r f o r CO i s b e t t e r t h a n 1 p p b r w i t h a p r e c i s i o n o f 1.5% and an accuracy o f about 2.5%. Another a u t o m a t i c method f o r measuring CO a t ambient l e v e l s has been developed by Goldan e t a l . ( 1 3 7 ) . I t c o n s i s t s o f a two-dimension a l GC f o r s e p a r a t i n g halocarbons and COP f r o m methane and CO. The f o r m e r a r e r e t a i n e d on a Poropak Q precolumn whereas t h e l a t t e r a r e i n j e c t e d , f r a c t i o n a t e d i n t o a m o l e c u l a r s i e v e column and CD i s d e t e c t e d b y a 1420-sensitized ECD.

238

Enhanced s e n s i t i v i t y f o r C O Y which p e r m i t s d e t e c t i o n l i m i t s as l o w as 1

pg w i t h 2% p r e c i s i o n , i s o b t a i n e d by c a t a l y t i c c o n v e r s i o n o f CO t o C02 i n t h e p r e sence o f N20 on t h e h o t d e t e c t o r w a l l s . A l t h o u g h e x p e n s i v e and r a t h e r c o m p l i c a ted, t h e s e i n s t r u m e n t r e p r e s e n t t h e b e s t achievements i n t h i s f i e l d f o r as t h e y a r e c o m p e t i t i v e w i t h o t h e r s o p h i s t i c a t e d chemical methods and f u l l y s p e c i f i c .

6.1

PHOTOCHEMICAL OXIDANTS W i t h t h e t e r m photochemical o x i d a n t s a r e designed t h o s e p r o d u c t s formed du-

r i n g a c o m p l i c a t e d sequence o f sun1 i g h t - i n d u c e d o x i d a t i o n processes o c c u r r i n g when c e r t a i n species i n d i c a t e d as p r e c u r s o r s a r e r e l e a s e d i n t o t h e atmosphere. The i n c r e a s e i n c o n c e n t r a t i o n o f photochemical o x i d a n t s above c e r t a i n l e v e l s i s r e p o r t e d as a "photochemical smog" episode. The occurrence o f t h i s e v e n t i s ass o c i a t e d w i t h p h y s i c a l , b i o l o g i c a l and chemical e f f e c t s such as r e d u c t i o n o f v i sibility,

i r r i t a t i o n o f eyes and t h r o a t and w e a t h e r i n g o f m a t e r i a l s ( e s p e c i a l l y

r u b b e r ) . On t h e chemical v i w x p o i n t , photochemical smog r e s u l t s i n a s u b s t a n t i a l i n c r e a s e i n t h e c o n c e n t r a t i o n o f ozone, aldehydes, hydrogen peroxyde and p a r t i c u l a t e m a t t e r and w i t h t h e f o r m a t i o n o f a f a m i l y o f compounds whose more import a n t t e r m i s peroxy a c e t y l n i t r a t e (PAN). "Smog chamber" experiments c a r r i e d w i t h o l e f i n s and n i t r o g e n o x i d e s i n t h e presence o f U.V.

r a d i a t i o n y i e l d v a r i o u s compounds such as chetones, b i a c e t i l ,

g l y o x a l (138) ; w i t h a1 k y l benzene a1 so n i t r o p h e n o l and n i t r o c r e s o l have been observed. These i n v e s t i g a t i o n s suggested t h a t photochemical smog o c c u r s when s u b s t a n t i a l amounts o f hydrocarbons HC and n i t r o g e n o x i d e s a r e r e l e a s e d i n t h e atmosphere i n t h e presence o f i n t e n s e s o l a r r a d i a t i o n . Photochemical smog e p i s o des t o a v a r i o u s e x t e n t t a k e p l a c e almost i n e v e r y p a r t o f t h e w o r l d i n h o t and sunny days. The f o r m a t i o n o f atmospheric o x i d a n t s , l i k e most p h o t o l i t i c r e a c t i o n s proceeds m a i n l y t h r o u g h r a d i c a l s i n t e r m e d i a t e s . A s i m p l i f i e d diagram i l l u s t r a t i n g t h e f o r m a t i o n o f photochemical o x i d a n t s and t h e O3 b u i l d - u p i n t h e atmosphere i s shown i n F i g u r e 3. Among t h e v a r i o u s compounds shown i n t h e f i g u r e , aldehydes and PAN c e r t a i n l y r e p r e s e n t t h e most i m p o r t a n t c l a s s e s o f compounds formed by photochemical r e a c t i o n s which can be m o n i t o r e d by GC. The m o n i t o r i n g o f adehydes i s i m p o r t a n t because t h e i r photochemical decomposition l e a d s t o an i n c r e a s e o f t h e OH r a d i c a l s and t h e r e f o r e t h e i r

concentrations

nay

be

i n t e r p r e t e d as an i n d i r e c t

measurement o f t h e atmospheric r e a c t i v i t y . PAN, which i s p h y t o x i c , can be d e t r i mental t o p l a n t growth and causes f o r e s t s d e g r a d a t i o n as i t can be t r a n s p o r t e d as f a r as s e v e r a l k i l o m e t e r s f r o m t h e emission source.

239

T i g u r e 3.

6.2

S i m p l i f i e d diagram showing t h e f o r m a t i o n o f photochemical o x i d a n t s i n t h e atmosphere.

Chromatographic Techniques f o r t h e D e t e r m i n a t i o n o f Aldehydes and PAN The d e t e r m i n a t i o n o f aldehydes ( e s p e c i a l l y formaldehyde and a c e t a l d e h y d e )

by chromatographic techniques i s a r e l a t i v e l y r e c e n t approach and i s p r e f e r r e d because i s v e r y s e n s i t i v e , s p e c i f i c , a c c u r a t e and a l l o w s t h e i n d i v i d u a l q u a n t i t a t i o n o f d i f f e r e n t compounds i n a s i n g l e r u n . The aldehydes p r e s e n t i n a i r a r e u s u a l l y sampled i n a l i q u i d s o l u t i o n (139) o r t h r o u g h a s o l i d a d s o r b e n t s ( 1 4 0 ) c o n t a i n i n g 2-4 n i n i t r o p h e n y l h y d r a z i n e (DNPH). Q u a n t i t a t i v e c o n v e r s i o n i n t o t h e c o r r e s p o n d i n g dinitrophenyl-hydrazones i s o b t a i n e d on a a c i d s u b s t r a t e ( s u l p h u r i c o r phosphoric a c i d ) k e p t a t pH 2.4;

t h e a n a l y s i s i s t h e n c a r r i e d o u t by GC

o r HPLC. A s a t i s f a c t o r y GC s e p a r a t i o n has been o b t a i n e d on a 1.5 m column packed w i t h Chromosorb

AW-HMDS coated w i t h 4% DV 17 (139) b y u s i n g as a d e t e c t o r a FID.

The a n a l y s i s o f a i r samples, c o l l e c t e d f r o m a u t o m o b i l e exhausts, show a s a t i s f a c t o r y s e p a r a t i o n o f aldehydes f r o m C, t o Cl0.

I n spite o f i t s simplicity,

t h e GC t e c h n i q u e has some l i m i t a t i o n s due t o t h e p a r t i a l d e c o m p o s i t i o n o f some d e r i v a t i v e s and t h e r a p i d d e t e r i o r a t i o n o f t h e column. F o r t h e s e reasons HPLC i s now p r e f e r r e d

f o r t h e d e t e r m i n a t i o n o f DNPH d e r i v a t i v e s . T h e a n a l y s i s

o f t h e d e r i v a t i z e d sample can be performed on a s t a n d a r d DDS column ( 2 5 cm x 4 mm) u s i n g a water-methanol m i x t u r e (35%, 65%) as e l u e n t and a U.V.

detector set

a t 254 nm. The s e n s i t i v i t y o f t h i s method a l l o w s t h e q u a n t i t a t i o n o f formaldehy-

240

de down t o p p t v l e v e l s . Measurements o f aldehydes by HPLC have been r e p o r t e d i n urban c i t i e s as w e l l i n t h e t r o p o s p h e r e up t o 8 Km ( 1 4 1 ) . A GC method i n t e n d e d f o r t h e d e t e r m i n a t i o n o f a c r o l e i n , a s t r o n g eyes and t h r o a t i r r i t a n t formed d u r i n g photochemical smog, and formaldehyde has been r e c e n t l y d e s c r i b e d by Kennedy e t a l . ( 1 4 2 ) . A c r o l e i n p r e s e n t i n t h e atmosphere a t p p b v l e v e l s i s made t o r e a c t w i t h 2-(hyd r o x y m e t h y l ) p i p e r i d i n e supported on a XAD-2 r e s i n . The r e a c t i o n p r o d u c t s a r e e x t r a c t e d f r o m t h e c a r t r i d g e by t o l u e n e and separated f r o m formaldehyde and acet a l d e h y d e d e r i v a t i v e s on a 2 m column packed w i t h 5% SP-2401-DB on S u p e l c o p o r t . Thermoionic d e t e c t i o n s p e c i f i c f o r n i t r o g e n compounds i s used t o d i f f e r e n t i a t e a c r o l e i n and formaldehyde d e r i v a t i v e s from t h e o t h e r s p e c i e s c a p t u r e d hy t h e trap. The 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 o f PAN i n a i r a t ppbv l e v e l s can be accomplished by d i r e c t i n j e c t i o n o f 2-5 cc o f a i r i n t o a 2 m column packed e i t h e r w i t h Chromosorb WHP coated w i t h 10% Carbowax 600 o r w i t h any s t a t i o n a r y phase o f s i m i l a r p o l a r i t y . P e r o x y a c e t y l n i t r a t e and p e r o x y p r o p i o n i l n i t r a t e a r e c o m p l e t e l y separated f r o m CC14 and o t h e r c h l o r i n a t e d hydrocarbons, CH3N03 and w a t e r ( 1 4 3 ) . S e l e c t i v e d e t e c t i o n o f PAN i s accomplished by u s i n g an e l e c t r o n - c a p t u r e d e t e c t o r working e i t h e r i n t h e p u l s e d - f r e q u e n c y o r c o n s t a n t - c u r r e n t mode. The d e t e c t o r response i s l i n e a r and good r e p r o d u c i b i l i t y i s o b t a i n e d f r o m ppmvtu p p b v l e v e l s . I t i s r e q u i r e d , however, t o keep t h e o p e r a t i n g temperature o f t h e column and

t h e d e t e c t o r i n t h e range o f 50°C t o p r e v e n t thermal decomposition o f PAN. D i sappearence o f PAN peak above 11)O"C can be used as d i a g n o s t i c means t o c o n f i r m t h e i d e n t i t y o f t h i s compound. F i g . 4a shows a t y p i c a l chromatographic r u n o b t a i n e d d u r i n g t h e d i r e c t anal y s i s o f a i r sampled i n a suburban a r e a 30 Km downwind o f Rome. F i g .

4b shows

t h e d i u r n a l v a r i a t i o n o f O3 and PAN measured i n t h e same s i t e d u r i n g a two days photochemical smog episode when t h e O3 c o n c e n t r a t i o n exeeded 1 0 0 p p b v . T h e r e s u l t s shown i n t h i s F i g u r e e v i d e n t i a t e c l e a r l y t h e c l o s e dependence between O3 b u i l t - u p and PAN p r o d u c t i o n . D e t e c t i o n by e l e c t r o n - c a p t u r e has been e x t e n s i v e l y used f o r t h e m o n i t o r i n g o f PAN i n d i f f e r e n t c o u n t r i e s i n t h e l a s t decade, (144) and t h e r e s u l t s o b t a i n e d i n d i c a t e t h a t t h e r e p o r t e d procedure i s q u i t e r e l i a b l e a l t h o u g h some problems may a r i s e i n t h e p r e p a r a t i o n o f standard m i x t u r e s necessary f o r an a c c u r a t e c a 1 i b r a t i o n o f t h e system ( 1 4 5 ) . C r y o t r a p p i n g t e c h n i q u e (146, 147) combined w i t h GC and ECD has been d e v e l o ped f o r t h e d e t e r m i n a t i o n o f PAN

i n remote areas and i n t h e t r o p o s p h e r e up t o

an a l t i t u d e o f 6 Km where t h e average c o n c e n t r a t i o n i s i n t h e p p t v range. A l t e r n a t i v e l y t o t h i s method, a chemiluminescent d e t e c t o r f o r NO has been r e c e n t l y m o d i f i e d f o r t h e a n a l y s i s o f PAN ( 1 4 8 ) . The e l u a t i s f r o m t h e column, pass o v e r a GCB-molibdenum c a t a l y s t k e p t a t 330°C i n o r d e r t o q u a n t i t a t i v e l y

241 c o n v e r t PAN i n t o NO, which i s t h u s sensed by t h e d e t e c t o r . A l t h o u g h t h e c h e m i l u minescent response i s l i n e a r and can be rendered h i g h l y s p e c i f i c , t h e s e n s i t i v i t y i s one o r d e r of magnitude l o w e r t h a n t h a t o b t a i n e d by u s i n g a ECD.

I n s p i t e o f t h e s e s u c c e s s f u l a p p l i c a t i o n s , t h e p o t e n t i a l i t y o f chromotograp h i c t e c h n i q u e f o r t h e d e t e r m i n a t i o n o f photochemical o x i d a n t s i s s t i l l l a r s e l y unexplored and new methods need t o be developed f o r 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 o f many compounds found i n "smog chamber" experiments b u t n o t y e t measured i n t h e atmosphere.

F i g u r e 4.

7.1

a ) GC a n a l y s i s o f p e r o x i l n i t r a t e s i n a i r ; t h e c o n c e n t r a t i o n o f PAN r e p o r t e d i n t h e f i g u r e corresponds t o 4 ppbv. b ) d i u r n a l v a r i a t i o n o f O3 and PAN observed d u r i n g a photochemical smog episode.

PARTICULATE MATTER P a r t i c u l a t e m a t t e r i s , by f a r , t h e more complex p o l l u t a n t s as, i n g e n e r a l

sense, i t may c o n t a i n any s o l i d o r l i q u i d s p e c i e s d i s p e r s e d i n t h e atmosphere. From a p h y s i c a l stand p o i n t , i t c o v e r s a l l m a t e r i a l m o l e c u l a r l y d i s p e r s e d w i t h a s i z e range between 10 and 0.01 pm. I t i s w e l l known t h a t a e r o s o l s f o r m i n two ways: by c o n d e n s a t i o n i n t o p r i m a r y p a r t i c l e s , which c o a g u l a t e t o f o r m aggregat e s o r t h r o u g h chemical r e a c t i o n s between v o l a t i l e substances, which y i e l d an homogeneous n u c l e a t i o n w i t h t h e g r o w t h o f n u c l e i which i n t u r n can c o a g u l a t e . Major a n t h r o p o g e n i c sources o f p a r t i c u l a t e m a t t e r a r e a s s o c i a t e d w i t h b u r n i n g o f f o s s i l f u e l s and r e s i d e n t i a l f i r e p l a c e s , a u t o m o b i l e s exhaust, r u b b e r t y p e wear as w e l l w i t h a v a r i e t y o f i n d u s t r i a l a c t i v i t i e s . F i n e p a r t i c l e s a r e a l s o produced d u r i n g photochemical smog episodes. Volcanoes, wood f i r e s f r o m f o r e s t and m a r i n e a e r o s o l s a r e t h e m a j o r n a t u r a l sources f o r p a r t i c u l a t e m a t t e r . A e r o s o l s i n f l u e n c e t h e weather, p a r t e c i p a t e t o chemical r e a c t i o n s i n t h e atmosphere and have adverse h e a l t h e f f e c t s . On occount o f t h e v a r i e t y o f t h e r e a c t i o n s i n which p a r t i c u l a t e m a t t e r i s i n v o l v e d , a tremendous amount o f work has been c a r r i e d o u t t o i n v e s t i g a t e i t s chemical f e a t u r e s and chromatographic

242

techniques have been found t o be v a l u a b l e means t o e l u c i d a t e i t s c o m p o s i t i o n namely i n terms o f those compounds which may have a d e f i n i t e b i o l o g i c a l i m p a c t . The more i m p o r t a n t f a m i l i e s o f compounds p r e s e n t i n t h e o r g a n i c f r a c t i o n o f a i r b o r n e p a r t i c u l a t e m a t t e r a r e t h e p o l y n u c l e a r a r o m a t i c hydrocarbons (PAH) ( i n c l u d i n g n i t r a t e d and oxygenated PAH) and t h e c h l o r i n a t e d s p e c i e s such as c h l o r o d i b e n z o d i o x i n s , c h l o r o d i b e n z o f u r a n s and p o l y c h l o r i n a t e d b y p h e n i l s (PCB ) . A l k a nes i n general and p a r t i c u l a r l y c i c l i c alkanes, p h t a l a t e e s t e r s and f a t t y a c i d s a r e o t h e r c l a s s e s which have been i n v e s t i g a t e d m a i n l y w i t h t h e aim t o i d e n t i f y t h e e m i s s i o n source o f p a r t i c u l a t e m a t t e r .

A g e n e r a l procedure f o r t h e a n a l y s i s p a r t i c u l a t e m a t t e r r e q u i r e s t h e f o l l o wing s t e p s 1 ) Sampling o n f i l t e r s , adsorbents o r a c o m b i n a t i o n o f b o t h

2) E x t r a c t i o n o f t h e s o l u b l e o r g a n i c f r a c t i o n (SOF) w i t h s o l v e n t s 3) P r e f r a c t i o n a t i o n o f t h e SOF i n t o s e p a r a t e c l a s s e s by means o f l i q u i d and t h i n l a y e r chromatography o r l i q u i d p a r t i t i o n

4) D e t e r m i n a t i o n o f t h e c o n s t i t u e n t s p r e s e n t i n each c l a s s by h i g h r e s o l u t i o n GC o r LC u s i n g s p e c i f i c d e t e c t i o n .

7.2

Sampling o f p a r t i c u l a t e m a t t e r Sampling o f p a r t i c u l a t e m a t t e r i s u s u a l l y c a r r i e d o u t on g l a s s and q u a r t z

f i b e r f i l t e r s connected t o a h i g h volume pump. The procedure i s n o t f r e e f r o m a r t i f a c t s due t o t h e v o l a t i l i z a t i o n o f t h e l o w b o i l i n g components (149, 150), so t h a t t r a p s packed w i t h Tenax GC (151), Carbopack B ( 1 5 2 ) , o r p o l y m e r i c r e s i n s such as p o l y u r e t a n e foam (153) o r XAD r e s i n s (151) have been p l a c e d i n s e r i e s t o t h e f i l t e r t o r e t a i n t h e compounds e v e n t u a l l y r e l e a s e d . Another sampling a r t i f a c t may a r i s e f r o m t h e prolonged exposure o f c o l l e c t e d p a r t i c u l a t e d m a t t e r t o some a i r p o l l u t a n t s such as ozone, and n i t r o g e n d i o x i d e (154). A l t h o u g h experiments c a r r i e d o u t i n s i m u l a t e d atmospheres (155, 156) c o n t a i n i n g h i g h l e v e l s o f t h e s e compounds i n d i c a t e t h e o c c u r r e n c e o f t h e s e r e a c t i o n s , i t i s d i f f i c u l t t o e v a l u a t e t o what e x t e n t t h e s e e f f e c t s may change t h e q u a l i t y

of t h e sample. However t h e s e l i m i t a t i o n s m i g h t be overcome by p e r f o r m i n g t h e samp l i n g by means o f t h e

7.2.2

denuders p r e v i o u s l y d e s c r i b e d .

E x t r a c t i o n o f S o l u b l e Organic F r a c t i o n (SOF) E x t r a c t i o n o f p a r t i c u l a t e m a t t e r can be c a r r i e d o u t i n a So>.hlet apparatus

u s i n g t e t r a h y d r o f u r a n , methanol, methanol-benzene m i x t u r e s , benzene, acetone, cyclohexane (157) and methylene c h l o r i d e (158) which a r e r e p o r t e d t o be n e a r l y 100% e f f i c i e n t i n t h e e x t r a c t i o n o f a1 kanes and PAH. Since methylene c h l o r i d e

and cyclohexane have a s l i g h t l y l o w e r e f f i c i e n c y t h a n benzene, b u t a r e l e s s hazardous, t h e i r use has been endorsed by s e v e r a l o f f i c i a l agencies. Methanol

243

and benzene-methanol m i x t u r e s have been p r e f e r r e d when p o l a r o r g a n i c f r a c t i o n s (such as oxygen and n i t r o g e n c o n t a i n i n g PAH) need t o be r e c o v e r e d q u a n t i t a t i v e l y ( 1 5 2 ) . U l t r a s o n i c v i b r a t i o n e s p e c i a l l y i n t h e presence o f h y d r o f l u o r i c a c i d ,

i s an e f f i c i e n t a l t e r n a t i v e t o S o x h l e t e x t r a c t i o n b u t i t s u s e i s l i m i t e d t o c e r t a i n components and t o t h o s e m a t r i c e s which do n o t s t r o n g l y adsorb t h e o r g a n i c m a t e r i a l (159). Vacuum s u b l i m a t i o n (160) has been a l s o a t t e m p t e d f o r t h e e x t r a c t i o n o f p o l a r and n o n - p o l a r PAH, b u t e v a p o r a t i v e l o s s e s have been observed f o r compounds w i t h t h r e e - f o u r benzenic r i n g s whereas components c o n t a i n i n g more t h a n one oxygen atom were d e f i n i t e l y degradated. S p e c i a l a t t e n t i o n has t o be p a i d i n t h e S o x h l e t e x t r a c t i o n when c h l o r i d i b e n z o d i o x i n s (PCDD) and c h l o r o d i b e n z o f u r a n s (PCDF) need t o be d e t e r m i n e d as t h e s e compounds can be a c t u a l l y formed f r o m PCB and c h l o r o p h e n o l s i f t h e t e m p e r a t u r e i s t o o h i g h and a l o n g e x t r a c t i o n t i m e i s used. E x t r a c t i o n t e m p e r a t u r e s a t about 100°C, t h e u s e o f benzene as e l u e n t and an e x t r a c t i o n t i m e n o t exceeding 16 hours can p r e v e n t a r t i f a c t u a l problems a r i s i n g f r o m u n d e s i r e d c o n d e n s a t i o n reactions.

7.2.3

P r e f r a c t i o n a t i o n o f S o l u b l e Organic F r a c t i o n

There i s no

u n i q u e procedure f o r t h e p r e f r a c t i o n a t i o n o f t h e SOF e x t r a c -

t e d f r o m a i r b o r n e p a r t i c u l a t e m a t t e r as t h e i s o l a t i o n o f a s p e c i f i c c l a s s o f c o w pounds i s n o t always c o m p a t i b l e w i t h an e f f i c i e n t s e p a r a t i o n o r f u l l r e c o v e r y o f t h e o t h e r c o n s t i t u e n t s . I n p r a c t i c e , i t has t o be decided, i n advance, what a r e t h e s p e c i e s o f a c e r t a i n i n t e r e s t and f u r t h e r l y t o d e s i g n a s e p a r a t i o n scheme s p e c i f i c a l l y t a i l o r e d t o t h e d e t e r m i n a t i o n o f t h e s e l e c t e d components. More species have t o be determined, more complex i s t h e s e p a r a t i o n scheme t o be adopt e d and, consequently, h i g h e r a r e t h e sample l o s s e s which may t a k e

place.

Since airborne p a r t i c u l a t e c o n t a i n predominantly neutral materials, t h e steps i n v o l v i n g s e p a r a t i o n o f s t r o n g a c i d i c and b a s i c components by l i q u i d p a r t i t i o n can, i n many i n s t a n c e s , be avoided and a s i m p l i f i e d procedure adopted. I f t h e i n t e r e s t i s focused on source-immission c o r r e l a t i o n s and t h e f i n g e r p r i n t a n a l y s i s o f t h e m a j o r components can be c a r r i e d o u t e f f e c t i v e l y on h i g h r e s o l u t i o n GC columns connected t o MS c a p a b l e t o y i e l d mass fragmentograms, i t i s even p o s s i b l e t o e l i m i n a t e t h e p r e f r a c t i o n a t i o n s t e p ( 1 6 1 ) . The g e n e r a l procedure f o r t h e s e p a r a t i o n o f t h e n e u t r a l f r a c t i o n s o f e n v i r o n mental samples i n v o l v e s s e p a r a t i o n on adsorbents w o r k i n g i n b o t h s t e r i c and ads o r p t i o n mode. B o t h t h i n - l a y e r (162, 163) and l i q u i d column (164, 165) chromatography a r e s u i t a b l e f o r s e p a r a t i n g a l i p h a t i c , a r o m a t i c s and p o l a r compounds on s i l i c a and alumina adsorbents. I f HPLC columns a r e used, d e t e c t i o n o f t h e e l u i t e s can be c a r r i e d o u t b y U.V. a b s o r p t i o n o r induced f l u o r e s c e n c e and t h e f r a c t i o n on i n t e -

244

r e s t i s ready f o r t h e GC o r LC a n a l y s i s . For components h a v i n g s i m i l a r p o l a r i t y (such as PAH, nitro-PAH,

keto-PAH and azaarenes) i t may be necessary t o remove

f i r s t t h e alkanes and p o l a r components from t h e e x t r a c t by l o w r e s o l u t i o n LC,

and

t h e n submit t h e f r a c t i o n o f i n t e r e s t t o a f u r t h e r s e p a r a t i o n on a HPLC

lumn w o r k i n g i n g r a d i e n t e l u t i o n (152,167).

CO-

Up t o f o u r d i f f e r e n t f r a c t i o n s

s u i t a b l e f o r GC o r LC a n a l y s i s can be o b t a i n e d w i t h t h i s procedure. 0

Non aqueous s i z e - e x c l u s i o n chromatography on Bio-beds (167), Spherogel 50 A

(168) o r s i m i l a r m a t e r i a l s i s a n o t h e r u s e f u l t e c h n i q u e f o r t h e p r e f r a c t i o n a t i o n o f SOF as i t p r o v i d e s an e f f i c i e n t s e p a r a t i o n o f p o l y a r o m a t i c compounds a c c o r ding

to

t h e i r r i n g number.

A more e f f i c i e n t method f o r s e p a r a t i n g c l a s s e s o f compounds h a v i n g d i f f e r e n t p o l a r i t y and m o l e c u l a r s i z e i n v o l v e s e l u t i o n on Sephadex LH-20 columns ( 1 6 9 ) . By u s i n g a methanol-hexane m i x t u r e as e l u e n t t h e l i p o p h i l c and h y d r o p h i l i c

f r a c t i o n can be separated from l o w p o l a r components. A f t e r t h e e l u t i o n , p a r a f f i n i c substances can be removed from t h e column w i t h i s o p r o p a n o l whereas p o l y a r o m a t i c compounds can be f u r t h e r separated a c c o r d i n g t o t h e i r r i n g number.

Lee,

Novotny and B a r t l e (170) r e p o r t e d a v e r y e f f i c i e n t f r a c t i o n a t i o n o f p o l y a r o m a t i c compounds e x t r a c t e d from a i r p a r t i c u l a t e s by u s i n g a column packed w i t h Sephadex LH-20. Seven f r a c t i o n s were c o m p l e t e l y separated and c o l l e c t e d by o p e r a t i n g t h e column a t v e r y l o w f l o w r a t e s 2,500 and 3,000 t h e o r e t i c a l

( 6 m l / h ) which had an e f f i c i e n c y r a n g i n g between p l a t e s . The same a u t h o r s have e x t e n s i v e l y reviewed

and d e s c r i b e d a l l f r a c t i o n a t i o n schemes adopted f o r t h e i s o l a t i o n o f polyaromat i c compounds from complex m a t r i c e s ( 1 7 1 ) . F u r t h e r d e t a i l s on t h i s s u b j e c t can be

found i n t h e r e f e r e n c e s c i t e d by these a u t h o r s . Sample clean-up and f r a c t i o n a t i o n schemes o f p o l y c h l o r i n a t e d dibenzo-D- d i o -

xins

(PCDD) and p o l y c h l o r i n a t e d d i b e n z o f u r a n s (PCDF) f o l l o w s two d i f f e r e n t

procedural t r e n d s depending upon t h e t y p e o f i n f o r m a t i o n requested and

the

a n a l y t i c a l s t e p s e l e c t e d ; one, proposed by Buser e t a l . (172), i n v o l v e s t h e s e p a r a t i o n o f PCDD and PCDF f r o m o t h e r c h l o r i n a t e d a r o m a t i c s and p o l a r

compo-

nents. I t i s based on t h e subsequent s e p a r a t i o n o f t h e SOF on s i l i c a and alumina columns.

S e p a r a t i o n on s i l i c a spheres f r a c t i o n a t e s a l l s l i g h t l y - p o l a r c h l o r i -

nated compounds, alkanes and p o l y a r o m a t i c compounds f r o m phenols and o r g a n i c a c i d s whereas t h e e l u t i o n on alumina a l l o w s t h e s e p a r a t i o n o f PCDD, PCDF and PAH from p o l y c h l o r i n a t e d b y p h e n i l s (PCB), c h l o r i n a t e d benzenic HC and p o l y c h l o r o p h e n i l e t h e r s . The f r a c t i o n s e l u t e d f r o m t h e alumina column a r e r e a d y f o r t h e GC

analysis. According t o t h e o t h e r p r e f r a c t i o n a t i o n scheme f o r PCDD a n a l y s i s developed

by Lamparski e t a l . (173), t h e benzene e x t r a c t i s separated on a m u l t i - l a y e r column f i l l e d w i t h s i l i c a and s i l i c a coated w i t h a l k a l i and s u l f u r i c a c i d . I n t e r f e r i n g compounds c o e l u t e d i n t h e PCDD f r a c t i o n a r e d e f i n i t e l y removed b y LC u s i n g two columns: one packed w i t h s i l i c a coated w i t h s i l v e r n i t r a t e and t h e

245

o t h e r w i t h b a s i c alumina. The r e s u l t i n g f r a c t i o n c o n t a i n i n g a l l PCDD i s t h u s ready f o r t h e a n a l y s i s . F r a c t i o n a t i o n o f SOF e x t r a c t e d f r o m t h e adsorbents i n s e r t e d a f t e r t h e f i l t e r can be performed w i t h t h e same procedures adopted f o r p a r t i c u l a t e m a t t e r o r , when i t i s p o s s i b l e , b y thermal d e s o r p t i o n . 7.2.4 Chromatographic D e t e r m i n a t i o n o f t h e Components p r e s e n t i n t h e v a r i o u s Fractions Although t h e d e s c r i b e d procedures f o r t h e p u r i f i c a t i o n and sample enrichment are q u i t e effective t o separate

d i f f e r e n t species from a i r b o r n e p a r t i c u l a t e

m a t t e r , n o t always i t i s p o s s i b l e t o e l i m i n a t e i n t e r f e r e n c e s from u n d e s i r e d components. I t i s

thus

advisable t o perform the analysis by using h i g h r e s o l u t i o n

columns p o s s i b l y connected t o s e l e c t i v e d e t e c t o r s . r y GC f o r t h i s purpose i s now undisputed.

The s u p e r i o r i t y o f c a p i l l a -

Wall-coated o r cross-linked c a p i l l a -

r i e s made o f g l a s s o r f u s e d - s i l i c a o f f e r t h e p o s s i b i l i t y t o r e s o l v e almost any complex m i x t u r e .

For t h e h i g h r e s o l u t i o n which can be reached, many i s o m e r i c

components can be c o m p l e t e l y separated and i d e n t i f i e d .

T h i s aim i s o f a key i m -

p o r t a n c e i n some cases, such as t h e a n a l y s i s o f PCDD and p o l y a r o m a t i c compounds, where t h e d e t e r m i n a t i o n o f s p e c i f i c isomers e x e r t i n g a s t r o n g e r adverse e f f e c t

on human h e a l t h , has t o be c a r r i e d o u t . HPLC

has been a l s o s u c c e s s f u l l y a p p l i e d t o t h e d e t e r m i n a t i o n o f t h e o r g a n i c s

d e p o s i t e d on a i r b o r n e p a r t i c u l a t e m a t t e r .

I t s a p p l i c a t i o n i s , however, more li-

m i t e d than c a p i l l a r y GC because o f t h e lower s e n s i t i v i t y a f f o r t e d by t h e convent i o n a l d e t e c t i o n systems.

On t h e o t h e r hand, HPLC i n general and p a r t i c u l a r l y

reversed-phase l i q u i d chromatography (RPLC) o f f e r s some r e a l advantages which can be summarized as f o l l o w s : t h e q u a l i t y o f t h e f r a c t i o n s c o n t a i n i n g t h e r m a l l y u n s t a b l e components can be f u l l y preserved, t h e s e p a r a t i o n o f a g i v e n m i x t u r e can be t a i l o r e d f o r t h e s o l u t i o n o f a s p e c i f i c problem by s i m p l y changing t h e e l u e n t composition,

t h e i n d i v i d u a l components can be recovered and f u r t h e r ana-

l y z e d by s p e c t r o s c o p i c methods a n d , f i n a l l y , h i g h m o l e c u l a r w e i g h t components which a r e sometimes i r r e v e r s i b l y adsorbed on GC columns, can be e l u t e d and i d e n t i f i e d . The complementary f e a t u r e s o f b o t h t e c h n i q u e s have been found e x t r e m e l y usef u l f o r t h e a n a l y s i s o f what i s c o n s i d e r e d t h e most i m p o r t a n t and complex c l a s s o f compounds p r e s e n t i n t h e SOF o f p a r t i c u l a t e m a t t e r , i . e . t h e f a m i l y o f p o l y a I t i n c l u d e s a wide number o f d i f f e r e n t c o n s t i t u e n t s

r o m a t i c compounds (PAC).

a l l c h a r a c t e r i z e d by t h e presence i n t h e i r m o l e c u l e o f a number o f b e n z e n i c - r i n g s r a n g i n g between 2 and 14.

The importance o f PAC i s r e l a t e d t o t h e f a c t t h a t

some o f them a r e known t o have s t r o n g mutagenic and c a r c i n o g e n i c p r o p e r t i e s (174). perties

The c l o s e r e l a t i o n e x i s t i n g between s t r u c t u r e o f PAC and t h e i r t o x i c pror e q u i r e s t h a t many c o n s t i t u e n t s as p o s s i b l e

positively identified.

have t o be separated and

A l t h o u g h c a r c i n o g e n i c i t y o f PAC has m a i n l y be observed

246

f o r compounds w i t h a number o f r i n g s between 3 and 6, t h e number o f isomers i s so h i g h t h a t t h e maximum r e s o l u t i o n i s i m p e r a t i v e f o r b o t h i d e n t i f i c a t i o n and s e p a r a t i o n purposes. The most abundant PAC p r e s e n t i n a i r b o r n e p a r t i c u l a t e m a t t e r a r e polyaromat i c hydrocarbons (PAH). T h e i r presence i n urban d u s t e x t r a c t s has been e s t a b l i shed as e a r l y as 1964 when L i b e r t i e t a l . (175) r e p o r t e d t h e f i r s t s e p a r a t i o n on a c a p i l l a r y column.

The same a u t h o r s a l s o r e c o g n i z e d t h e importance o f

elec-

t r o n c a p t u r e d e t e c t i o n f o r t h e s e l e c t i v e d e t e r m i n a t i o n o f t h e s e compounds ( 1 7 6 ) . The t e c h n o l o g y o f c a p i l l a r y columns i s grown t o a p o i n t t h a t , today, 209 PAH can be i d e n t i f i e d o n a column s h o r t e r t h a n t h e one used two decades ago. The p o s s i b i l i t y o f u s i n g t h e r e t e n t i o n i n d e x w i t h 95% c o n f i d e n c e i n t e r v a l (177) f o r s t r u c t u -

r a l i d e n t i f i c a t i o n o f PAH i n many complex m i x t u r e s g i v e s an i d e a o f t h e g r e a t power o f c a p i l l a r y GC. for the whereas

F a c t o r s a f f e c t i n g t h e r e s o l u t i o n o f c a p i l l a r y columns

s e p a r a t i o n o f PAH have been c r i t i c a l l y reviewed by Lee e t a l . (178) a more comprehensive i n f o r m a t i o n c o n c e r n i n g t h e occurrence, c h e m i s t r y

and t o x i c o l o g y o f PAH as w e l l as t h e methods f o r t h e i r e x t r a c t i o n , p r e f r a c t i o n a t i o n and a n a l y s i s can b e found i n t h e book w r i t t e n by Lee, Novotny and B a r t l e (171). A wide number o f s e p a r a t i o n s o f PAH e x t r a c t e d f r o m atmospheric d u s t has been

reported

i n t h e l i t e r a t u r e m a i n l y r e p o r t i n g t h e d e t e r m i n a t i o n o f compounds r a n -

g i n g between phenanthrene and coronene. Among them, we would l i k e t o mention those o b t a i n e d

by Lee e t a l . (170), B i d r s e t h e t a l . (179), Grimmer e t a l . ( 1 8 0 ) ,

Giger e t a l . (181) and, more r e c e n t l y , by Ramdahl e t a1.(182), Romanowski e t a l . (104).

N i e l s e n (183) and

These c o n t r i b u t i o n s p r o v i d e a c l e a r p i c t u r e o f t h e ty-

p i c a l d i s t r i b u t i o n o f PAH i n d i f f e r e n t e n v i r o n m e n t a l a i r samples. A d e t a i l e d i n v e s t i g a t i o n on t h e o r g a n i c f r a c t i o n o f p a r t i c u l a t e m a t t e r and t h e r e l a t i o n e x i s t i n g between p a r t c l e s i z e d i s t r i b u t i o n , t o x i c i t y and PAH c o m p o s i t i o n has been c a r r i e d o u t by review

C a u t r e l s e t a1.(185) and B r o d d i n e t a l . ( 1 8 6 ) . An e x c e l l e n t

on t h e same s u b j e c t has been p u b l i s h e d by Van Cauwenberghe e t a l . ( 1 7 3 ) .

A l t h o u g h i t i s recognized t h a t t h e amount and d i s t r i b u t i o n o f PAH i n a i r b o r ne p a r t i c u l a t e m a t t e r e x e r t some d e f i n i t e e f f e c t s on human h e a l t h ( 1 8 7 ) , t h e comp l e x i t y of b i o l o g i c a l f a c t o r s i n v o l v e d i n t h e i r metabolism makes d i f f i c u l t t o def i n e a i r q u a l i t y standards i n terms o f PAH c o n c e n t r a t i o n and t h e i r d e t e r m i n a t i o n i n a i r samples i s more f r e q u e n t l y used t o assess t h e impact o f c e r t a i n anthropog e n i c sources on t h e environment. To r e a c h t h i s aim, i t i s r e q u i r e d t o have i n f o r m a t i o n on t h e s t r u c t u r a l c h a r a c t e r i z a t i o n o f t h e v a r i o u s i s o m e r i c species pres e n t i n a g i v e n sample.

A l t h o u g h some c l u e s f o r i d e n t i f i c a t i o n can be o b t a i n e d

from t h e r e t e n t i o n time, a d d i t i o n a l i n f o r m a t i o n a c q u i r e d by t h e use o f v a r i o u s s p e c t r o m e t r i c techniques and s e l e c t i v e GC d e t e c t i o n i s r e q u i r e d . W h i l e computer a s s i s t e d GC-MS systems can s u p p l y i n f o r m a t i o n upon t h e number o f isomers h a v i n g t h e same number of benzenic r i n g s and a l k y l a t e d s u b s t i t u e n t s (161,188),

electron

capture

d e t e c t i o n p r o v i d e s good s e l e c t i v i t y f o r c e r t a i n

can be i d e n t i f i e d

s p e c i f i c isomers which

a c c o r d i n g t o t h e i r ECD/FID r a t i o ( 1 8 9 ) .

Recently, a d d i t i o n

o f oxygen t o t h e c a r r i e r gas has been found a s u i t a b l e t e c h n i q u e t o e v i d e n t i a t e some

s t r u c t u r a l f e a t u r e s o f PAH d e t e c t e d by e l e c t r o n c a p t u r e d e t e c t i o n ( 1 9 0 ) .

P h o t o i o n i z a t i o n d e t e c t i o n i n c o m b i n a t i o n w i t h FID

can be

f e r e n t i a t e PAH from alkanes and o l e f i n s ( 1 9 1 ) i n t h e SOF.

,

i n s t e a d , used t o d i f -

Less c o n v e n t i o n a l dete-

c t i o n techniques f o r PAH i n c l u d e n e g a t i v e i o n chemical i o n i z a t i o n mass s p e c t r o m e t r y (192,193),

F o u r i e r t r a n s f o r m i n f r a r e d s p e c t r o m e t r y (194), l a s e r induced

m u l t i p h o t o n i o n i z a t i o n mass s p e c t r o m e t r y (195) and f l u o r e s c e n c e s p e c t r o m e t r y on cryogenic i s o l a t e d

m a t r i x (196).

W h i l e t h e f o r m e r two t e c h n i q u e s have been

a p p l i e d t o t h e s t r u c t u r a l c h a r a c t e r i z a t i o n o f PAH i n complex

fractions, mufti-

photon mass s p e c t r o m e t r y has made p o s s i b l e t h e d e t e c t i o n o f c e r t a i n components a t l e v e l s as l o w as ZOO femtograms. Fluorescence s p e c t r o m e t r y on i s o l a t e d m a t r i x has been proven q u i t e s e l e c t i v e t o p e r m i t d i r e c t i d e n t i f i c a t i o n o f some PAH i n t h e SOF. S e p a r a t i o n o f PAH on RPLC columns w o r k i n g i n g r a d i e n t e l u t i o n o r i s o c r a t i c conditions vides

and s e l e c t i v e d e t e c t i o n b y U . V .

induced f l u o r e s c e n c e (197,198) p r o -

a d d i t i o n a l i n f o r m a t i o n on sample c o m p o s i t i o n .

factors

measured

by U.V.

induced f l u o r e s c e n c e , U.V.

By comparing t h e response

a b s o r p t i o n (199) o r peroxy-

o x a l a t e chemiluminescence ( Z O O ) d e t e c t i o n p o s i t i v e i d e n t i f i c a t i o n o f some s p e c i f i c components can be o b t a i n e d . I n t h e l a s t few y e a r s , i n c r e a s i n g a t t e n t i o n has been p a i d on PAC c o n t a i n i n g heteroatoms

i n t h e i r m o l e c u l e and p a r t i c u l a r l y i n t h e f o r m o f n i t r o , c a r b o n y l

and h y d r o x y l groups. the determination o f

A g r e a t amount o f e f f o r t s has been e s p e c i a l l y devoted t o PAH c o n t a i n i n g

n i t r o groups

(nitro-PAH)

as t h e y have

been i d e n t i f i e d i n t h e p a r t i c u l a t e m a t t e r e m i t t e d f r o m d i e s e l engines and a l u m i num

s m e l t e r s and

monella

proven t o g i v e a p o s i t i v e response t o t h e Ames t e s t w i t h S a l -

Typhimurium s t r a i n s .

The

analysis

of

nitro-PAH i n environmental

samples i s a d i f f i c u l t t a s k t o be achieved as these components a r e p r e s e n t a t v e r y low c o n c e n t r a t i o n s and t h e y cannot be c o m p l e t e l y separated f r o m o t h e r i n t e r f e r i n g compounds d u r i n g t h e p r e f r a c t i o n a t i o n s t e p . variety

o f d e t e c t i o n systems has

F o r t h e s e reasons, a wide

been connected t o t h e c a p i l l a r y columns t o

a l l o w a s e l e c t i v e d e t e r m i n a t i o n o f n i t r o - P A H i n t h e presence o f i n t e r f e r i n g species.

They i n c l u d e e l e c t r o n c a p t u r e ( 1 6 6 ) , a l k a l i f l a m e (152,166,201),

t h e r m o i o n i c (202,203)

and chemiluminescence d e t e c t i o n u s i n g thermal energy

a n a l y z e r s (TEA) (204,205).

W h i l e t h e ECD can p r o v i d e an a c c u r a t e d e t e r m i n a t i o n

o f n i t r o - P A H i n v e r y d i l u t e d samples which do n o t c o n t a i n o t h e r s t r o n g e l e c t r o p h i l l i c PAC i n a p p r e c i a b l e amounts, nitrogen-phosphorus d e t e c t o r s (NPD) have t o be

p r e f e r r e d f o r t h e a n a l y s i s o f more c o n c e n t r a t e d samples which a r e d i f f i c u l t

t o be p r e f r a c t i o n a t e d by LC.

An example o f t h e s e l e c t i v e d e t e r m i n a t i o n by NPD

o f n i t r o - P A H e x t r a c t e d f r o m an atmospheric sample c o l l e c t e d i n a suburban a r e a

248

i s shown i n F i g u r e E

1

2 5

I

’

1

0

LLI t(min) I

I

I

30

20

10

4’0

F i g u r e 5. GC a n a l y s i s o f t h e HPLC f r a c t i o n c o n t a i n i n g n i t r o - P A H e x t r a c t e d f r o m a sample c o l l e c t e d i n a suburban area. The compounds were d e t e c t e d by an a1 k a l i f l a m e d e t e c t o r . 1 ) n i t r o n a p h t a l e n e , Z ) n i t r o f l u o r e n e , 3 ) n i t r o a n thracene ,4) n i t r o f 1u o r a n t hene, 5) unknown ,6) 1 - n i t r o p y r e n e ( 206).

A t t h e p r e s e n t time, chemiluminescence u s i n g TEA appears t o be t h e most p r o m i s i n g GC d e t e c t i o n method

f o r nitro-PAH

as i t p r o v i d e s s u f f i c i e n t s e n s i t i v i t y

f o r d e t e c t i n g components p r e s e n t a t t r a c e l e v e l s (10-20 pg) and i s r e l a t i v e l y f r e e from interferences. sitivity

Good performances i n terms o f s e l e c t i v i t y and sen-

can be a l s o o b t a i n e d

by c o n n e c t i n g c a p i l l a r y columns t o a MS opera-

t i n g i n e l e c t r o n impact (152,207,209)

as w e l l as i n n e g a t i v e i o n chemical i o n i -

z a t i o n u s i n g methane as r e a g e n t gas (166,207,210,211). t a t i v e e v a l u a t i o n o f nitro-PAH decomposition can

I n any case, t h e q u a n t i -

i n atmospheric samples i s r a t h e r d i f f i c u l t as

e a s i l y occur d u r i n g a l l the a n a l y t i c a l steps i n c l u d i n g the

f i n a l GC s e p a r a t i o n ( 2 1 2 ) .

Since t h e a d d i t i o n o f d e u t e r a t e d s p e c i e s can o n l y

p a r t l y a l l e v i a t e such a r t i f a c t u a l problems, Campbell e t a l . (213) have proposed an a l t e r n a t i v e method where n i t r o - P A H a r e f i r s t reduced w i t h KBH4 and t h e n d e r i vatized w i t h pentafluoropropionic anhydride.

This l a s t step y i e l d s products

which can be submitted t o GC a n a l y s i s on c a p i l l a r y columns and e a s i l y i d e n t i fied

by

GC-MS

o r s e l e c t i v e GC d e t e c t i o n .

The q u a l i t y o f t h e a i r sample can be b e t t e r p r e s e r v e d by u s i n g LC t e c h n i ques

i n the

f i n a l a n a l y t i c a l step.

A

c l e v e r method i n v o l v i n g s e p a r a t i o n o f

nitro-PAH on a RPLC rnicrobore column connected t o an amperometric d e t e c t o r e q u i pped w i t h a g l a s s y carbon e l e c t r o d e The method

has been r e p o r t e d by J i n e t a l . ( 2 1 4 ) .

i s s u i t a b l e f o r t h e s e l e c t i v e d e t e c t i o n o f nitro-PAH p r e s e n t i n t h e

e x t r a c t a t sub-nanogram l e v e l s . Fluorescence quenching d e t e c t i o n a f t e r t h i n - l a y e r chromatography has been, i n s t e a d , proposed by J i g e r (215) f o r t h e e v a l u a t i o n o f nitro-PAH. A l t h o u g h t h e

249

method i s s i m p l e and c o s t - e f f e c t i v e ,

t h e s e n s i t i v i t y i s poor

and t h e d e t e c t i o n

m i g h t be s u b j e c t t o i n t e r f e r e n c e s . Other PAC which can be found i n a i r b o r n e p a r t i c u l a t e m a t t e r a r e azaarenes, keto-PAH and hydroxy-PAH.

Azaarenes, which can be p a r t l y separated f r o m n i t r o -

PAH d u r i n g t h e p r e f r a c t i o n a t i o n o f SOF(152,167),

v a r i o u s methods developed f o r nitro-PAH.

can be determined w i t h t h e

T h e i r p o s i t i v e i d e n t i f i c a t i o n can be

f a c i l i t a t e d w i t h t h e procedure d e s c r i b e d by Novotny e t a1 . ( 2 1 6 ) who combined t h e p r e c i s e measure o f r e t e n t i o n i n d i c e s w i t h mass s p e c t r a l i n f o r m a t i o n . An HPLC method f o r t h e r a p i d d e t e r m i n a t i o n o f azaarenes e x t r a c t e d from urban samples has been d e s c r i b e d

by Bresson e t a l . ( 2 1 7 ) . The components,frac-

t i o n a t e d by g r a d i e n t e l u t i o n on a ODS column, a r e i d e n t i f i e d by U.V.

induced

fluorescence. PAC c o n t a i n i n g c a r b o n y l o r h y d r o x y l groups can be d e t e c t e d by e l e c t r o n cap-

t u r e d e t e c t i o n (166) and i d e n t i f i e d by mass s p e c t r o m e t r y o p e r a t i n g i n e l e c t r o n impact (152,166,207)

o r n e g a t i v e i o n chemical i o n i z a t i o n u s i n g methane as r e a -

gent gas (152,166,218). O t h e r c l a s s e s o f PAC which r e c e i v e d s p e c i a l a t t e n t i o n a r e PCDD and PCDF. T h e i r d e t e r m i n a t i o n i n a i r b o r n e p a r t i c u l a t e m a t t e r can be c a r r i e d o u t GC o r HPLC t e c h n i q u e s .

The GC a n a l y s i s

e i t h e r by

i s u s u a l l y performed on c a p i l l a r y

columns connected t o a MS which can be rendered s e l e c t i v e f o r these compounds. T h i s aim i s p o s s i b l e because o f t h e i n t e n s e m o l e c u l a r i o n c l u s t e r s generated by p o l y c h l o r i n a t e d s p e c i e s .

and t y p i c a l i s o t o p i c

A p r e l i m i n a r y i n f o r m a t i o n on

t h e presence and d i s t r i b u t i o n o f PCDD and PCDF can be o b t a i n e d i n a s i n g l e GC r u n by o p e r a t i n g t h e MS i n programmed S I D (173,219,220) chromatograms w i t h computerized systems ( 2 2 1 ) .

o r by g e n e r a t i n g mass

The r e s u l t s a r e r e p o r t e d i n

terms o f c o n t e n t o f d i o x i n s and f u r a n s c o n t a i n i n g t h e same number o f c h l o r i n e a toms. Another approach c o n s i s t s i n t h e s e p a r a t i o n and d e t e r m i n a t i o n o f t h e isomer i c s p e c i e s which a r e known t o be p a r t i c u l a r l y t o x i c .

Since the highest b i o l o -

g i c a l a c t i v i t y i s observed w i t h PCDD and PCDF c o n t a i n i n g 4 t o 6 c h l o r i n e atoms i n t h e l a t e r a l p o s i t i o n s , a column should be a b l e t o s e p a r a t e , a t l e a s t , one t e t r a , one penta

and t h r e e hexa-COD from t h e o t h e r 22, 13 and 7 isomers respec-

t i v e l y as w e l l as a g r e a t e r number o f PCDF i s o m e r i c species. separation o f 2,3,7,8

Since t h e f i r s t

tetra-CDD from t h e o t h e r isomers r e p o r t e d by Buser and

Rappe (222) i n 1980, t h i s t a s k has been c o n s t a n t l y pursued so t h a t , when a l l i s o m e r i c compounds were s y n t h e t i z e d , i t has been a l s o p o s s i b l e t o s e p a r a t e t h e more t o x i c penta and hexa-CDD f r o m t h e o t h e r isomers ( 2 2 3 ) .

Two c a p i l l a r y

columns a r e p r e s e n t l y a v a i l a b l e t o p e r f o r m such f r a c t i o n a t i o n ( 2 2 4 ) : one i s a

22 m g l a s s column i n t e r n a l l y coated w i t h S i l a r 10 C, t h e o t h e r i s a 60 m f u s e d s i l i c a column coated w i t h SP 2330.

By u s i n g t h e l a t t e r column, t h e a n a l y s i s o f

a l l d i o x i n s isomers w i t h 4-6 c h l o r i n e atoms can be performed i n l e s s t h a n one

250

hour.

Good

s e p a r a t i o n s o f many PCDF isomers have been r e p o r t e d on t h e same

column (224) b u t a complete q u a n t i t a t i o n o f a l l t o x i c s p e c i e s r e q u i r e s , a t l e a s t , another column h a v i n g a d i f f e r e n t p o l a r i t y .

A more complex, y e t s u c c e s s f u l procedure f o r t h e d e t e r m i n a t i o n o f many t o x i c d i o x i n s i s t h a t d e s c r i b e d by Lamparski and N e s t r i c k ( 1 7 4 ) . These a u t h o r s e x p l o i t e d t h e d i f f e r e n c e i n r e t e n t i o n t h a t v a r i o u s isomers e x h i b i t on a d s o r p t i o n and reversed-phase LC. P o s i t i v e i d e n t i f i c a t i o n o f t h e s p e c i e s i s o l a t e d i n t h e HPLC f r a c t i o n s was c a r r i e d o u t by GC-MS working i n S I D mode. Other c h l o r i n a t e d compounds o f t e n a s s o c i a t e d w i t h PCDD and PCDF i n t h e p a r t i c u l a t e m a t t e r a r e c h l o r i n a t e d benzenes, c h l orophenol s ,chl o r i n a t e d p e s t i c i d e s and

PCB.

The a n a l y t i c a l s t e p s necessary f o r t h e i r d e t e r m i n a t i o n a r e s i m i l a r

t o those u t i l i z e d f o r PCDD and PCDF.

According t o S a u t e r e t a l . (225) t h e use

o f a f u s e d - s i l i c a c a p i l l a r y column connected t o a MS can p r o v i d e a s u i t a b l e det e r m i n a t i o n o f t h e f o r m e r t h r e e c l a s s e s o f c h l o r i n a t e d compounds even i n t h e presence o f s e v e r a l d i f f e r e n t p r i o r i t y p o l l u t a n t s p r e s e n t i n t h e SOF. An e x c e l l e n t s e p a r a t i o n and r a p i d d e t e r m i n a t i o n o f PCB can,instead,

be p e r -

formed on w a l l - c o a t e d g l a s s c a p i l l a r y columns w i t h small i n t e r n a l d i a m e t e r

(100ym)capable t o y i e l d up t o 275,000 t h e o r e t i c a l p l a t e s on a 25 m column ( 2 2 6 ) . Phtalate esters, o f t e n present i n airborne p a r t i c u l a t e matter c o l l e c t e d i n p o l l u t e d areas, can be e a s i l y e v a l u a t e d by s u b m i t t i n g t h e SOF t o GC-MS a n a l y s i s . T h e i r presence can be e v i d e n t i a t e d by s e l e c t i v e l y r e c o r d i n g t h e i o n c o r r e s p o n d i n g t o a v a l u e o f m/z=149. The same p r i n c i p l e can be s u c c e s s f u l l y adopted f o r t h e a n a l y s i s o f t h e f r a e t i o n s c o n t a i n i n g a1 kanes, f a t t y a c i d s , f a t t y a l c o h o l s , a l i p h a t i c aldehydes and e s t e r s (162,185,227,228).

Q u a n t i t a t i v e d a t a on these s p e c i e s combined w i t h

t h e d i s t r i b u t i o n o f PAH can be o f u t i l i t y f o r t r a c i n g a i r masses f r o m p a r t i c u l a r geographic r e g i o n s as w e l l as f o r t a g g i n g v e h i c u l a r o r combustion emissions. By c o n s i d e r i n g t h a t s e v e r a l i n o r g a n i c species, such as s u l f a t e s , c h l o r i d e s ,

n i t r a t e s and carbonates, can be a l s o r o u t i n e l y analyzed and q u a n t i f i e d

by c h r o -

matographic methods, i t appears c l e a r l y t h a t chromatography covers t h e w i d e s t range o f a p p l i c a t i o n today a c h i e v a b l e by a s i n g l e a n a l y t i c a l t e c h n i q u e i n t h e characterization o f airborne p a r t i c u l a t e matter. It n o t d i f f i c u l t t o predict, t h e r e f o r e , t h a t t h e answer t o some u n r e s o l v e d q u e s t i o n s

d e a l i n g w i t h t h e forma-

t i o n , t r a n s p o r t and environmental e f f e c t s o f p a r t i c u l a t e m a t t e r i s s t r i c l y r e l a t e d t o t h e advances i n chromatography t h a t w i l l be r e a l i z e d i n t h e n e a r f u t u re.

251

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16,

ERRATUM Ref. 19 should T h i r d European t a n t s " , Varese del Publisher,

read: M. P a y r i s s a t , B. N i c o l l i n , H. Stangl, Proceedings o f t h e Symposium on "Physico-Chemical Behaviour o f Atmospheric P o l l u 10-12 A p r i l 1984, B. Versino and G. A n g e l e t t i E d i t o r s , D. Rei1984, p. 90.

257

THE FUSED SILICA GLASS CAPILLARY COLUMN FOR GAS CHROMATOGRAPHY

-

THE ANATOMY

OF A REVOLUTION

S.R. LIPSKY D i r e c t o r , S e c t i o n o f P h y s i c a l Sciences New Haven, CT 06510 (U.S.A.)

-

Yale U n i v e r s i t y School o f M e d i c i n e

INTRODUCTION I t was t h e l a s t week o f A p r i l , 1979 and t h e f i r s t morning s e s s i o n o f t h e

T h i r d I n t e r n a t i o n a l Symposium on C a p i l l a r y Chromatography h e l d i n t h e b e a u t i f u l l i t t l e town o f Hindelang, Germany, had begun. One o f t h e papers t h a t m o r n i n g was by a q u i e t , unassuming young man who r e c e n t l y r e c e i v e d h i s d o c t o r a l degree and was b e g i n n i n g h i s f i r s t f o r m a l p o s i t i o n w i t h t h e H e w l e t t Packard Company. I n a l u c i d and d r a m a t i c p r e s e n t a t i o n , Dandeneau and h i s c o l l e a g u e s (1, 2 ) , des c r i b e d t h e i r experiences w i t h t h e u s e o f q u a r t z and f u s e d s i l i c a g l a s s e s i n t h e development o f c a p i l l a r y columns f o r gas chromatographic a p p l i c a t i o n s .

Figure 1 Schematic diagram o f appar a t u s used f o r d r a w i n g f u sed s i l i c a c a p i l l a r y t u b i n g and t h e o n - l i n e coat i n g o f the t h i n walled tube w i t h a polymeric sheath

.

258

I t was t h e i r c o n c l u s i o n f r o m s e l e c t e d s t u d i e s t h a t t h i s m a t e r i a l p r o v i d e d a de-

gree o f chemical i n e r t n e s s unmatched by o t h e r glasses. Moreover, when drawn f r o m a p r e f o r m i n a s t a n d a r d drawing tower used i n t h e f i b e r o p t i c s i n d u s t r y ( F i g . l ) , t h e c a p i l l a r y t u b i n g had a v e r y t h i n w a l l and was c o a t e d e x t e r n a l l y a t t h a t t i m e w i t h a s i l i c o n e polymer t o p r o t e c t i t s o u t e r s u r f a c e f r o m m o i s t u r e and contaminants. Under t h e s e circumstances, t h e pure f u s e d s i l i c a g l a s s e x h i b i t e d a h i g h degree o f f l e x i b i l i t y and s t r e n g t h which g r e a t l y f a c i l i t a t e d i t s h a n d l i n g as a c a p i l l a r y column i n t h e gas chromatographic apparatus. I n c l o s e l y examining t h e d a t a f r o m t h i s e p i c study, t h e r e s u l t s o b t a i n e d from t h e a n a l y s i s o f m i x t u r e s o f phenols, amines, a l c o h o l s , f r e e f a t t y a c i d s . sulphur

-

c o n t a i n i n g compounds, and t h e l i k e , were t h e most o u t s t a n d i n g ones

t h a t I have ever observed f r o m some t w e n t y y e a r s o f e x p e r i e n c e i n t h e f i e l d a t t h a t t i m e . Q u a n t i t a t i v e a n a l y s i s , where performed was e x c e l l e n t and t h e ' t a i l i n g ' o f p o l a r compounds on n o n p o l a r columns r a r e l y o c c u r r e d . I t became q u i t e obvious t h a t t h e use o f t h e s e new g l a s s e s would r e p r e s e n t a ' b r e a k t h r o u g h ' i n t h e f a b r i c a t i o n o f c a p i l l a r y columns. A l l o f t h i s n o t w i t h standing, I was s t r u c k by what I c o n s i d e r e d t o be c e r t a i n a m b i g u i t i e s t h a t were p r e s e n t i n t h i s most f a s c i n a t i n g s t u d y . These were: A) t h e need t o p r e c o a t t h e s u r f a c e o f t h e f u s e d s i l i c a g l a s s c a p i l l a r y column w i t h Carbowax 20M p r i o r t o t h e p r e p a r a t i o n o f a n o n p o l a r c a p i l l a r y column. T h i s seemed q u i t e odd t o me s i n c e t h e s u r f a c e o f t h e fused s i l i c a c a p i l l a r y was s t a t e d t o have a metal o x i d e c o n t e n t o f l e s s t h a n one ppm (TABLE I ) . C e r t a i n l y ,

Metal Content of Various Types of Fused Silica Tubing in ppm

TYPES Natural Quartz

Ca

Al

Cu

Fe

20-33

Regular Soda Lime Glass

30%

60%

Borosilicote Glass

2 0%

0 5%

B

P

3

20

20

03

05

001

05

10

-

-

0004

02

01

001

-

004

0001

001

01

0 01

003

-

-

-

0004

0005

-

-

16%

0.5%

01

-

K

003

-

003

Na

10

08

0 I

01

Ti

03

10

I - 10

SynlhehcFusedSilico

Mn

05

30-50

Purified Natural Quartz

High Purity Fused Silica (Synthetic)

Mg

4 0%

4%

TABLE I

13 %

259

under t h e s e circumstances, one d i d n o t have t o be concerned w i t h Lewis a c i d s i t e s and t h e i r d e l e t e r i o u s e f f e c t s on chromatographic performance. Moreover, i t had been n o t e d by o t h e r s (3, 4) as w e l l as t h o s e i n o u r own l a b o r a t o r i e s , t h a t when much o f t h e m e t a l o x i d e s p r e s e n t i n soda l i m e g l a s s were removed by p r o p e r l e a c h i n g w i t h H C I , t h e r e s u l t i n g s u r f a c e s when p r o p e r l y t r e a t e d , p r o v i d e d one w i t h e x c e l l e n t ' d e a c t i v a t e d ' columns which c o u l d be r e a d i l y used f o r h i g h temp e r a t u r e work w i t h o u t p r e t r e a t m e n t w i t h Carbowax 20M. F u r t h e r , i t has been l o n g r e c o g n i z e d t h a t t h e procedure o f " u n d e r c o a t i n g " n o n p o l a r columns w i t h a p o l a r phase, w h i l e e f f e c t i v e t o a c e r t a i n e x t e n t i n " d e a c t i v a t i n g " t h e g l a s s s u r f a c e , o f t e n gave r i s e t o c e r t a i n u n d e s i r a b l e consequences. Thus, f o r many substances, i t produced c o n s i d e r a b l e d i s t o r t i o n s o f Kovats R e t e n t i o n I n d i c e s . O f t e n a c o l u mn so f a b r i c a t e d behaved l i k e one c o n t a i n i n g a m i x t u r e o f phases, and a c c o r d i n g l y a t times, i t became somewhat d i f f i c u l t t o compare d a t a o b t a i n e d f r o m d i f f e r e n t l a b o r a t o r i e s due t o . s u b t l e d i f f e r e n c e s i n t h e h a n d l i n g o f t h e Carbowax 20M p r e c o a t i n g procedure d u r i n g column f a b r i c a t i o n , Columns so prepared a l s o l a c k e d t h e r m a l s t a b i l i t y above t h e 230-240°C range. O p e r a t i o n above t h e s e temperatures r e s u l t e d b o t h i n a s i g n i f i c a n t i n c r e a s e of " b l e e d i n g e f f e c t s " as w e l l as s u r f a c e a c t i v i t y .

B) The a f o r e m e n t i o n c d s t u d y ( 1 ) a l s o c o n t a i n e d s e v e r a l o m i s s i o n s t h a t were p u z z l i n g t o me. These were: t h e l a c k o f d a t a on f u s e d s i l i c a g l a s s c a p i l l a r y columns coated w i t h phases more p o l a r t h a n Carbowax 20M, namely, t h e v e r y usef u l cyanopropyl s i l i c o n e s (OV-225, OV-275, S i l a r 5C, and 1OC).

I

suspected pos-

s i b l e d i f f i c u l t i e s here, f o r again, e x p e r i e n c e i n t h e p a s t has shown t h a t chem i c a l l y i n e r t columns had s u r f a c e s which were n o t f a v o r a b l e f o r w e t t i n g w i t h t h e n a v a i l a b l e h i g h s u r f a c e t e n s i o n p o l a r phases u n l e s s some p h y s i c a l o r chemic a l method was b r o u g h t i n t o b e i n g t o s u i t a b l y a l t e r t h e s u r f a c e o f t h e g l a s s under c o n s i d e r a t i o n . Another p u z z l i n g o m i s s i o n was t h e conspicuous absence o f d i m e t h y l a n i l i n e (DMA) i n c e r t a i n o f t h e i r " p o l a r i t y t e s t " m i x t u r e s . The chromatographic behav i o u r of t h i s compound a l o n g w i t h d i m e t h y l p h e n o l (DMP) has been l o n g used by many i n v e s t i g a t o r s ( 5 ) as a measure o f t h e degree o f n e u t r a l i t y o f t h e s u r f a c e o f t h e coated g l a s s c a p i l l a r y column. C) The presence o f a s i l i c o n e polymer as an o u t e r p r o t e c t i v e sheath w i t h an upper temperature l i m i t o f about 230-240°C mentioned by Dandeneau ( 1 ) as a t h e n present l i m i t a t i o n

-

u n t i l a more s u i t a b l e polymer came a l o n g - was a l s o a n i m -

260

mediate concern t o me

-

a l o n g w i t h one o t h e r m a j o r one a t t h a t moment. Succin-

c t l y , t h i s was q u i t e s i m p l y , how do I o b t a i n s u f f i c i e n t f u s e d s i l i c a c a p i l l a r y tubing

- to

n o t o n l y c o n f i r m f i r s t hand, t h e r e s u l t s t h a t were p r e s e n t e d b u t t o

answer t h e many p e r p l e x i n g q u e s t i o n s t h a t r a c e d t h r o u g h my mind a t t h e t i m e . To me, t h e r e was no doubt whatsoever t h a t t h i s was a development o f m a j o r importance. Q u i t e o b v i o u s l y , t h e r e were many v o i d s t o b e f i l l e d b e f o r e i t s magnitude c o u l d be determined. Then and t h e r e I made t h e d e c i s i o n t o f o r e g o f u r t h e r p a r t i c i p a t i o n i n t h e Synlposium as w e l l as a n o t h e r f o u r days i n t h e b e a u t i f u l German A l p s , and t o h u r r y back t o my l a b o r a t o r y and p r e p a r e f o r t h i s new u n d e r t a k i n g .

I t h e n asked Dr. L e s l i e E t t r e w i t h whom I was rooming w i t h a t t h e H o t e l t o i n q u i r e by phone about t h e t r a i n schedule t o Z u r i c h . My i n t e n t i o n s were t o f i n d l o d g i n g s near t h e a i r p o r t and t h e n f l y back t o t h e S t a t e s t h e n e x t day. D u r i n g lunch, I sought o u t t h e one i n d i v i d u a l who I t h o u g h t may possess some v i t a l i n f o r m a t i o n which would h e l p me save s i g n i f i c a n t t i m e . I had remembered t h a t some t i m e ago,

I

had e i t h e r r e a d about o r heard P r o f e s s o r Dennis Desty d i s c u s s t h e

drawing o f q u a r t z t u b i n g i n a m o d i f i e d h i g h t e m p e r a t u r e v e r s i o n o f h i s now f a mous c a p i l l a r y drawing machine (6, 7 ) . A f t e r t a k i n g Dennis aside, I q u i c k l y i n formed him about my f e e l i n g s about t h e Dandeneau development and my p l a n s t o t r y t o o b t a i n q u a r t z o r f u s e d s i l i c a g l a s s c a p i l l a r y columns as soon as p o s s i -

b l e . A f t e r about two hours o f i n t e n s e d i s c u s s i o n and s e v e r a l l o n g d i s t a n c e phone c a l l s by b o t h of us, t h e f o l l o w i n g i n f o r m a t i o n emerged. Indeed, ( a ) he had m o d i f i e d h i s u n i t f o r h i g h t e m p e r a t u r e work by d e s i g n i n g a b u r n e r assembly t h a t used a propane oxygen m i x t u r e t o o b t a i n t h e h i g h temperatures (2000°C) necessar y t o draw q u a r t z , ( b ) he would a r r a n g e f o r me t o p i c k up a s e t o f b l u e p r i n t s

f o r t h e u n i t a t London A i r p o r t t h e n e x t day, and, ( c ) I would r e q u i r e a 'bendi n g ' t u b e ( t o c o i l t h e c a p i l l a r y ) t h a t c o u l d t o l e r a t e 1250-1350"C.With

this in-

f o r m a t i o n i n hand, I c a l l e d one o f my colleagues, D r . Leon T a l a l a y , and reques t e d him t o f i n d sources f o r a l l t h e necessary m a t e r i a l s we would r e q u i r e and t o prepare on o f t h e two home b u i l t drawing machines ( F i g . 2) i n o u r l a b o r a t o r y i n o r d e r t o house t h e new b u r n e r assembly, ( F i g . 3 ) which had t o be f a b r i c a -

ted.

261

F i g u r e 2 - In-house d r a w i n g machine m o d i f i e d t o accept b u r n e r assembly.

Figure 3

-

Desty b u r n e r assembly.

The n e x t day a f t e r p i c k i n g up D e s t y ' s b l u e p r i n t s i n London, t h e States

-

I

was back i n

most a n x i o u s t o g e t t h e p r o j e c t underway. Several h e c t i c weeks l a -

t e r ¶ we f a b r i c a t e d about a dozen 25 m e t e r permanently c o i l e d , t h i c k w a l l e d 0.22-0.27

mm i . d . c a p i l l a r y columns f r o m q u a r t z and f u s e d s i l i c a t u b i n g by u s i n g

o u r m o d i f i e d d r a w i n g machine. W i t h t h e e x c e p t i o n o f an o c c a s i o n a l b r e a k i n t h e c o i l d u r i n g t h e r u n and some d i f f i c u l t y i n h o l d i n g t h e t o l e r a n c e s we wanted f o r t h e i n t e r n a l diameters, t h e Desty b u r n e r performed m a g n i f i c a n t l y ( F i g . 3 ) . Soon thereafter,

we q u i c k l y v e r i f i e d Dandeneau's r e s u l t s and were a b l e t o c a r r y o u t

a few o f o u r own experiments w i t h t h i s f a s c i n a t i n g m a t e r i a l . A t t h i s p o i n t , o u r g r e a t enthusiasm was tempered by two f a c t s , f i r s t , o u r t h i c k w a l l e d c o i l s were n o t f l e x i b l e and second, each expensive p r e f o r m we used o n l y y i e l d e d about 50 t o 75 m e t e r s o f c a p i l l a r y t u b i n g

-

a most c o s t l y p r o p o s i t i o n . It r a p i d l y became

obvious t h a t we had t o f i n d someone i n t h e v e r y busy f i b e r o p t i c s i n d u s t r y t o draw f u s e d s i l i c a c a p i l l a r y t u b i n g f o r u s . B u t

-

where t o b e g i n ? A n o v e l scheme

was hatched. We decided t o f i n d t h o s e companies t h a t made s p e c i a l h i g h temperat u r e f u r n a c e s f o r t h e f i b e r o p t i c s i n d u s t r y and i n q u i r e , as p o t e n t i a l purchasers, about t h e i r u s e r s i n t h e N o r t h e a s t e r n p a r t o f t h e U n i t e d S t a t e s . Luck was w i t h us on t h i s p a r t i c u l a r day. The f i r s t company t h a t we came a c r o s s was t h e Centour

262

C o r p o r a t i o n o f New Hampshire. A f t e r a l o n g t e l e p h o n e c o n v e r s a t i o n , we l e a r n e d t h a t one u s e r o f t h e i r f u r n a c e was t h e G a l i l e o C o r p o r a t i o n i n S t u r b r i d g e , Massac h u s e t t s , about one h o u r ' s d r i v e f r o m New Haven. Flushed w i t h t h i s b i t of good f o r t u n e , I phoned them and was p l a c e d i n c o n t a c t w i t h a Mr. Ron Anderson. I p r o ceded t o t e l l him a b c u t my r e q u i r e m e n t s f o r a c o n t i n u o u s l e n g t h o f 0.25 mm i . d . e x t e r n a l l y coated fused s i l i c a t u b i n g . He seemed most c o o p e r a t i v e and t o l d me t o c a l l back i n s e v e r a l days and perhaps he c o u l d have something t o show me a t t h a t t i m e . T h i s I d i d a few mornings l a t e r and much t o my amazement. I was t o l d t o come up t h a t day f o r a 250 meter l o n o sample. When I a r r i v e d , a p i e c e was und e r a measuring microscope f o r me t o view. I t was p r e c i s e l y 0.25 mm i . d . and had an o u t e r p r o t e c t i v e c o a t i n g o f a b o u t 15 microns. When I i n q u i r e d a b o u t t h e n a t u r e o f t h e c o a t i n g and i t s upper temperature range, I was t o l d t h a t i t was a UV c u r a b l e m a t e r i a l ( a u r e t h a n e - a c r y l a t e polymer) good t o about 125-150°C. crestfallen,

I was

p a r t i c u l a r l y a f t e r h e a r i n g t h a t t h e y d i d n o t have f a c i l i t i e s a v a i -

l a b l e t o a p p l y h i g h e r t e m p e r a t u r e c o a t i n g s 'on l i n e ' t o t h e c a p i l l a r y t u b i n g as i t was b e i n g drawn. As I d r m e back toward New Haven, I remembered a s i m p l e ex-

periment t h a t I d i d y e a r s b e f o r e w i t h my c o l l e a g u e P r o f e s s o r H o r v a t h w h i c h i n v o l v e d some methyl m e t h a c r y l a t e i n o r d e r t o o b t a i n carbon b l a c k c o a t e d g l a s s beads f o r HPLC. I n t h e l a b o r a t o r y t h a t a f t e r n o o n , I t o o k o f f s e v e r a l meter l e n g t h s o f t h e f l e x i b l e g l a s s t u b i n g f r o m t h e spool, t i e d them i n t o c o i l s w i t h a t e f l o n s t r i n g and p l a c e d them i n t o s e v e r a l d i f f e r e n t gas chromatographic ovens programmed t o 250°C a t d i f f e r e n t r a t e s . When I r e t u r n e d t h a t evening, much t o my astonishment s e v e r a l c o i l s had a b r i g h t b l a c k , u n i f o r m o u t e r c o a t i n g . The t u b i n g m a i n t a i n e d i t s s t r e n g t h and f l e x i b i l i t y . The s u r f a c e s o f two o t h e r c o i l had f l e c k s o f d u l l b l a c k m a t e r i a l which had peeled away, exposing a b r i t t l e g l a s s surface.

I n subsequent days, I r e p e a t e d t h e s e experiments many t i m e s and soon

found t h e t h e p r o p e r temperature program t o c o n v e r t t h e o u t e r c o a t i n g t o a ' c a r bonized' p o l y m e r i c m a t e r i a l t h a t would subsequently p e r m i t us t o use t h e f l e x i b l e fused s i l i c a c a p i l l a r y columns up t o 25O"C-26O0C w i t h o n l y an odd spontaneous break i n t h e c o i l , f o r many weeks w i t h o c c a s i o n a l use a t 300°C p r o v i d e d t h e o u t e r s u r f a c e was n o t exposed t o t h e s e h i g h e r temperatures f o r any p r o l o n g e d per i o d o f t i m e . A t t h i s p o i n t , I now o b t a i n e d 13 k i l o m e t e r s o f 0.25 mn i . d . s i l i c a t u b i n g coated w i t h t h e

fused

UV c u r a b l e m a t e r i a l , a t a most r e a s o n a b l e p r i c e

f r o m G a l i l e o . W i t h t h i s and o u r c o n v e r s i o n method a t hand, we were now r e a d y t o f u l l y e x p l o r e t h e use o f f u s e d s i l i c a g l a s s f o r t h e p r e p a r a t i o n o f c a p i l l a r y

263 columns f o r gas chromatographic a n a l y s i s . THE FUSED SILICA GLASS SURFACE W i t h i n a r e l a t i v e l y s h o r t p e r i o d o f time, we began t o r e a l i z e t h a t we were c o n f r o n t e d w i t h a f u s e d s i l i c a s u r f a c e t h a t was q u i t e d i f f e r e n t f r o m t h a t encountered w i t h o t h e r t y p e s o f g l a s s e s ( 8 ) . The r e l a t i v e l y few s i l a n o l groups p r e s e n t here rendered t h e s u r f a c e m i l d l y a c i d i c . To c o m p l i c a t e m a t t e r s somewhat, w i t h e x p e r i e n c e i t became obvious t h a t t h e degree o f a c i d i t y v a r i e d f r o m b a t c h t o batch. Several phenomena were r e s p o n s i b l e f o r t h i s e f f e c t . A p p a r e n t l y , as t h e fused s i l i c a p r e f o r m was heated i n t h e drawing tower, as t h e temperatures approached 800-1000°C

-

on t h e way t o t h e f i n a l d r a w i n g t e m p e r a t u r e range o f 1950-

-21OO"C, t h e s i l a n o l m o i t i e s p r e s e n t on t h e s u r f a c e condensed. Water vapour i s v o l a t i l i z e d o f f and h i g h l y s t r a i n e d , v e r y r e a c t i v e s i l o x a n e b r i d g e s form. A f t e r t h e c a p i l l a r y t u b i n g i s formed and drawn, t h e l o w e r temperatures cause t h i s r e a c t i o n t o be r e v e r s i b l e once again. I n t h e presence o f room a i r (and m o i s t u r e ) , a r e l a t i v e l y s m a l l number o f s i l a n o l grouos once a g a i n f o r m on t h e s u r f a c e . U n f o r t u n a t e l y , even a t t h e p r e s e n t time, we do n o t possess a simple, a c c u r a t e method o f d e t e r m i n i n g t h e r a t i o s o f s i l a n o l groups t o s i l o x a n e b r i d g e s i n a c a p i l l a r y tube. Another o b s e r v a t i o n a l s o h e l p e d e x p l a i n t o some e x t e n t , t h e presence o f an a c i d i c s u r f a c e i n t h e f u s e d s i l i c a c a p i l l a r y tube. By happenstance, we n o t e d t h a t s i g n i f i c a n t q u a n t i t i e s o f an a c i d i c vapour c o u l d be blown o u t o f l o n g l e n g h t s o f t h e newly drawn f u s e d s i l i c a c a p i l l a r y t u b i n g Nhen a stream o f

N, was

a p p l i e d t o one end w h i l e t h e e x i t was b e i n g m o n i t o r e d by l i t m u s paper. T h i s mat e r i a l , which l a t e r proved t o be p r e d o m i n a n t l y H C I , would egress f o r a p e r i o d o f

5 t o 10 m i n u t e s b e f o r e n e u t r a l c o n d i t i o n s were noted. A p p a r e n t l y , t h i s was due t o r e s i d u a l q u a n t i t i e s o f h i g h p u r i t y s y n t h e t i c SiC1,

which i n c o m p l e t e l y r e a c t e d

w i t h oxygen a t h i g h t e m p e r a t u r e s d u r i n g t h e manufacture o f t h e f u s e d s i l i c a g l a s s preform. DEACTIVATION OF SILANOL GROUPS W i t h t h i s knowledge i n t h e background, we t h e n began an i n t e n s i v e e f f o r t t o e x p l o r e v a r i o u s t e c h n i q u e s i n o r d e r t o assess t h e i r e f f e c t i v e n e s s i n removing t h e r e s i d u a l a c t i v i t y . We q u i c k l y r u l e d o u t t h e use o f Carbowax 20M h e r e f o r reasons a l r e a d y c i t e d . V a r i o u s s i l y a t i o n procedures, (9-11) e f f e c t i v e w i t h soda

264

lime o r b o r o s i l i c a t e glass l e f t much t o be d e s i r e d here. T h e i r o v e r a l l e f f e c t i v e n e s s and r e p r o d u c i b i l i t y i n o u r hands i n these e a r l y t i m e s v a r i e d c o n s i derably. F i n a l l y , since these s u r f a c e s were n o t e d t o resemb l e t h a t which was found i n certain states o f s i l i c a gel, we t h e n t u r n e d o u r a t t e n t i o n t o t h e s e as w e l l as o t h e r areas i n order t o obtain a s o l u t i o n t o t h i s problem. In t h e f i r s t i n s t a n c e , a l c o h o l s were u t i l i zed i n an a t t e m p t t o f o r m s t a ble, e f f e c t i v e e s t e r linkages w i t h the s i l a n o l moieties. Using v e r y s i m p l e l a b o r a t o r y procedures f o r c r e a t i n g d i f f e r e n t a l k o x y l i n k a g e s by means o f hydrogen bonding o f S i O H groups, n e u t r a l , we1 1 d e a c t i vated f u s e d s i l i c a g l a s s s u r -

Figure 4

faces were o b t a i n e d which wer e t h e n e a s i l y w e t t e d by c o m m e r c i a l l y a v a i l a b l e n o n p o l a r methyl (OV-101, SE-30) ( F i g . 4) and methyl 5 % phenyl p o l y s i l o x a n e (SE-52, SE-54) s t a t i o n a r y phases. Such columns showed good e f f i c i e n c y and thermal s t a b i l i t y up t o 300°C ( 1 2 ) . But a n o l appeared t o p r o v i d e t h e b e s t r e s u l t s here. A p p a r e n t l y , as f i r s t n o t e d w i t h s i l i c a ( 1 3 ) , t h e a l c o h o l m o i e t y r e a c t s w i t h t h e OH groups o f s i l a n o l s under f a v o u r a b l e c o n d i t i o n s t o f o r m b u t o x y groups ( e s t e r s i l s ) , which on a f u l l y e s t e r i f i e d s u r f a c e proved t o be remarkably s t a b l e , o r g a n o p h i l i c and hydrophophobic CH3(CH,),CH,0H

+

SiOH-R'OSi

+

HO,

A p r i o r casual o b s e r v a t i o n made d u r i n g o u r i n v e s t i g a t i o n o f t h e use o f a l c o h o l s and d i o l s ( 1 2 ) i n b i n d i n g s u r f a c e s i l a n o l s prompted us t o c o n c e n t r a t e o u r

265

e f f o r t s i n t h i s area. T h i s i n v o l v e d t h e r e s u l t s o b t a i n e d w i t h one o f a s e r i e s o f s p e c i a l l y prepar e d d i o l p o l y s i l o x a n e polymers. T h i s semi-viscous m a t e r i a l was c o a t e d dynamic a l l y o n t o an u n t r e a t e d 0.25 mm i . d . f u s e d - s i l i c a g l a s s c a p i l l a r y column, t h e s o l v e n t removed, t h e ends o f column were s e a l e d and t h e column was heated t o 325-350°C o v e r n i g h t . A t t h i s p o i n t i n t i m e , t h e o u t e r sheath o f t h e f u s e d s i l i ca c a p i l l a r y was now coated w i t h a polyimic'e polymer (DuPont) good t o 370°C. Such o u t e r c o a t i n g s were f i r s t i n t r o d u c e d by t h e Hewlett-Packard Company. The column was a l l o w e d t o c o o l t o room temperature, t h e s e a l e d ends were removed and t h e column was c o n d i t i o n e d by s l o w l y r a i s i n g t h e t e m p e r a t u r e t o 300°C. An e f f i c i e n t , t h e r m a l l y s t a b l e m e t h y l p o l y s i l o x a n e column ( F i g . 5) was produced, which showed s l i g h t t a i l i n g o f c e r t a i n p o l a r compounds.

Figure 5 Chromatogram o b t a i n e d w i t h a 25 M.xO.25 mm i . d . f u s e d s i l i c a g l a s s c a p i l l a r y column w i t h a h i g h m o l e c u l a r w e i g h t pol y s i l o x a n e d i o l polymer coated d i r e c t l y on t h e u n t r e a t e d s u r f a c e . Temperature, 100°C; pressure, 18 p . s . i . (He); c h a r t speed, 60 cm/hr. Peaks: I=C-9; 2=2-octanone; 3=C-10; 4 = l - o c t a n o l ; 5=2,6-dimet h y l p h e n o l ; 6 = C - l l ; 7=2,4-dimethylanil i n e ; 8=naphthalene; 9=C-12. Note t h e or-li/ s i g n o f a c t i v i t y i s t h e presence o f s l i g h t t a i l s on peaks 2, 4, and 7 . The peak r a t i o s a r e e x c e l l e n t .

W i t h t i m e and experience, i n most i n s t a n c e s c o m p l e t e l y i n e r t s u r f a c e s were r o u t i n e l y o b t a i n e d . A p p a r e n t l y , under t h e s e circumstances, i t i s p l a u s i b l e t o

266

b e l i e v e t h a t w i t h h e a t t h e OH groups o f t h i s p a r t i c u l a r s i l i c o n e polymer r e a d i l y condensed w i t h t h e s l i g h t l y a c i d i c s u r f a c e s i l a n o l groups, which may have

a c t e d as a c a t a l y s t i n t h i s r e a c t i o n . I n c o n t r a s t t o r e l a t i v e l y s h o r t - c h a i n a l c o h o l s o r d i o l s , i n t h i s case, t h e s i l o x a n e l i n k a g e t o t h e g l a s s s u r f a c e i n v o l ved a v e r y l o n g - c h a i n s i l i c o n e polymer. Here, a s t a b l e , u n i f o r m l y

distributed

p o l y m e r i c f i l m (which we l a t e r found c o u l d be e a s i l y c r o s s l i n k e d ) w i t h a l l t h e chromatographic p r o p e r t i e s o f t h e m e t h y l s i l i c o n e s , OV-101, SE-30 o r OV-1

, was

produced on s u r f a c e s t h a t now appeared t o be n e u t r a l . Hence, t h e r e was no need t o go t h r o u g h t h e usual second s t e p , namely, c o a t i n g w i t h t h e c o n v e n t i o n a l s t a t i o n a r y phase. T h i s f i n d i n g t u r n e d o u t t o be one o f m a j o r importance t o us as we c o n t i n u e d t o pursue t h i s l i n e o f study. A s i m i l a r r e s u l t was o b t a i n e d w i t h a high

m o l e c u l a r w e i g t h v i n y l c o n t a i n i n g m e t h y l 20% phenyl p o l y s i l o x a n e p o l y -

mer a l s o coated d i r e c t l y on t o t h e u n t r e a t e d f u s e d s i l i c a s u r f a c e . The mechanism by which t h e d e a c t i v a t i o n o c c u r s here remains s p e c u l a t i v e a t t h i s p o i n t . T h i s r e s u l t prompted us t o i n v e s t i g a t e a g a i n two areas o f i n t e r e s t . The first,

i n v o l v e d those f a c t o r s w h i c h govern t h e successful w e t t i n g o f t h e fused-

- s i l i c a g l a s s s u r f a c e by a wide ranae o f n o n p o l a r and p o l a r p o l y m e r i c f i l m s , and t h e second, t h o s e r e a c t i o n s which may be r e s p o n s i b l e f o r t h e s t r u c t u r a l changes i n v o l v i n g e i t h e r t h e b i n d i n g o r t h e e f f e c t i v e c o v e r i n g o f t h e s i l a n o l groups, t h u s l e a d i n g t o t h e n e u t r a l i z a t i o n o f t h e s l i g h t l y a c i d i c s u r f a c e o f f u s e d - s i l i c a g l a s s . Obviously, i n t e r e s t h e r e t o o c e n t e r e d around t h e f a s c i n a t i n g p o s s i b i l i t y o f g r e a t l y extending the a b i l i t y t o " n e u t r a l i z e " t h e surface and c o a t e f f i c i e n t l y t h i s v e r y v e r s a t i l e g l a s s w i t h a wide v a r i e t y o f d i f f e r e n t polymeric f i l m s i n a s i n g l e step. CHANGING THE PHYSICAL CHEMICAL PROPERTIES OF THE STATIONARY PHASE

When i t was f i r s t observed t h a t c e r t a i n c o m m e r c i a l l y a v a i l a b l e s t a t i o n a r y phases ( p a r t i c u l a r l y s i l i c o n e polymers w i t h s u b s t a n t i a l phenyl o r cyanopropyl s u b s t i t u t i o n s ) c o u l d n o t be p r o p e r l y w e t t e d on t h e s u r f a c e o f f u s e d - s i l i c a g l a s s , we a t t h a t t i m e i m p r o p e r l y assumed t h a t t h i s e f f e c t was due t o t h e r e l a t i v e l y low surface energy of t h i s p a r t i c u l a r t y p e o f u n t r e a t e d g l a s s . A c c o r d i n g l y , we t h e n p o s t u l a t e d t h a t t h e s e s u r f a c e s had t o be a l t e r e d by e i t h e r p h y s i c a l o r chemical means i n o r d e r t o i n c r e a s e t h e s u r f a c e energy t o t h e l e v e l wher e t h e s e m a t e r i a l s can r e a d i l y wet t h i s a l t e r e d s u r f a c e . L a t e r , when s t u d i e s by c o n t a c t a n g l e measurements ( 1 4 ) r e v e a l e d t h e r e l a t i v e l y h i g h s u r f a c e e n e r g i e s

261

of 50-72 dyn/cm for fused-silica glass, we still found difficulty in reconciling our laboratory experience with these data. With time, however, it appeared that both sets of laboratory observations were valid, but certain assumpt ons were inappropriate. Evidence was mounting i n our laboratory to make us be ieve that those specific commercial stationary phases used in our studies, i.e , OV-3, OV-7, OV-11, OV-17 (10-50%methyl-phenylsiloxanes) and SP-2340 and S lar 1OC (75-100% cyanopropylmethylpolysiloxane) , did not possess the appropriate physical chemical properties for effective use with fused-silica surfaces. They were relatively low molecular weight liquid polymers and, in these instances, the particular intermolecular forces between the stationary phase molecules and

Figure 6 Chromatogram obtained with 25 M. x 0.25 mm i.d. fused silica glass capillary column coated with high molecular weight vinyl containing methyl 20% phenyl polysiloxane polymer on the untreated surface. Temp: 110°C; press: 20 p.s.i. (He); chart: 60 cm/hr Film thickness = 0.25pV; Peaks: 1=2-octanone,2=l-octanol, 3=2,6-dimethylphenol, 4=C-12, 5=2,4-dimethylaniline, 6=naphthalene. 7=C-13.

Figure 7 Chromatogram obtained with same phase as in Figure 6 except length = 100 M. and film thickness = O.lpm. Temp: llO"C, press: 45 p.s.i. (H2); chart: 60 cm/hr. Peaks as in Fig. 6.

268

t h e e x i s t i n g s u r f a c e m o i e t i e s on f u s e d - s i l i c a g l a s s were l e s s t h a n t h e f o r c e s between t h e molecules o f t h e s t a t i o n a r y f i l m s themselves. T h i s r e s u l t e d i n d r o p l e t f o r m a t i o n w i t h a l l i t s consequences. When c e r t a i n p h y s i c a l chemical m o d i f i c a t i o n s were made i n some o f t h e s p e c i a l l y prepared h i g h - m o l e c u l a r - w e i g h t s i l i cone polymers used i n w e t t i n g experiments on f u s e d - s i l i c a g l a s s , i t was n o t e d f o r t h e f i r s t t i m e t h a t , f o r example, t h o s e made w i t h h i g h phenyl o r cyanopropyl s u b s t i t u t i o n ( b i s c y a n o p r o p y l as we1 1 as c y a n o p r o p y l m e t h y l ) n o t o n l y wet v e r y w e l l b u t a l s o gave r i s e t o w e l l d e a c t i v a t e d s u r f a c e s . Some e a r l y r e s u l t s o f t h i s s t u d y a r e shown i n F i g s . 6 and 7. The p a r t i c u l a r m a t e r i a l used t o c o a t t h e s e f u s e d - s i l i c a columns d i r e c t l y i n a s i n g l e s t e p by t h e s t a t i c t e c h n i q u e w i t h o u t p r i o r d e a c t i v a t i o n procedures was a spec a l l y synt h e s i z e d h i g h - m o l e c u l a r - w e i g h t , v i s c o u s , v i n y l - c o n t a i n i n g m e t h y l p o l y s l o x a n e polymer w i t h 20% phenyl s u b s t i t u t i o n .

4 Column

25M 75% CYANOPROPYL SILICONE (DIRECT)

Temp Press Chart

110°C 18 p s I He 60 cmlhr

Peaks 1 c.12 2. cyclooctanone 3. 1.octanol 4 C.16 5 naphlhalene 6 2 6 dirnethvlDhenol 7 2.4 dimethylanlllne 8 C.18

1

3

: 5 7

9

Figure 8

269

The deactivation was almost perfect for both the 25M and the thin-film lOOM columns. The only imperfection was a barely perceptible tail on the peaks representing octanol and 2,4-dimethylaniline, Similarly, a very highly polar f u sed-silica glass capillary column was obtained by the single-step direct application of one o f a series o f specially prepared high molecular weight vinyl-containing methylpolysiloxanes with, in this instance, 75% cyanopropylmethyl substitution (15). Fig. 8 again shows that another very efficient, thermally stable film (27OOf') was produced on a completely deactivated surface. CROSSLINKING OF THE NEW STATIONARY PHASES During the course o f these studies, considerable progress was being made in a particular field of gas chromatography by Madini et al. (16-19)and Blomberg and Wannman (20, 21) by pioneering the development of methods for the in situ production of efficient, thermally stable, insoluble silicone polymeric stationary phases from , -hydroxvpolymethylsiloxane type prepolymers on the treated surfaces of either sodalime or borosilicate glass capillary tubes. Film stability, the exceedingly low level o f

I'

bleeding" phenomena at elevated temperatu-

res, the imperviousness o f cross-linked stationary phases to exposure to large volumes of solvent, the ability to rinse away residual sample and film debris by appropriate solvents, and the relative ease by which thick films could be fabricated, were considered by these investigators to be the outstanding advantages of this technique.

In an attempt to achieve similar goals, Grob and co-workers (22, 23) and Sandra et al. (24) utilizing methods well known in the field of silicone polymer chemistry used peroxides to insolubilize certain vinyl containing high molecular weight polydimethylsiloxane gums commonly employed as stationary phases for glass capillary columns. Both of these groups were impressed with the relative ease by which their preliminary attempts to cross-link these nonpolar silicone phases succeeded in producing satisfactory capillary columns. Interestingly, Grob et al. (22) who carried out crosslinking experiments on regular glass capillary column surfaces which were first subjected to persilanization (25) readily obtained well deactivated columns. In contrast, in a similar study, Sandra et al. (24) using fused-silica glass capillary tubing which was first deactivated either by o c t a m e t h y l c y c l o t e t r a s i l o x a n e (26) or by the polysiloxane technique of Schomburg et al. (27) was also successful with in situ crosslinking

210

of t h e s t a t i o n a r y phase. However, no d a t a was p r o v i d e d h e r e c o n c e r n i n g t h e s t a t u s o f t h e a c t i v i t y o f t h e s u r f a c e s under t h e c o n d i t i o n s o f t h e i r experiment. The aforementioned a t t r i b u t e s o f t h e c r o s s l i n k e d s t a t i o n a r y phase seemed so overwhelmingly f a v o r a b l e , t h a t we decided t o a t t e m p t t o i n c o r p o r a t e t h i s conc e p t f o r t h w i t h i n t o o u r ongoing i n v e s t i g a t i o n s i n v o l v i n g t h e a p p l i c a t i o n o f f u sed s i l i c a g l a s s c a p i l l a r y columns f o r gas chromatographic a n a l y s i s . When we sought t o m o d i f y t h e methods o f M a d i n i e t a l . (16-19) and Blomberg e t a l . (20, 21) as w e l l as t h o s e o f Grob and co-workers (22, 23) and Sandra e t a l . ( 2 4 ) t o i n s o l u b i l i z e n o n p o l a r s i l i c o n e polymers on f u s e d - s i l i c a g l a s s c a p i l l a r y surfaces, s e v e r a l problems were encountered i n o u r e a r l y s t u d i e s (28, 2 9 ) . These p r i m a r i l y c e n t e r e d around ( a ) t h e r e s i d u a l s u r f a c e a c t i v i t y t h a t r e mained f o l l o w i n g v u l c a n i z a t i o n and ( b ) t h e e r r a t i c w e t t a b i l i t y o f t h e s e surfaces by p o l y m e r i c s o l u t i o n s c o n t a i n i n g c e r t a i n p e r o x i d e s . I n an e f f o r t t o overcome t h e s e and o t h e r t e c h n i c a l problems t h a t developed under t h e s e c i r c u m s t a n ces, we t h e n s t u d i e d i n d e t a i l , t h e v a r i o u s f a c t o r s t h a t a f f e c t e d c r o s s - l i n k i n g , u n i f o r m f i l m f o r m a t i o n , thermal s t a b i l i t y , and s u r f a c e d e a c t i v a t i o n .

A t t h e o u t s e t , i t was a l s o r e c o g n i z e d t h a t t h e c r o s s - l i n k i n g r e a c t i o n s t h a t o c c u r r e d w i t h t h e t y p e o f s i l i c o n e polymer produced by t h e methods d e s c r i b e d by Madini and co-workers (19) and Blomberg and Wannman (20, 21), d i f f e r e d s i g n i f i c a n t l y f r o m t h a t b r o u g h t about by t h e a c t i o n o f f r e e r a d i c a l s formed d u r i n g t h e thermal decomposition o f v a r i o u s p e r o x i d e s upon t h o s e v i n l y c o n t a i n i n g s i l i c o n e gum phases u s u a l l y employed i n gas chromatography. I n t h e f i r s t i n s t a n c e , c r o s s l i n k i n g w i t h t e t r a c h l o r o s i l a n e produced polymers w i t h S i - 0 - S i l i n k a g e s . I n t h e l a t t e r case, t h e f r e e r a d i c a l s r e a d i l y a t t a c k e d t h e d o u b l e bond i n t h e v i n y l m o i e t i e s , g i v i n g r i s e t o polymer l i n k a g e s o f t h e Si-C-C-Si

t y p e . B o t h c l a s s e s o f polymers, when p r o p e r l y prepared, produced ex-

c e l l e n t fused s i l i c a c a p i l l a r y columns showing e x c e l l e n t e f f i c i e n c y , thermal s t a b i l i t y and r e s i s t a n c e t o c e r t a i n s o l v e n t s . C h r o m a t o g r a p h i c a l l y , t h e y were i n d i s t i n g u i s h a b l e f r o m one a n o t h e r . O f a l l t h e p e r o x i d e s u s e d i n o u r s t u d y (30), dicumyl p e r o x i d e produced t h e b e s t r e s u l t s . W i t h t h e a v a l a i b i l i t y o f a z 0 - E -butane (31), we subsequently found t h a t by u s i n g t h i s m a t e r i a l i n t h e gas phase w i t h a simple, c o n t i n u o u s f l o w t e c h n i q u e (30) we c o u l d r e a d i l y and e f f i c i e n t l y c r o s s l i n k c e r t a i n n o n p o l a r v i n y l c o n t a i n i n g phases a f t e r t h e s u c c e s s f u l chromatographic t e s t i n g of t h e nonvulcanized prepolymer f i l m l a i d down on t h e

271

fused s i l i c a c a p i l l a r y s u r f a c e . However, d i f f i c u l t i e s were s t i l l encountered here i n m a n t a i n i n g w e l l d e a c t i v a t e d s u r f a c e s under t h e c o n d i t i o n s o f t h e exper i m e n t . F i r s t , we found t h a t i f b u t a n o l was used t o d e a c t i v a t e t h e s u r f a c e s , t h e c r o s s l i n k i n g r e a c t i o n was d i s t u r b e d . Second, d e a c t i v a t i n g w i t h o c t a m e t h y l c y c l o t e t r a s i l o x a n e ( 2 6 ) o r s l i g h t v a r i a t i o n s o f t h e p o l y s i l o x a n e method o f Shomburg e t a l . ( 2 7 ) , on o c c a s i o n gave r i s e t o e r r a t i c r e s u l t s . T h i r d , t r e a t m e n t w i t h aqueous H C I o r H20 a t e l e v a t e d temperatures t o i n c r e a s e s u r f a c e s i l a n o l s f o l l o wed by s i l y l a t i o n , i n c r e a s e d t h e f r a g i l i t y o f t h e f u s e d s i l i c a t u b i n g . Because o f t h e s e problems and r e c a l l i n g o u r p r i o r o b s e r v a t i o n s o f s i n g l e s t e p ' p o l y m e r d e a c t i v a t i o n ' and c o a t i n g , we d e c i d e t o c o n c e n t r a t e on t h i s t e c h n i q u e t o deac t i v a t e t h e s u r f a c e and p e r m i t c r o s s l i n k i n g o f t h e s t a t i o n a r y phase. T h i s i n v o l v e d t h e development o f a s e r i e s o f d i f f e r e n t p o l y m e r i c s t a t i o n a r y phases t h a t possessed t h e f o l l o w i n g p h y s i c a l chemical c h a r a c t e r i s t i c s :

--

1 . t h e polymers can be r e a d i l y c r o s s l i n k e d i n s i t u 2. c r o s s l i n k i n g can be r e a d i l y accomplished by h e a t o r f r e e r a d i c a l forma-

tion 3. t h e polymers r e a d i l y wet t h e f u s e d s i l i c a g l a s s s u r f a c e

4. t h e polymers may a i d i n t h e d e a c t i v a t i o n o f t h e s u r f a c e 5. t h e c r o s s l i n k e d polymers can be r i n s e d w i t h s o l v e n t s 6 . t h e c r o s s l i n k e d polymers have e x c e l l e n t f i l m and thermal s t a b i l i t y 7 . t h e polymers can be r e a d i l y d e p o s i t e d o n t o t h e s u r f a c e as t h i n o r v e r y thick films

8. t h e c r o s s l i n k e d polymers e x h i b i t McReynold's Constants s i m i l a r t o t h o s e n o t e d w i t h commercial s t a t i o n a r y phases 9. t h e c r o s s l i n k e d polymers can be c o n v e n i e n t l y used w i t h a l l a v a i l a b l e i n j e c t i o n techniques.

As a r e s u l t o f t h i s e f f o r t ( 3 2 ) , i t was f o u n d t h a t a s e r i e s o f h i g h molecul a r w e i g h t , h i g h v i s c o s i t y , n o n p o l a r s i l a n o l t e r m i n a t e d ( F i g . 9-11) o r v i n y l c o n t a i n i n g p o l y d i m e t h y l s i l o x a n e polymers w i t h o r w i t h o u t phenyl s u b s t i t u t i o n , when a p p l i e d i n a s i n g l e s t e p t o u n t r e a t e d f u s e d s i l i c a g l a s s c a p i l l a r y t u b i n g , e f f i c i e n t l y d e a c t i v a t e d and w e t t e d t h e s e s u r f a c e s . W i t h t h e s i l a n o l t e r m i n a t e d polymers, upon h e a t i n g , t h e r e was i n d i r e c t evidence t o i n d i c a t e t h a t hydrogen bonding o c c u r r e d between t h e s i l a n o l m o i e t i e s o f t h e polymer and t h o s e o f t h e f u s e d s i l i c a s u r f a c e . T h i s p r o b a b l y formed a c o v a l e n t l i n k a g e between t h e p o l y mer and t h e g l a s s . Concomitantly, c o n d e n s a t i o n o f t h e polymer o c c u r r e d , e f f e -

272

cting crosslinking o f the Si-0-Si-type.

Figure 9

Figure 10

Very successful single step deactivation and uniform wetting o f untreated fused silica capillary tubing was also noted (32) with a wide variety of new medium molecular weight generally viscous polar cyanopropyl silicone polymers with vinyl and/or phenyl groups ( Fig. 12, 13). In several instances, however, difficulties were encountered in the cross linking of these polymers in the presence o f either free radical generator: or heat. Similar problems were also noted by Blomberg (34) and Lee (35). VERY THICK CROSSLINKED FILVS ON WIDE BORE FUSED SILICA COLUMNS Finally, following the observations of Grob et al. (33) that crosslinked films greater than 1.0 micron can be readily placed on the prepared surfaces of regular glass capillary tubing with i.d.'s greater than 0.25 mm, provided that a special methyl silicone stationary phase with appropriate properties can be used, we decided to attempt to achieve the same results on fused silica

273

F i g u r e 11 Column 25 M. x 0.25 mm 1.0. f u s e d s i l i c a g l a s s c a p i l l a r y column. C r o s s l i n k e d w i t h methyl 501 phenyl s i l i c o n e . A p p l i e d d i r e c t l y t o u n t r e a t e d s u r f a c e . Temp=llO°C; press=14 p . s . i . (H2); c h a r t = 1 8 0 cm/hr. Heat bonded. Note e x c e l l e n t d e a c t i v a t i o n and thermal s t a b i l i t y t o 325-350°C.

F i g u r e 12 For F i g u r e 12, n o t e t h e r a p i d emergence o f hydrocarbons C-15 t o C-19 on t h i s v e r y p o l a r phase.

F i g u r e 13 F o r F i g u r e 13, n o t e t h e emergence o f t h e C-20 m e t h y l e s t e r b e f o r e t h e C-18:3 on t h i s v e r y p o l a r phase.

274

g l a s s c a p i l l a r y columns

-

u s i n g o u r growing l i s t o f t a i l o r made polymers. We

found t h a t we c o u l d f a b r i c a t e w i d e b o r e (0.32 mm and 0.53 mm i . d . )

fused s i l i c a

c a p i l l a r y columns c o n t a i n i n g a number o f s p e c i a l l y prepared s t a t i o n a r y phases w i t h f i l m t h i c k n e s s o f 3 t o 5 m i c r o n s . We p r o v i d e d some o f o u r e a r l y columns (August, 1983) t o D r . L e s l i e E t t r e a t t h a t t i m e ; who t h e n proceeded t o do an o u t s t a n d i n g s e r i e s o f i n v e s t i g a t i o n s (36-39) on t h e t h e o r e t i c a l and p r a c t i c a l aspects o f t h i c k f i l m c a p i l l a r y columns. S h o r t l y t h e r e a f t e r , Ryder e t a l . ( 4 0 ) made t h e o b s e r v a t i o n t h a t i f t h e t h i c k f i l m e d columns a r e used w i t h v e r y h i g h gas v e l o c i t i e s (140 cm/sec. o r more, He o r Hz) (Packed Column Mode), t h e y have many o f t h e c h a r a c t e r i s t i c s o f a packed column, i . e .

h i g h sample c a p a c i t y , f a s t

a n a l y s i s times, modest column e f f i c i e n c y and good r e s o l u t i o n t h e disadvantages o f t h e packed column

-

-

w i t h o u t any o f

namely, a c t i v e s i t e s on t h e s o l i d sup-

port, I t was n o t e d by E t t r e ( 3 9 ) t h a t by s u b s t a n t i a l l y i n c r e a s i n g t h e f i l m t h i c k -

ness t o f i v e microns

-

as w e l l as i n c r e a s i n g t h e i n n e r d i a m e t e r o f t h e columns,

one can s i g n i f i c a n t l y i n c r e a s e t h e sample c a p a c i t y o f t h e c a p i l l a r y column w h i l e l i m i t i n g t h e r e d u c t i o n i n column e f f i c i e n c y and r e s o l u t i o n . Thus, by decreas i n g t h e phase r a t i o (

) , one i n c r e a s e s t h e c a p a c i t y f a c t o r k ' , and r e q u i r e s

l e s s t h e o r e t i c a l p l a t e s ( n ) f o r a g i v e n r e s o l u t i o n . G e n e r a l l y speaking, we found t h a t a one m i c r o n f i l m on 0.53 mm i . d .

f u s e d s i l i c a c a p i l l a r y column

had a sample c a p a c i t y o f about 1.2 micrograms p e r component b e f o r e o v e r l o a d i n g became apparent. On t h e o t h e r hand, a l o a d o f up t o 8.5 microgram p e r component c o u l d be placed on a s i m i l a r i . d . column w i t h a 5 m i c r o n f i l m b e f o r e a 10% i n crease i n peak w i d t h became apparent. When optimum gas v e l o c i t i e s were used ( a b o u t 20 cm/sec.) ( 3 t o 5 m i c r o n s ) on 0.32 mm o r 0.53 mm i . d .

with thick films

fused s i l i c a glass t u b i n g ( C a p i l l a -

r y Column Mode), t h e s e columns e x h i b i t e d h i g h sample c a p a c i t i e s (50-80% o f t h a t

found w i t h a l i g h t l y coated ( 5 % ) packed column), good column e f f i c i e n c y , r e l a t i v e l y good speed o f a n a l y s i s and good column r e s o l u t i o n . The f o l l o w i n g c h r o matograms i l l u s t r a t e some o f t h e v e r s a t i l e q u a l i t i e s o f t h e s e columns:

275

Temp. Chill:

140. Wcnvhr

Psak.: I . C 9 hydrocarbon 2. 2.mClanone 3. C10 hydrocarbon 4. I.Ccll"Ol 5. 2 8 dimethylphenol 6. Cl! hydrocarbon 1. 2C dime!hylanlllne 8 naphlhalsns 9. GI2 hydrocarbon 2

i

-1

17.0 mln.

i

Figure 14 Figure 15 Figure 16 Col: 25 M. x 0.53 mm I.D., 5.0 micron film. Crosslinked methyl s i l i c o n e . Figure 14 gas v e l o c i t y = 27.8 cm/sec, Figure 15 = 59.5 cm/sec, Figure 16 = 83.3 cm/sec. Note increased speed of analysis as one approaches optimal v e l o c i t i e s f o r operating in t h e packed column mode, Figure 16. Also observe complete absence of t a i l i n g of t h e component bands.

tl.omln.

Figure 17

Figure 18

a

Figure 1 7 : Pestcides. Column : 15 M. x 0.53 mm I.D., 3.0 micron film. Crosslinked methyl 50% phenyl s i l i c o n e . Temp=24O0C ; press=3 p . s . i . (He); chart=60 W h r ; t o t a l sample injected onto column - 45 micrograms. Figure 18: Free f a t t y acids i n water. Column : 25 M. x 0.53 mm 1.0.3.0 micron film, crosslinked FFAP. Temp=17O0C; velocity=84cm/sec ( H 2 ) ; attn=X32; chart=60 cm/hr; t o t a l sample injected onto column - 40 micrograms.

216

Figure 19 Phenols: Column: 15 M. x 0.53 mm I.D., 3.0 micron film. Crosslinked methyl 50% phenyl silicone. Temp=lOO"C/l min. hold, 210°C at 8"C/min.; press=3 p.s.i. (He); chart=60 cm/hr

15

Figure 20 Mixture of drugs. Column: 25 M. x 0.53 mm I.D., 5.0 micron film. Crosslinked methyl silicone. Temp=24OoC-270"C/8"C hold at 270°C; press=4 p.s.i. (He); chart=60 cm/hr. Peaks: l=Amobarbitol, 2~Pentobarbitol, 3=Secobarbitol , 4=Phenobarbitol , 5=Methadone, 6=Cocaine, 7=Codeine, 8=Morphine.

2

4

7

.1

L>

L 18.3 mln.

)I

277

Figure 21 Alcohols. Column: 25 M. x 0.53 mm I.D., 3.0 micron film. Crosslinked Carbowax 20M. Temp=7O0C; press=2 p.s.i. (H2); attn=X64, chart=60cm/hr. Peaks: l=methanol, Z=Z-methyl-Z-propanol 3=2-propanol, 4=ethanol, 5=2-methyl-Z-butanol, 6=2-butanol, 7=l-propanol, 8=2-methyl-l-propanol, 9=3-propanol lO=Z-pentanol, 11=1 -butanol, 12=2-methyl-l-butanol, 13=3-methyl-l-butanoly 14=l-pentanol.

1

a

11

10

1 These columns make an excellent addition to the standard capillary columns (0.25 mn I.D.) with 'standard' films (0.25 microns) as well as small bore (100

micron) columns with thin films (0.1 microns) as noted in Figure 22.

NON-POLARTEST MIXTURE Column Tamp. Press. Chart

ISM BONDED METHYL SILICONE 100 MICRON ID FUSED SILICA

im'c 38p.r.i. HS 240 Crmhr

Peaks I n.nooedecane 2 2-oclanone 3 mdecane 4 1.ocImol 5 2.8 dimethylphenol 6 n.undscane

7 2.4 dlmelhylDnlllne 8 naphthalene 9 ndodecane

F i g u r e 22 Note t h e r a p i d a n a l y s i s t i m e on t h i s excellently deactivated t h i n f i l m column.

X

I--

2.61nina.

*I

F i n a l l y , i t i s apparent t h a t a small group o f s c i e n t i s t s across t h e w o r l d were most i n s t r u m e n t a l i n p r o p a g a t i n g t h e e x c e e d i n g l y r a p i d advances made i n t h i s area. F i r s t and foremost, o f course was t h e H e w l e t t Packard Group l e d by Dandeneau and h i s c o l l e a g u e s Zerenner (1, 2, 41), Bente, Rooney and Hiskes, soon t o be j o i n e d by S t a r k , L a r s o n (26, 31, 42) and Ryder e t a1.(40).

Lars

Blomberg (20, 21, 34, 43-52) and h i s young s t a f f , q u i e t l y w o r k i n g away f o r y e a r s on t h e s y n t h e s i s o f new polymers t o be used as c r o s s l i n k e d s t a t i o n a r y phases f o r c a p i l l a r y columns soon found t h e i r areas o f r e s e a r c h t o be i n t h e f o r e f r o n t o f t h i s e f f o r t . S i m i l a r l y , L e e ' s group (4, 14, 35, 53-56) a t Utah, Sandra and V e r z e l e (24, 57-60) i n Belgium, and Schomberg (27, 61) i n Germany, a l l made i m p r e s s i v e c o n t r i b u t i o n s which h e l p e d p r o p e l t h e r e v o l u t i o n . Many key developments were based d i r e c t l y o r i n d i r e c t l y on t h e many v e r y i m p o r t a n t observ a t i o n s made by t h e Grobs (3, 5, 10, 22, 23, 25, 33, 62) who became i n v o l v e d i n s t u d y i n g t h e f a b r i c a t i o n and t h e use o f g l a s s c a p i l l a r y columns

-

over a

span o f many y e a r s . F i n a l l y , t r i b u t e must be p a i d t o t h e H e w l e t t Packard Comp a n y ' s Management and t h e i r e n l i g h t e n e d p a t e n t p o l i c y which p e r m i t t e d t h e f r e e

279

e n t e r p r i s e system t o f l o u r i s h . Without i t , t h e i n t e n s e good natured r i v a l r y amongst i n d i v i d u a l s c i e n t i s t s a l l over t h e world, which f u e l e d these r a p i d developments i n t h e r e l a t i v e l y s h o r t p e r i o d o f f i v e years, would n o t have occurred. Thus, another f i n e chapter i s being w r i t t e n i n t h e f i e l d o f gas chromatography. With t h e passing of time, those o f us who have been a c t i v e i n t h e f i e l d f o r many y e a r s

-

o f t e n t h i n k t h a t much

o f t h e m a j o r advances have a l r e a d y co-

me t o pass. Needless t o say, we a r e most p l e a s a n t l y s u r p r i s e d t o once again f i n d t h e emergence o f y e t another major advance t h a t c a p t i v a t e s our i n t e r e s t . Some t h i r t y years l a t e r , Professor M a r t i n ' s i n v e n t i o n o f Gas Chromatography s t i l l continues t o p r o v i d e enormous challenges t o c o u n t l e s s numbers o f r e s t l e s s souls w o r l d wide. Those o f us, g i v e n t h e o p p o r t u n i t y t o p a r i c i p a t e i n t h e perpetual quest f o r f u r t h e r knowledge here, are, indeed, enormously g r a t e f u l t o him f o r t h i s wonderful legacy. SUMMARY Approximately f i v e years a f t e r t h e announcement o f t h e use o f f l e x i b l e fused s i l i c a g l a s s t u b i n g f o r c a p i l l a r y column gas chromatography, evidence developed from a group o f l a b o r a t o r i e s i n Europe and i n t h e U n i t e d S t a t e s , now leads us t o b e l i e v e t h a t t h e fused s i l i c a g l a s s s u r f a c e can be e x p e d i t i o u s l y coated by an ever i n c r e a s i n g v a r i e t y o f new, p r o p e r l y designed nonpolar and p o l a r s t a t i o n a r y phases. When these phases a r e r e a d i l y c r o s s l i n k e d and bonded t o t h e s u r f a c e of t h i s unique glass, these columns e x h i b i t o u t s t a n d i n g chromat o g r a p h i c p r o p e r t i e s . When t h e q u a l i t y o f these c a p i l l a r y columns and t h e

=

o f t h e i r f a b r i c a t i o n a r e taken i n t o account, they have t o be considered t o r e -

-

present t h e ' i d e a l ' c a p i l l a r y

co=

-

f o r a l o n g t i m e t o come. Moreover, r e -

cent developments i n v o l v i n g t h i c k , c r o s s l i n k e d f i l m s o f a new g e n e r a t i o n o f s t a t i o n a r y phases on wide bore fused s i l i c a columns, l e a d us t o b e l i e v e t h a t

--

they, too, can be looked upon as t h e i d e a l column f o r f i n a l l y r e p l a c i n g , where a p p l i c a b l e , t h e conventional packed column and i t s many f a u l t s . Indeed, c o n s i d e r i n g t h e r e l a t i v e l y s h o r t t i m e i t took t o reach t h i s stage o f development, t h e dramatic r e s u l t s t h a t have been o b t a i n e d t o d a t e has caused a r e v o l u t i o n i n t h e f i e l d o f gas chromatography.

280

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283 DISCONTINUOUS SYSTEMS I N THE COUNTER CURRENT D I S T R I B U T I O N (CDD).The

G.B.

use o f d i s c o n t i n u o u s m o b i l e phases

M A R I N I BETTOLO

Oipartimento d i Biologia Vegetale U n i v e r s i t a d i Roma " L a S a p i e n z a " Centro Chimica d e i R e c e t t o r i e d e l l e Molecole Biologicamente Attive, Cuore, C.

I s t i t u t o d i Chimica, U n i v e r s i t a C a t t o l i c a d e l Sacro Roma ( I t a l y )

GALEFFI

L a b o r a t o r i o d i C h i m i c a d e l Farmaco, Sanita,

I s t i t u t o Superiore d i

Roma ( I t a l y )

C h r o m a t o g r a p h i c m e t h o d s h a v e made p o s s i b l e i n t h e l a s t f i f t y y e a r s t h e s e p a r a t i o n o f c o m p l e x m i x t u r e s b o t h o n a n ana-

l y t i c a l and a p r e p a r a t i v e scale. Column c h r o m a t o g r a p h y b a s e d on a b s o r p t i o n s t a t i o n a r y phase, whereas

uses a s o l i d

n t h a t based on p a r t i t i o n between

two l i q u i d phases t h e l i q u d s t a t i o n a r y phase i s s u p p o r t e d on a solid. I n b o t h cases t h e s o l i d - s o l u t e v e r s i b l e a b s o r p t i o n and/or

i n t e r a c t i o n may c a u s e i r r e -

c h e m i c a l m o d i f i c a t i o n s o f t h e com-

ponents o f t h e m i x t u r e submitted t o separation. Moreover t h e technique r e q u i r e s a l o n g time and d i s p l a y s a r e l a t i v e l y l o w r e s o l u t i o n power. These d i s a d v a n t a g e s do n o t o c c u r i f t h e s e p a r a t i o n i s performed b y two-phase systems i n c o u n t e r c u r r e n t ' processes s u c h as t h a t p r o p o s e d b y C r a i g a n d C r a i g ( 1 ) .

There are d i f -

f e r e n t a p p a r a t u s w h i c h a c h i e v e mu1 t i s t e p d i s t r i b u t i o n s o p e r a t i n g e i t h e r by s i n g l e withdrawal counter-current

distribu-

t i o n (CCD) o r b y a l t e r n a t i v e w i t h d r a w a l c o u n t e r - c u r r e n t s t r i b u t i o n ( 2 ) o r by counter double c u r r e n t d i s t r i b u t i o n (CDCD) ( 3 ) .

di-

284

D r o p l e t c o u n t e r - c u r r e n t c h r o m a t o g r a p h y (DCC) i s a r e l a t i v e l y r e c e n t m e t h o d w h i c h u t i l i z e s t h e u p w a r d o r d o w n w a r d movement o f d r o p l e t sequences o f one phase ( l i g h t e r o r h e a v i e r , respective1y)through

a s t a t i o n a r y phase i n t h i n tubes ( 4 ) .

I n t h e p r e s e n t p a p e r we w i l l s u r v e y t w o p a r t i c u l a r a s p e c t s o f t h e CCD w h i c h have been a p p l i e d s u c c e s s f u l l y b y us d u r i n g the l a s t f i f t e e n years t o t h e preparative separation o f i o n i c a n d n e u t r a l s u b s t a n c e s f r o m many c o m p l e x m i x t u r e s , plant extracts, ges,

u s i n g a Post-model

generally

C r a i g apparatus (200 sta-

10 m l v o l u m e o f b o t h u p p e r a n d l o w e r p h a s e ) .

Separation o f b a s i c and a c i d i c substances by d i s c o n t i n u o u s pH c h a n g e o f t h e m o b i l e p h a s e T h e s i n g l e w i t h d r a w a l 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 (CCD) i s p a r t i c u l a r l y c o n v e n i e n t f o r t h e s e p a r a t i o n o f b a s i c and a c i d i c s u b s t a n c e s when a n o r g a n i c s t a t i o n a r y p h a s e i s u s e d a n d a b u f f e r s o l u t i o n w i t h pH g r a d i e n t c o n s t i t u t e s t h e m o b i l e p h a se

(*I. I n 1 9 6 9 ( 5 ) we c o u l d d e m o n s t r a t e t h a t t h e u s e o f s u c h a

system c o u l d enable us t o separate a v e r y complex m i x t u r e o f a l k a l o i d s a n d e v e n t o i s o l a t e f o u r new s u b s t a n c e s f r o m t h e e x t r a c t o f a p l a n t , S t r y c h n o s nux vomica, In red

s t u d i e d s i n c e 1817.

he case o f t h i s procedure two e q u i l i b r i a must be consideone o f p a r t i t i o n o f t h e u n d i s s o c i a t e d species between

t h e o r g a n i c and t h e aqueous phases,

t h e o t h e r of d i s s o c i a t i o n

due t o t h e i o n i c n a t u r e o f t h e s o l u t e . The f u n c t i o n t h a t c o n t r o l s t h e d o u b l e d i s t r i b u t i o n a n d t h e d i s s o c i a t i o n e q u i l i b r i u m i n t h e c a s e o f a weak b a s e g e n e r a l l y a non q u a t e r n a r y a l k a l o i d

-

-

i s t h e f o l l o w i n g when

t h e volumes o f t h e two phases a r e e q u a l : 1o g

11 1

where K

i s t h e p a r t i t i o n c o e f f i c i e n t , i.e. t h e r a t i o of t h e r c o n c e n t r a t i o n s o f t h e und s s o c i a t e d s p e c i e s between t h e aqueous

and t h e o r g a n i c phase,

Kg

i s the dissociation constant o f the

285

Logarithm of the reciprocal of the extraction codficient ct - [B+]/ [B+] as a function of the p H corresponding t o unitary' values of thc tcrm ]fix : /<,,,/ (K,.Kb)}.From ref. 5 .

3

4

5

6

7

PH

8

Concentration of the aqueous phase [B+]as a function of the pH corresponding to unitary values of the term log {Ii,(,/ (h','Kb)}. From ref. 5 .

286

base;

c

W

and c

0

a r e r e s p e c t i v e l y t h e c o n c e n t r a t i o n s o f t h e so-

l u t e i n t h e aqueous and o r g a n i c phase. The f o l l o w i n g e x p r e s s i o n o b t a i n e d b y s t a t i s t i c a l

calcu-

l a t i o n s f o r C C D f o r a weak b a s e c o r r e l a t e s t h e n u m b e r o f transfers

1, t h e

p o s i t i o n o f t h e d i s t r i b u t i o n c u r v e maximum

r , w i t h t h e pH ( 6 ) log

r

= n-r

-

Kr Kg pH + l o g ___ kW

The a b o v e e x p r e s s i o n a p p l i e s toanaqueous m o b i l e p h a s e , w h e r e a s f o r an o r g a n i c m o b i l e phase i t i s :

131

m

Thus t w o bases w i t h a K .K product i n a 1:lO ratio r B s h o u l d b e e l u t e d a t c o n s t a n t pH w i t h a n u m b e r o f t r a n s f e r s i n t h e same r a t i o . T h e r e f o r e i t i s p o s s i b l e p r a c t i c a l l y t o e n v i age t h e p r e parative separation o f a mixture o f t e r t i a r y alka oids,

i .e.

weak b a s e s b y : ( 7 ) 1

-

t h e use o f a steady b u f f e r phase and an upper m o b i l e organ i c p h a s e , whose c o m p o s i t i o n i s p r o g r e s s i v e l y m o d i f i e d ( b y v a r y i n g t h e percentages o f two o r more o r g a n i c s o l v e n t s ) i n order t o vary the K

2

-

r'

t h e bases b e i n g e l u t e d i n o r d e r

o f increasing K K ; r B t h e use o f a s t e a d y o r g a n i c phase and a n aqueous m o b i l e p h a s e c o n s t i t u t e d b y a b u f f e r , w h o s e pH i s d i s c o n t i n u o u s l y varied from n e u t r a l i t y t o increasing a c i d i c values,

in

s u c h a way a s t o e x t r a c t t h e a l k a l o i d s i n o r d e r o f d e c r e a sing K K

r B'

( * ) M c I l v a i n p h o s p h a t e - c i t r a t e b u f f e r has b e e n u s e d as r o u t i n e . I f t h e a l k a l o i d s p r e c i p i t a t e a c e t a t e b u f f e r i s employed. ( * * ) T h e 1 maximum h a s b e e n d e t e r m i n e d e i t h e r b y U . V . m e a s u r e m e n t s o r b y d i r e c t i n s p e c t i o n o f t h e TLC p l a t e s .

m Thus t w o a l k a l o i d s w i t h a K K p r o d u c t i n 1 : 1 0 r a t i o are r B e l u t e d i n a n e q u a l n u m b e r o f t r a n s f e r s a t pH v a l u e s w h i c h d i f f e r by

m.

S i n c e t h e pH r a n g e m u s t b e l i m i t e d b e t w e e n 7 a n d 3 i n o r d e r t o a v o i d t h a t t h e a l k a l o i d s decompose o v e r l o n g p e r i o d s of c o n t a c t w i t h b a s i c o r t o o a c i d i c s o l u t i o n s and t o guarantee t h e absence o f e m u l s i o n s , f o r counter-current separation,

the product o f

K K

r B’ must be i n t h e range between

l o m 7 and F o r t h e s e p a r a t i o n o f v e r y weak b a s e s i t i s n e c e s s a r y t o h a v e a relatively high K

v a l u e ( u n f a v o u r a b l e o r g a n i c s o l v e n t ) whir ( v e r y f a v o u r a b l e o r g a n i c s o l v e n t ) i s necessa-

l e a very low K r r y f o r more b a s i c compounds. For the procedure 2-,

i t was f o u n d t h a t t h e m o r e s u i t a b l e

o r g a n i c s o l v e n t f o r a s t a t i o n a r y p h a s e was c h l o r o f o r m o r m i x t u Yes w i t h o t h e r c h l o r i n a t e d

w a t e r and l e s s f a v o u r a b l e ,

solventsorwithsolvents 1i g h t e r than provided the s p e c i f i c g r a v i t y o f t h e

m i x t u r e i s always higher than 1 ( a t l e a s t 1.3). The p r o c e d u r e d e s c r i b e d a t p o i n t 1 - , o r g a n i c phase,

i.e.

h a s n e v e r b e e n u t i l i z e d by u s ,

an upper mobile f i r s t l y because

l i g h t organic solvents generally give K higher than chloroform,

values f o r a l k a l o i d s r a n d t h u s make i t n e c e s s a r y t o w o r k i n

t h e a l k a l i n e pH r a n g e , a n d s e c o n d l y b e c a u s e i t r e q u i r e s v e r y l a r g e v o l u m e s o f o r g a n i c s o l v e n t s as m i x t u r e s . The p r o c e d u r e d e v i s e d b y u s f o r t h e s e p a r a t i o n o f c o m p l e x a l k a l o i d m i x t u r e s based on t h e use o f b u f f e r t r a i n s o f decreas i n g pH ( t r a n s i e n t p a r a m e t e r ) f o r t h e m o b i l e u p p e r p h a s e h a s been a p p l i e d t o l a r g e s c a l e p r e p a r a t i o n o f t h e m a j o r components as w e l l a s t o t h e i s o l a t i o n o f m i n o r compounds.

The s e p a r a t i o n

i s monitored by t h i n l a y e r chromatography and according t o t h e

r e s u l t s pH v a l u e i s e i t h e r m a i n t a i n e d o r d e c r e a s e d . The p r o c e dure thus allows t h e q u a n t i t a t i o n o f the separation, c u l a t i o n of

t h e K,KB

the cal-

p r o d u c t s and a p e r f e c t p r e p a r a t i v e sepa-

r a t i o n of a l l t h e components i n a s i n g l e o p e r a t i o n .

288 TWO

alkaloids,

o n e o f whose K K

rB

p r o d u c t s i s 50% h i g h e r

t h a n t h e o t h e r , c a n b e s e p a r a t e d a t 9 9 % i n o n e day by a 200 tube (theoretical plates) apparatus

i n 400 t r a n s f e r s .

I n o r d e r t o r e a l i z e t h e l a r g e p o s s i b i l i t i e s of t h i s proc e d u r e i t i s s u f f i c i e n t t o m e n t i o n t h a t t h e r a n g e o f KB o f t h e seventy a l k a l o i d s o f m a r a n t h u s roseus i s between

lo-''

and t h a t t h e r a n g e o f K K

r B

and

i s e v e n much g r e a t e r .

I n t h e r a r e c a s e s when t h e d i f f e r e n c e i n K K

r B

between

t w o a l k a l o i d s i s l e s s t h a n 5 0 % , t h e s e p a r a t i o n may b e a c h i e ved e i t h e r by r e c y c l i n g o r by changing t h e o r g a n i c s o l v e n t . This l a t t e r operation usually a f f e c t s the K

r

values o f t h e

for

two a l k a l o i d s i n d i f f e r e n t p r o p o r t i o n s . Thus c h a n g i n g example t h e l o w e r phase f r o m c h l o r o f o r m t o t h e m i x t u r e (65:35,

v:v) chloroform-ethyl acetate,

c l o s e l y r e l a t e d compounds,

the e x i t order o f the

as b r u c i n e , w a n d 8 - c o l u b r i n e s ,

i s reversed (5). T he f o r m u l a w h i c h p e r m i t s t h e c a l c u l a t i o n tage o f a substance

px i n

every tube a t a distance

maximum o f t h e d i s t r i b u t i o n c u r v e a f t e r 1owing :

--

= X

where

2 and

m

X

of t h e percen-

n

from the

transfers i s the fol-

2

- e L m

141

2.rrnms

a r e t h e s o l u t e f r a c t i o n s a f t e r one p a r t i t i o n i n

t h e s t a t i o n a r y a n d m o b i l e p h a s e , r e s p e c t i v e l y . F o r a weak b a s e they a r e expressed by t h e f o l l o w i n g equations ( 6 ) :

where C

t

i s the total concentration.

T h e o r e t i c a l d i s t r i b u t i o n c u r v e s and t h e e x p e r i m e n t a l ones f o r d i l u t e d s o l u t i o n o v e r l a p i n g o o d a g r e e m e n t (6)(8). S i m i l a r c o n s i d e r a t i o n s c a n b e a p p l i e d t o t h e s e p a r a t i o n s o f weak

acids,

f o r which the mathematical expressions o f t h e p a r t i t i o n a r e q u i t e s i m i l a r t o t h o s e o f weak b a s e s . T h e r e f o r e we w i l l n o t

289

TABLE 1

KrKB VALUES OF ALKALOIDS PARTITIONED BETWEEN AN ORGANIC STATIONARY PHASE

AND MOBILE PHASE CONSTITUTED BY BUFFERS Alkaloid

Source ( " )

Statppy

S t r y c hnos nux vomi ca 15- hydroxy- s t r y c hn ine I, Isos t r y c hni ne (X2 1 ,I Strychnine I, a-colubrine (*) I1 B-colubrine ( * ) I, Brucine (*) Pseudostrychnine ,I 11 Pseudo-u -colubrine(X ) 3 II Pseudo-8-col u b r i n e (X4) Pseudobruci ne I, I c aj i n e II Vomi c ine I1 Novaci ne I, I s o s t r y c h n i ne Acetyl is o s t r y c h n i ne II 15- hydroxys t r y c h n ine 15-acetoxystrychnine 11-methoxydiaboline Strychnos romeu-belemi 11-methoxy-Wieland-Gumlich aldehyde Isocondensamine 1 1 ,I1 '-dimethoxycaracurine Tabascanin Strychnos tabascana 0-methyl ,N-acetylI, strychnospl end i n e I, Strychnobrasil ine I1 Acety 1taba scan in 10-methoxystrychnoI1 brasiline Strychnos medeol a 11-methoxydiabol i n e ,I Normacusi ne 0-acetylnormacusine Strychnine Strychnos panamensis I, Brucine 11-methodiabol i n e Strychnos amazonica I, Normacusi ne

K r KB

References 5

CHCl

I1

II

,I 'I

8.1 X I 0-l'

12 II

5 I1 ,I

I' 'Ij

4. Ox 10-1 2.5~10-'I

8, I, ,I

I1

II

,I

I,

I1

" " I,

I' 'I

I1

-9 8 . 0 ~ 1 0 ~ ~13~ I, 1 . 5 ~ 0-8 1 2.0xl o_l 14 I, 5 . 4 ~1 0 -8 6.0~10 15

-8 9.0x10_9 (**) 4.0~10_~ CHC13 2 . 5 ~ 1 0 - ~ " 1 .ox10 II

I, ,I

(***I

I1

16

I,

17 I1 I,

18 I1

I,

"

I1 I,

4 . 2 ~ 1-90 - ~ ~ I 1 I, 7.1~10-~~ ,I 5.0~10

(***) 2.6~10:: CHC13 6 . 0 ~ 1 0 _ ~ " 1 .5x10m1 " 2.0x10

'I

I1

-8 6 . 3 ~1 0-8 1.2x10

19 ,I

The q u o t a t i o n marks i n t h e column o f t h e Source are m i s s i n g when a d e r i v a t i v e i s reported. (*) Recovered non-separated from C H C l and subsequently separated by 3 C H C l -EtOAc: (65:35) i n t h e f o l l o w i n g o r d e r : brucine, a - c o l u b r i n e , 3 8-colubrine. (**) CHC13-EtOAc 7:3 (***) CHC13-EtOAc 6 5 ~ 3 5 ( O )

290

A1 k a l o i d

KrKB References Stationary phase -7 Strychnos brachiata C H C l 2 . 7 ~ 1 0 - ~ 19 I, II 6 . 0 ~ 10-8 Strychnos f e n d l e r i 9x1 0 20,21 Source

Wieland-Gum1 i c h aldehyde 11-methoxydiaboline Strychnofendl e r i ne (") diaboline ( O )

II

11-methoxystrychnoferdlerine 12-hydroxy-1 l-methoxystrychnofendl e r i ne N ( a ) - a c e t y l s t r y c h n o s p l endine Spermostrychnine ( 0 0 ) N ( a 1-ace t y 1 12- hydroxy-

-

11-methoxystrychnosplendine Henningsamine N( a)-desacetyl strychnof e n d l e r i ne 10 -hydroxyni g r i t a n i n 18-dehydro-10-hydroxyn i g r itanin Nigritani n 18-dehydronigri t a n i n 18-dehydro- 1O-methoxyn ig r it a n .i n 10-acetoxynigritanin 10-hydroxyakageri ne Akagerine K r i b i ne Anhydroakageri ne 21 - d i hydroakagerine 1 1-methoxydiabol ine Akaqerine Diabol ine Akageri ne Akageri ne 11-methoxydiabol i n e Normacuri ne Strychnorubi g i ne 0-acetyl s t r y c h n o r u b i g i n e Strychnohirsutine

I,

5 . 2 ~10-8

0,

"

I,

-7 7 . 0 ~ 10-9 7.3~10

Strychnos n i g r i t a n a

,I

22 II

-9 1 . 8 ~ 1 0 - ~" ~ 5.9~10 'I 1 .4x1O-l0 'I

,I II II

Strychnos spinosa I1 I,

Strychnos gardneri I,

Strychnos j o b e r t i a n a ,I

" ,I

Strychnos p a r v i f o l ia Strychnos r u b i g i nosa

'I

I,

I1

II

I1

#I

Strychnos h i r s u t a "

I' I1

(*-*I

-10 I1 2.0xl o-l 5 . 4 ~ 10-7 'I 1.8~10 23 'I 7 .Ox1 0 I 1 1 . 8 ~ 1 0 - ~" ~ 6.2 x 10" 7 . 0 ~ 1 0 - ~ 'I 6 . 0 ~ 1 0 - ~ 24 7 . 0 ~ 1 0 - ~ 'I 9.0x10-8 " 7 . 0 ~ 1 0 - ~ 'I 7. Ox 10 'I 6 . 0 ~ 1 0 - ~ 25 1 .2x10-9 " 1 .4x10m12 " 8 . 8 ~ 1 0 - ~ I' 1 . 5 ~ 1 0 - ~26~ I' 6.3~10 4.5~10-l~

Separated by CCD between b u f f e r a t pH 766 and EtOAc phase: s t r y c h n o f e n d l e r i n e , K K 6 . 4 ~ 1 0 - , d i a b o l i n e r B Separated by CCD on r e c y c l e between b u f f e r a t pH 7 as mobile phase N(a)-acetylstrychnosplendine, KrKB -7 and spermostrychnine, K K 2 . 0 ~ 1 0 r B CClq-cyclohexane 3:2

.

(*-*I

,I

-9 1 . 5 ~ 1 0 - ~" ~ 5.0~10 I'

I,

O-acetyltetradehydrostrychnohi r s u t i ne

( O 0 )

I,

II

I1

I,

Tetradehydrostrychnohirsutine

("1

"

-8 4 . 5 ~ 10-9 4x1 0

II

(OO)

I,

as m o b i i e 1.3~10-

26

.

and EtOAc 2.5~10-7

291

A1 k a l o i d

S t a t iona r y phase Strychnos castelnaeana C H C l Source

Diabol i n e 3-hydroxydiabol i n e

I,

II

a-0-acetyl-3-hydroxy d i a b o l ine

-8 9. ox10-9 4.0~10

2 . 3 ~ 1-11 0-~

N-methyl-sec-pseudodiaboline

,I

S t r y c hnos a1 v i m i ana Ta bascani n ,I A l v i m i ne ,I Strychnobrasi 1 ine I, Acetyl tabascanin I, Alviminine ,I S t r y c hnosi 1 ine Strychnos henningsi i Diaboline ,I Hol s t i i n e " N( a ) - a c e t y l s trychnospl endine I, N ( a ) - a c e t y l - l I -methoxyI, strychnospl endine 0-ace t y l hol s t ine D i h y d r o e r g o c o r n i n e D i h y d r o e r g o t o x ne (hydergi ne) I, D i h y d r o e r g o c r y p t i ne It D ihydroergocristine Chanocl a v i ne I Claviceps purpur a

I, I, I,

II $1 ,I I, I, I,

8, I#

"

I,

1.3~10-~ " 1 . 4 ~ 1 0 - ~ 28 1 . 3 ~ 1 0 - ~" ~ 6 . 3 ~10" " 4. Ox 107.6~10-~ " 4.6~10-~ " 9.0x10-9 29 6.1 x l 0-9 " 1 . 5 ~ 1 0 - ~" ~ 2.9x 10 " 5.4~10 "

-1 0 CHC13-CC141 . 6 x 1 0 8 1 :1 -1 1 I, 5 . 0 ~ 1 0 - ~" 8, 3.2~1 "

c8

CHCl -n3 PrOH 2 : l

I#

CHC13-CC14 1 :1 I8 I, I, I,

Claviceps f u s i f o r m i s

27

,I

I,

Isochanoclavine E l ymocl a v i ne Agroclavin'e Ergotamine Ergocorni rte Ergotami n i ne Ergocryptine Ergoc r y p t in ine, Chanoclavine I

References

Kr KB

C H C l -n-

9x10

30

4 . 1 ~ -8 14~ " 3 . 2 ~ 10-1 1 . 4 ~ 1 0 - ~ "~ 7x10

I,

-12 1 .3x10-t2 4 ~ 1 0 - ~ ~,, 8.8~10" 7x1 0-8 I, 9.0~10 31 ,I

I,

-Pro$ 2:1 I ,I 9.0x10-8 " Chanoclavine I 1 I, I, 4.1 x l 0-9 -3 I, Isochanoclavine 3 . 2 ~ 1 0-1 I' fl y mo c 1av ine 6, 1 .4x10 ,I Agroclavi ne * ) Separated by r e c y c l i n g w i t h CHC13-EtOAc 1 : l and b u f f e r a t pH 8.3:chano~ chanoclavine I, K r K b 2 . 4 ~ 1 0 - 2 c l a v i n e 11, K KB 3 . 2 ~ 1 0 - and r

*

-

+) Separated by CCD w i t h buffer o f pH-7 and EtOAc as mobile phase:-8 7-O-demethylpeinamine, K * K 1 . 6 ~ 1 0 7 , and macolidine, KrKb 2x10 (Note t o t h e f o l l owi ng pabeb,.

.

292

R,R-isochondodendrine R ,R- 12 -0-methyl c u r i ne R ,R-0,O-dimethyl c u r i ne Bs~&&&&%bPYie R ,R-7 I -0-acetyl -1 1 4.0~10-~ -1 2 -0-methyl c u r i ne 1 .OxtO-10 Pei-nam6 curare Peinamine 5.0~10N-methyl peinamine 6 .Ox1 00-methylpeinamine 1 .2X1Oe9 N,O-dimethylpeinamine+ 3.5~10-~ Abuta g r i s e b a c h i i 7,0-demethyl pei nami ne I1 3 .5x10a9 macol i d i ne+ I1 1.Oxl o-l pei nami ne 6.3~10 N-methyl ,7-0-demethylpeinamine 0,O'-dimethylmacolidine 2- (p-hydroxyphenyl- B e t h y l ) - 4 , 5 -9 I1 5 . 4 ~ 10-1 dimethoxyphenyl -B-ethyl dimethylami ne 5.6~10-~ S-7-demethyl -O-methylarmepavi ne 35 I, 2.2x1 Oe9 Isochondodendrine Sciadotenia t o x i f e r a I1 I, 1 . 8 ~ 1 0 ~ ~ ~ S inome n ine 11 I, 9 .ox1 o-l I' Sciadenine ,I I, 7 . 0 ~ 1 0 - ~ I' S c i a d o f e r i ne II I, 8 . 0 ~ 1 0 - ~ , I' Sci ado1 ine ,I II ,I 2.Oxl Oe8 Unknown a1 k a l o i d I, 4 . 0 ~ 1 0 - ~ ,I Norarmepavi ne ,I 4 . 9 ~ 1 0 - ~ ~" Armepavi ne I, It 4.7~10 Cycl ea n ine I, -10 1 .ox1 o-8 0-methylsciadenine N,N-dimethyl-5-methoxytryptamine Yanodma d a r t poison ' I 4 . 0 ~ 1 0 - ~ 36 3 . 4 ~ 1 0 - ~ 11 Peruvian curare R,S-nor-Nb-chondrocurine 1 . 5 ~ 1 0 ~'I ~ ~ R,R-curine 5.3~10-~ I, R ,S-c hond rocu r ine ,I 5.5~ 10" R,S-dimethyl chondrocurine I1 3 . 5 ~ 1 0 ~ 37 ~ A l s t o n i a boonei N- (a)-formyl echi tami d i ne I, 11 2.3~10 I' Echi tamidine N- (a) f ormyl 1 2-me thoxy7.0x10:11 " echi tamidine 3.2~10 " 12-methoxyechi t a m i d i ne 0-acetyl N (a ) -f ormylI1 8.0~10::~ " 1 2-met hoxyec h i tam id ine 3.0~10 38 H y d r a s t i s canadensis 'I Unknown a1 k a l o i d Bis-0,O'-demethyl -S-9 I, II 1 .oxlo~lo tetrahydropalmat ine ( ?) II 0, 4 . 0 ~ 1 0 - ~ I'~ Canada1 ine ,I I1 1 . 6 ~ 1 0 - ~I' ~ Isohydrastidine I, I1 8 . 0 ~ 10-1 " Hydras t i d i ne 0, I, 3 Ox 10" S- isocorypalmi ne ,, I, 8.0~10I' S-corypalmi ne I, I, 6.0~10" B -hydrastine I, I, 2.0x10 Canadi ne

-

-

-

-

-

.

293 r e p o r t them h e r e b u t r e f e r t o t h e c i t e d l i t e r a t u r e ( 6 ) . I n conclusion

t h i s method,

by t h e proper choice o f t h e s t a t i o -

n a r y o r g a n i c s o l v e n t ( g e n e r a l l y c h l o r o f o r m and l e s s frequent l y i t s mixture with other solvents),

e n a b l e s us t o o p e r a t e

w i t h b u f f e r s b e t w e e n pH 7 a n d 3 a n d t o p e r f o r m t h e m o s t comp l e x m i x t u r e f r a c t i o n a t i o n o f a1 k a l o i d s on a s i n g l e o p e r a t i o n b y d e c r e a s i n g t h e pH o f t h e m o b i l e p h a s e d i s c o n t i n u o u s l y . I n o t h e r w o r d s t h e d i s c o n t i n u o u s c h a n g i n g o f t h e pH i n a s i n g l e o p e r a t i o n m o d i f i e s t h e s o l u t i o n p r o p e r t i e s o f t h e solute,

i.e.,

changes t h e i r e x t r a c t i o n c o e f f i c i e n t ,

which, d i f -

f e r e n t l y from t h e p a r t i t i o n c o e f f i c i e n t , i s t h e r a t i o between t h e sum o f t h e c o n c e n t r a t i o n s o f t h e d i s s o c i a t e d a n d u n d i s s o c i a t e d s p e c i e s o f t h e s o l u t e i n t h e u p p e r a n d l o w e r pha'ses. L i k e w i s e t h e change o f t h e s o l u t i o n p r o p e r t i e s o f a s o l u t e can be achieved by f o r m a t i o n o f complexes w i t h d i f f e r e n t agents.

I n t h i s case t h e e x t r a c t i o n c o e f f i c i e n t i s t h e r a t i o

b e t w e e n t h e sum o f t h e c o n c e n t r a t i o n s o f t h e c o m p l e x e d a n d non-complexed s p e c i e s i n t h e upper and l o w e r phases. S e p a r a t i o n b y d i s c o n t i n u o u s change o f t h e p o l a r i t y o f t h e m o b i l e phase The same v e r s a t i l i t y o f t h e C C D m a y b e u s e d i n t h e s e p a r a t i o n o f n a t u r a l mixtures o f substances n o t s i g n i f i c a n t l y d i s s o c i a b l e o r complexable. des o f t h e p r o b l e m .

I n t h i s c a s e we m u s t c o n s i d e r t w o s i -

Primarily,

the availability o f different

two-phase systems o f s o l v e n t s f o r t h e whole range o f p o l a r i t y , i.e.

from t h e l e a s t hydrophobic substances up t o t h e most hy-

d r o p h i l i c ones, which, by o r g a n i c s o l v e n t s , Secondly,

h o w e v e r may b e e x t r a c t a b l e f r o m w a t e r

f o r instance by higher alcohols.

the p o s s i b i l i t y o f changing the p a r t i t i o n c o e f f i -

c i e n t (here i d e n t i c a l w i t h the e x t r a c t i o n c o e f f i c i e n t ) i n s u c h a way a s t o c a r r y o n t h e s e p a r a t i o n i n a s i n g l e o p e r a tion,

a t l e a s t i n a l i m i t e d range o f p o l a r i t y . I n r e g a r d t o t h e f i r s t f e a t u r e t h e p r o p e r c h o i c e of

the

s o l v e n t s y s t e m m u s t a c h i e v e , a t l e a s t f o r o n e compound o f a

294

m i x t u r e , a comparable p a r t i t i o n o f t h e s o l u t e between t h e two phases, expressed by ! I! a n d

2,

i.e.

t h e f r a c t i o n a l amounts pre-

s e n t r e s p e c t i v e l y i n t h e m o b i l e and s t a t i o n a r y phase: K

m=

1

r -

and

Kr+l

; =s

161

Kr+l

I n f a c t i n t h e m u l t i s t e p separation o f two s o l u t e s w i t h close values o f the p a r t i t i o n coefficients,

t h e maximum s e l e -

n e a r one. The v a l u e r can be c a l c u l a t e d a f t e r n t r a n s f e r s by the f o l l o w i n g

c t i v i t y i s o b t a i n e d b y o p e r a t ng w i t h K o f Kr

expression:

r

-~ max "r n - r max K

where rmax i s t h e p o s i t i o n o f t h e maximum o f t h e d i s t r i b u t i o n c u r v e . The t h e o r e t i c a l d i s t r i b u t i o n c u r v e c a l c u l a t e d f r o m t h e e x p r e s s i o n [4! above r e p o r t e d corresponds t o t h e e x p e r i m e n t a l i s independent o f the solute concentration, i.e. i f r the p a r t i t i o n isotherm i s l i n e a r , a t l e a s t f o r low concentra-

one i f K

tions. The q u a s i - e q u a l

s o l v a t a t i o n o f t h e p o l a r and non-polar groups

o f a s o l u t e between two phases can be sometimes a c h i e v e d b y b i n a r y s o l v e n t s systems ( 9 , l O ) b u t even more e a s i l y b y t e r n a r y o r higher order s o l v e n t mixtures by changing t h e mutual r a t i o s . It i s ,

however, a d v i s a b l e t o o p e r a t e f a r f r o m t h e condi-

t i o n s o f complete m i x i n g ( p l a i t p o i n t ) o f t h e two-phase system because i t i s two s e n s i t i v e toward t h e changes o f temperature and C o n c e n t r a t i o n o f t h e s o l u t e ; m o r e o v e r t h e t o o c l o s e d e n s i t i e s o f t h e phases r e q u i r e a l o n g t i m e f o r s e p a r a t i o n . The l i s t o f t h e s y s t e m s u t i l i z e d b y u s i n o r d e r o f i n c r e a s i n g h y d r o p h i l i c c h a r a c t e r i s r e p o r t e d i n T a b l e 4. T he s o l v e n t s w e r e s e l e c t e d a c c o r d i n g t o t h e c r i t e r i a o f l o w e r t o x i c i t y , o f t h e s t a b i l i t y and o f t h e easy r e c o v e r y o f t h e s o l u t e by evaporation.

295

TABLE 2 KrKA VALUES OF PORPHYRINS (ACIDS) PARTITIONED BETWEEN A BUFFER (AT pH 6 , l ) AS STATIONARY PHASE AND AN ORGANIC MOBILE PHASE

PORPHY RI N

Coproporphyri n I I I 2t Zn Coproporphyrin I I I 2t Ni Coproporphyri n I I I

MOBILE PHASE Streptomyces sp. A-30 EtOAc-

SOURCE

0,

" I,

/I

" 6,

KrKA

REFERENCES

-6 1.2~10

-7 THF 2 : l 1 . 7 ~ 1 0 ,I I, 4'2x1 0-7

39 I

,I

TABLE 3

Kr VALUES OF NATURAL PRODUCTS FOR DIFFERENT SOLVENT SYSTEMS SUBSTANCE Isoquerci t r i n Q w r c e t i n 3 - a - ( 2-0-8- D -gluco-pyranosyl )-D-glucose

SOURCE

SOLVENT SYSTEM

Kr

Vestia l y c i o i d e s

H20-EtOAc 1:l H20-EtOAc-BuOH

2.3

,I

1.6

2:l:l

C1 eomi n

R itchiea l o n g i p e d i c e l 1a t a

Hypo xo s id e

Hypoxis obtusa

Dimethyl hypoxos i d e

REFERENCE

0.12

H20-CHC13 H20-EtOAc-BuOH 10:8:2 I,

,I

"

0.46 0.28

Hypoxoside aglycone

H20-EtOH-EtOAc Cycl ohexane 10:3:3:10

0.42

Tetramethyl -hypoxos i d e -aglycone

H 0-Acetone2 c y c l ohexane 4:6:7

0.66

I

0.20

Dimethyl -hypoxoside-agl ycone Diacetyl-dimethylhypoxoside aglycone

H20-EtOH-acetone-hexane 10:10:5:20

Dimethyl -hexahydrohypoxoside

H 0-EtOAc-n-BuOH 10:8:2

0.21

0.36

296

SOURCE

REFERENCES

SOLVENT SYSTEM

Kr

H 0-acetone2 c y c l ohexane 10:10:14

0.68

41

0.60

10

A c e t y l boonein

H20-cyclohexane- 0.30 acetone-EtOAc 10:10:10:1,5

I,

I-0x0-6 By7a,1 1 &H-eudesm- Artemi s i a barrelieri

H20-EtOH-nhexane-ace tone 10:1:18:10

SUBSTANCE Oimethyl-hexahydro-hypoxos ide-aglycone Boonei n

A l s t o n i a boonei

H20-CHC1 3

0.54

42

It

1-8-hydroxy-6B,7cr,lIBHeudesm-4-en-6,12-olide-

I1

I1

0.29

B a r r e l ine

,I

ll

0.11

Toddacul i n

Za n t hoxy 1um usambarense

H20-EtOH-nhexane 1:1:2 II

,)

0.65

43

0.43

II

0.73

It

0-methyl c e d r e l ops in

I,

Phellopterin

II

Pimpinell i n

II

I,

0.53

II

Toddal o l a c t o n e

I,

II

0.12

II

8 - a c e t y l shanzhis,ide methylester Shanzhiside methyl e s t e r

H 0-acetone2 -n-hexane 10:10:14

Mussaenda a r c u a t a H20-n-BuOH-EtOAc 0.26 5:4:1 II

P e n t a a c e t y l shanz h i s ide methyl e s t e r Tarennine I x o s i d e 11-methyl e s t e r

,I

44 ,I

0.13

H20-acetone0.8 cyclohexane-EtOAc 10: 1 O : l O : 1 Tarenna graveolens H20-EtOAc-nBuOH 10:9:1 8, H20-sec-BuOH

0.37

44

0.32

I,

1:l Ixoside

II

I,

0.19

I,

Pentaacetyl t a r e n n i ne

H 0-acetone0.48 $y c 1o hexa ne-EtOAc 5:5:5:1

,I

Hexaacetyl shanzhi s i d e

Cycl ohexane-H 0Et OAc- Et OH 0.61 9:8:4.5:4

-

,I

297

SUBSTANCE

SOLVENT SYSTEM

SOURCE

T e t r a a c e t y l i x o s i d e 11methyl e s t e r

Tarenna graveolens

Hal 1erone

Halleria lucida

Kr

Cyclohexane- 0.31 -H O-EtOH-

REFERENCES 44

-EZOA~

Halleridone A c e t y l ha1 1erone

I,

10:8:4:3 H 0-EtOH0.55 -ztOAc-Cycl ohexane 5:2:5:2 0, 0.25 H 0-EtOH0.70

45

I,

I,

E~OAC-

A c e t y l ha1 l e r i d o n e

Tubocurarine c h l o r i d e

Peruvian c u r a r e

Cyclohexane H20-EtOH-EtOAccyclohexane 0.60 5:2:4:3 CHC13-MeOH- 0.60 H20 5:5:2

II

11

TABLE 4 P A R T I T I O N SYSTEMS I N O R D E R OF INCREASING HYDROPHILIC C H A R A C T E R H20.EtOH.n.

Hexane

H20*Acetone.Cyclohexane H 2 0 - E t O A c - A c e t o n e . n . Hexane H 0.EtOH.Acetone.Cyclohexane

2

H20.EtOH-EtOAc.Cyclohexane

-

H20 CH C1

3 H20 * E tOAc H20.EtOAc .n.

BuOH

H 2 0 .CHC1 3. MeOH

The g e n e r a l p r o c e d u r e a d o p t e d b y u s f o r n a t u r a l e x t r a c t s i s the p a r t i t i o n o f the residue o f the methanolic extraction

between w a t e r and e t h y l a c e t a t e .

The a q u e o u s s o l u t i o n i s s u b s e -

q u e n t l y p a r t i t i o n e d w i t h n. b u t a n o l . T h i s procedure g i v e s r i s e t o two o r g a n i c e x t r a c t s , lic.

i.e.

t h e e t h y l a c e t a t e and t h e butano-

The c h o i c e o f a n a d e q u a t e t w o - p h a s e s y s t e m f o r t h e i r r e s o l u t i o n can n o t meet t h e i d e a l r e q u i r e m e n t s f o r e v e r y component, i.e.,

t o o b t a i n Kr

c l o s e t o one. T h i s c a n ,

however,

b e accom-

p l i s h e d by progressive o r discontinuous changing o f t h e p o l a r i t y o f t h e u p p e r m o b i l e p h a s e i n s u c h way a s t o c a r r y o n t h e s e p a r a t i o n o f a l l t,ie components i n a s i n g l e o p e r a t i o n .

A s a g e n e r a l r u l e we c a n s a y t h a t t h e m i x t u r e s r e p o r t e d i n Table 4

-

w h i c h c o n t a i n a c e t o n e d o n o t make i t p o s s i b l e

t o increase t h e p o l a r i t y o f t h e m o b i l e phase w i t h o u t simultaneous and s e n s i b l e change o f t h e volume o f t h e s t a t i o n a r y phase, p r e v i o u s l y s a t u r a t e d b y a l e s s p o l a r upper phase. On t h e o t h e r h a n d t h e m i x t u r e m o r e f l e x i b l e t o c h a n g e s o f p o l a r i t y o f t h e m o b i l e phase, w i t h o u t s e n s i b l e change o f t h e volumes o f t h e l o w e r phase, no1 ( 1 0 : 4 )

i s the following: water-etha-

e t h y l - a c e t a t e cyclohexane.

I n f a c t f o r t h i s system

t h e m u t u a l r a t i o o f e t h y l a c e t a t e and c y c l o h e x a n e c a n be i n c r e a sed d i s c o n t i n u o u s l y f r o m 0 : 1 4 t o 1 2 : 2 .

The w i d e r a n g e o f a p p l i -

c a b i l i t y o f t h i s s y s t e m makes i t e x c e e d i n g l y h e l p f u l f o r t h e s e p a r a t i o n o f t h e a f o r m e n t i o n e d e t h y l a c e t a t e e x t r a c t s o f nat u r a l source. F o r t h e above r e p o r t e d b u t a n o l i c e x t r a c t a good f l e x i b i l i t y i s displayed by the mixture water-ethylacetate-n-butanol, whereas t h e m i x t u r e chloroform-methanol - w a t e r

(used by us f o r

the purification o f quaternary alkaloids (11)) i s not f i t f o r any p r o g r e s s i v e change. When a c h o s e n s o l v e n t m i x t u r e d o e s n o t a l l o w t h e s e p a r a t i o n o f t w o compounds i n a s i n g l e w i t h d r a w a l s y s t e m s i n c e t h e r a t i o o f t h e i r p a r t i t i o n c o e f f i c i e n t s i s near 1 two a l t e r n a t i ve p o s s i b i l i t i e s can be considered: e i t h e r o p e r a t i n g by recyc l i n g o r f i n d i n g a d i f f e r e n t t w o p h a s e s y s t e m whose s e l e c t i v i t y cannot be evaluated o f course a p r i o r i .

299

E x a-m p l e s _ T o i l l u s t r a t e t h e a b o v e c o n s i d e r a t i o n s we r e p o r t h e r e a n u m b e r o f s e p a r a t i o n s made u s i n g t h e C C D w i t h d i s c o n t i n u o u s c h a n g e o f t h e m o b i l e p h a s e pH a n d c h l o r o f o r m a s u s u a l s t a t i o n a r y phase. The r a t h e r c o m p l e x s e p a r a t i o n o f t h e m i x t u r e o f S t r y c h n o s n u x vomica a l k a l o i d s ,

a c h i e v e d i n 1 9 6 9 , was t h e f i r s t a p p l i c a t i o n

o f t h i s technique (5).

I t was p o s s i b l e t o s e p a r a t e

on p r e p a -

r a t i v e s c a l e n o t o n l y t h e n i n e a l k a l o i d s a l r e a d y known, a l s o f o u r new a 1 k a l o i d s ,

pseudo-a-colubrine,

but

pseudo-B-colubri-

ne ( 1 2 1 , a l k a l o i d X 2 i d e n t i f i e d as i s o s t r y c h n i n e ( 1 3 ) and a l k a l o i d X 1 i d e n t i f i e d as 1 5 - h y d r o x y s t r y c h n i n e Then s e v e r a l

crude e x t r a c t s o f american Strychnos were submit-

ted t o t h i s procedure, (151, S .

i.e.

s.

romeu-belenii

( o n e new a l k a l o i d )

t a b a s c a n a ( 5 a l k a l o i d s o f w h i c h 4 n e w ) ( l 6 ) , S.

(2 a l k a l o i d s ) (17),

S.

fendleri

medeola

p a n a m e n s i s ( 2 a l k a l o i d s ) ( 1 8 1 , S.

z o n i c a ( 2 a l k a l o i d s ) a n d S. S.

(14).

8 alkaloids

brachiata (2 alkaloids)

( 6 o f them new)

(20,21)

ama-

( 1 9 ) . From

were o b t a i n e d

and f o r two n o n - s e p a r a t e d c o u p l e s o f them a d i f f e r e n t o r g a n i c s o l v e n t was u s e d t o a c h i e v e t h e s e p a r a t i o n . New i n d o l i c a l k a l o i d s w e r e a l s o i s o l a t e d f r o m A f r i c a n S t r y c h n o s , t h u s f o u r new f r o m S . S.

n i g r i t a n a (221,

o n e p l u s t w o known f r o m

s p i n o s a ( 2 3 ) a n d one p l u s t h r e e known f r o m S.

henningsii (29)

whereas o t h e r American Strychnos were i n v e s t i g a t e d i n t h e i r b a s i c components as S. l ia (241, S.

-.

( 2 7 ) and S.

gardneri,

r u b i g i n o s a (251,

>.

S.

j o b e r t i a n a and S.

h i r s u t a ( 2 6 1 , S.

parvifo-

castelnaena

alvimiana (28).

The same p r o c e d u r e was a l s o a p p l i e d t o m i x t u r e s o f o t h e r t y p e a1 k a l o i d s ,

i.e.

t h e m i x t u r e o f l y s e r g i c - p e p t i d e a1 k a l o i d s o f

H y d e r g i n e ( 8 ) a n d of. e r g o l i n e a l k a l o i d s o f C l a v i c e p s p u r p u r e a ( 9 a l k a l o i d s ) ( 3 0 ) and C.

fusiformis

( 5 a l k a l o i d s ) (311,

for

which t h e wide range o f p o l a r i t y compelled t h e use o f two d i f f e r e n t s y s t e m s a s s t a t i o n a r y p h a s e s , CHC13-n-PrOH e r g o l i n e s a n d CHC13-CC14

(1:l)

(2:l)

for

f o r peptides. Likewise bisbenzy-

l i s o q u i n o l i n e a l k a l o i d s were i s o l a t e d from G u a t t e r i a megalo-

300

p h y l l a ( 3 o f w h i c h 2 n e w ) , f r o m P e i - n a m o c u r a r e ( o n e new) (331, from Abuta g r i s e b a c h i i

( 4 and 2 r e s p e c t i v e l y ) (341, f r o m

=-

d o t e n i a t o x i f e r a ( 6 and 1 ) (35) and from a P e r u v i a n c u r a r e ( 3 and 1 ) ( 1 1 ) f r o w h i c h two d i a s t e r e o i s o m e r s , -chondocurine,

R,R-curine

and R,S-

w e r e e a s i l y s e p a r a t e d f r o m a new a l k a l o i d . A C -

c o r d i n g t o t h e above t e c h n i q u e a b i o l o g i c a l l y a c t i v e t r y p t a m i n e - d e r i v a t i v e was r e c e n t l y i s o l a t e d f r o m a n A m a z o n i a n a r r o w p o i s o n ( 3 6 ) a n d a new i n d o l e a l k a l o i d f r o m a n A f r i c a n A p o c y n a cea, A l s t o n i a b o o n e i , ( 3 7 ) whereas t e n b e n z y l i s o q u i n o l i n e a l k a l o i d s ( 2 o f them new) w e r e i s o l a t e d f r o m H y d r a s t i s c a n a d e n s i s (37).

A p a r t i c u l a r o r g a n i c m o b i l e p h a s e , A c O E t a n d THF,

was n e c e s s a r y f o r t h e s e p a r a t i o n o f a c i d i c p o r p h y r i n s w h o s e

K was c a l c u l a t e d ( T a b l e 2 ) . T h e i s o l a t i o n o f t h e n a t u r a l r A s u b s t a n c e s r e p o r t e d i n T a b l e 3 as w e l l t h e p u r i f i c a t i o n o f t h e i r

K

d e r i v a t i v e s have r e q u i r e d q u i t e d i f f e r e n t two-phase s o l v e n t s y s t e m s t o c o v e r a w i d e r a n g e o f p o l a r i t y . T h i s has a l l o w e d t o e s t a b l is h a s t h e m o s t adequate t h e systems H 0-EtOH-EtOAc-cyclohexane and 2 w a t e r - e t h y l a c e t a t e - n BuOH, w h o s e f l e x i b i l i t y m a k e s p o s s i b l e t o u t i l i z e t h e p r o g r e s s i v e change o f p o l a r i t y o f t h e

upper

phase f o r t h e s e p a r a t i o n of complex m i x t u r e s . N a t u r a l s u b s t a n c e s o f d i f f e r e n t t y p e have been t h u s i s o l a t e d : new d i g l y c o s i d e s f r o m l e s t i a l y c i o i d e s ( 4 0 ) a n d Hypox i s obtusa (411, eudesmanolides f r o m A r t e m i s i a b a r r e l i e r i (42),coumarins

from Zanthoxylon usambarense (431,

iridoids

f r o m T a r e n n a g r a v e o l e n s a n d Mussaenda a r c u a t a ( 4 4 1 , xenones f r o m H a l l e r i a l u c i d a ( 4 5 ) a

cyclohe-

s u l p h o r a t e d compound

from R i t c h i e a l o n g i p e d i c e l l a t a ( 9 ) and & - l a c t o n e from A l s t o n i a b o o n e i ( 1 0 ) and a l s o a w e l l known q u a t e r n a r y s a l t , d - t u b o c u r a r i ne c h l o r i d e , f r o m a P e r u v i a n c u r a r e ( 1 1 )

.

Concl us i o n s T h e a d v a n t a g e s o f t h e p r o p o s e d s y s t e m s i n C C D c a n b e summ a r i z e d as f o l l o w s : i

-

N a t u r a l m i x t u r e s do n o t need a n y p r e l i m i n a r y c l e a n u p : basic e x t r a c t s o f a l k a l o i d s and e t h y l a c e t a t e o r butanol i c e x t r a c t s f o r t h e n e u t r a l compounds c a n b e d i r e c t l y submitted t o CCD.

301 ii - E v e r y

two-phase system here reported,provided

the p l a i t point,

f a r from

can be p r o f i t a b l y used d i f f e r e n t l y f r o m

d r o p l e t c o u n t e r c u r r e n t c h r o m a t o g r a p h y , whose a p p l i c a t i o n i s l i m i t e d t o t h e few s o l v e n t systems f o r which t h e drop l e t formation i s possible without displacement o f the s t a t io n a r y p h a s e . i i i - T h e system i s p a r t i c u l a r l y f i t f o r a 24 hour run, t h e n i g h t time beeing s u f f i c i e n t f o r the f u l f i l m e n t o f t h e transfers i n a 200 t u b e u n i t , whereas t h e day t i m e a l l o w s t o estab l i s h - m a i n l y by t l c - t h e s t a t e o f t h e separation o f t h e various

components.This

makes p o s s i b l e t h e r e c o v e r y o f

t h e s e p a r a t e d s u b s t a n c e s a n d t o d e c i d e w h e t h e r t h e pH o r t h e p o l a r i t y o f t h e m o b i l e phase should be mantained o r changed. iv

-

The d i s c o n t i n u o u s e q u i l i b r i u m p r o c e s s a l l o w s a t a n y moment the survey o f the separation, t i o n o f t h e substances, calculation o f K

K

r B stances, r e s p e c t i vel y.

v

-

the prediction o f t h e posi-

t h e assessement o f t h e p u r i t y ,

or K

r

the

f o r weak b a s e s o r n e u t r a l sub-

The a p p a r a t u s i s r e l i a b l e , t h e

operationsare reproducible

and t h e r e s o l u t i o n and t h e c a p a c i t y a r e h i g h .

The f i l l i n g

o p e r a t i o n f o r t h e l o w e r phase i s r a p i d and t h e a u t o m a t i c a d j u s t m e n t o f t h e l e v e l o f e a c h t u b e may t a k e p l a c e o n l y a few tubes i n advance t o t h e t r a n s f e r o f t h e upper m o b i l e phase. REFERENCES L.C. C r a i g and D. C r a i g , P h y s i c a l Methods o f Organic Chemistry, v o l . 111, I n t e r s c i e n c e , New York, London, (1956), P a r t I , 149. H.A. Wilhelm and R.A. Foss, Ind. Eng. Chem., 51 (1959) 633. M. V e r z e l e and F. A l d e r w e i r e l d t , Nature 174 (1954) 702. T. Tanimura, 5.3. Pisano, Y. I t o and R.L. Bowman, Science 169 (1970) 54. C. G a l e f f i , M.A. Ciasca-Rendina, E. Miranda D e l l e Monache, A. V i l l a r d e l Fresno and G.B. M a r i n i B e t t o l o , J . Chromatography 45 (1969) 407. C. G a l e f f i , J . Chromatography 92 (1974) 1. C. G a l e f f i , J . Ethnopharmacol. 2 (1980) 129. C. G a l e f f i and E. Miranda D e l l e Monache, J . Chromatography, 88 (1974 413.

302 9 J.U. Oguakwa, M. Patamia, C. G a l e f f i , I . Messana and M. N i c o l e t t i , P l a n t a Med. 41 (1981) 410. 10 G.B. M a r i n i B e t t o l o , M. N i c o l e t t i , I . Messana, M. Patamia, C. G a l e f f i , J.U. Oguakwa, G. P o r t a l o n e and A. Vaciago, Tetrahedron 39 (1983) 323. 11 3 . L e m l i , C. G a l e f f i , I . Messana, M. N i c o l e t t i and G.B. M a r i n i B e t t o l o , P l a n t a Medica i n press. 12 G.B. M a r i n i B e t t o l o , E. Miranda D e l l e Monache, C. G a l e f f i , M.A. Ciasca-Rend i n a & V i l l a r d e l Fresno, Ann. Chim. (Rome) 60 (1970) 444. 13 C. G a l e f f i , E. Miranda D e l l e Monache and G.B. M a r i n i B e t t o l o , J. Chromatography, 88 (1974) 416. 14 C. G a l e f f i , M. N i c o l e t t i , I . Messana and G.B. M a r i n i B e t t o l o , Tetrahedron 35 (1979) 2545. 15 G.B. M a r i n i B e t t o l o , E. Miranda D e l l e Monache, S. Erazo G i u f f r a and C. Gal e f f i , Gazz. Chim. I t a l . , 101 (1971) 971. 16 C. G a l e f f i , M.A. Ciasca-Rendina, E. Miranda D e l l e Monache and G.B. M a r i n i B e t t o l o , Farmaco ed. S c i . , 26 (1971) 1100. 17 G.B. M a r i n i B e t t o l o , S. Erazo G i u f f r a , C. G a l e f f i and E. Miranda D e l l e Monache, Gazz. Chim. I t a l . , 103 (1973) 591. 18 G.B. M a r i n i B e t t o l o , M.A. Ciasca-Rendina, C. G a l e f f i , N.G. B i s s e t and B.A. K r u k o f f , Phytochemistry, 11 (1972) 381. 19 C. Galeffi,E. Miranda D e l l e Monache and G.B. M a r i n i B e t t o l o , Ann. Chim. (Rome) 63 (1973) 849. 20 C. G a l e f f i , A. Lupi and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . 106 (1976) 773. 21 C. G a l e f f i and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . 110 (1980) 81. 22 J.U. Oguakwa, C. G a l e f f i , I. Messana, R. La Bua, M. N i c o l e t t i and G.B. Mar i n i B e t t o l o , Gazz. Chim. I t a l . 108 (1978) 615. 23 J.U. Oguakwa, C. G a l e f f i , M. N i c o l e t t i , I.Messana, M. Patamia and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . , 110 (1980) 97. 24 G.B. M a r i n i B e t t o l o , I . Messana, M. N i c o l e t t i , M. Patamia and C. G a l e f f i , J. Nat. Prod. 43 (1980) 717. Messana, Phytochemistry, 25 G.B. M a r i n i B e t t o l o , C. G a l e f f i , M. N i c o l e t t i &I. 19 (1980) 992. 26 C. G a l e f f i and G.B. M a r i n i B e t t o l o , Tetrahedron 37 (1981) 3167. 27 C. G a l e f f i , M. Patamia, M. N i c o l e t t i , I . Messana and G.B. M a r i n i B e t t o l o , Phytochemistry, 21 (1982) 2393. 28 G.B. M a r i n i B e t t o l o , I . Messana, M. N i c o l e t t i , M. Patamia and C. G a l e f f i , Anales Asoc. Quim. Arg. 70 (1982) 263. 29 W.A. Chapya, C. G a l e f f i , M. Sperandei, J.D. Msonthy, M. N i c o l e t t i , I . MesSana and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . 113 (1983) 773. 30 C. G a l e f f i , S. Matos'i'd and A. Tonolo, A t t i Accad. Naz. L i n c e i C1. S c i . F i s . Mat. Nat. Rend. 56 (1974a) 951. 31 L. T u t t o b e l l o and C. G a l e f f i , A t t i Accad. Naz. L i n c e i C1. S c i . F i s . Mat. Nat. Rend. 64 (1978) 200. 32 C. G a l e f f i , G.B. Marini B e t t o l o and D. Vecchi, Gazz. Chim. I t a l . 105 (1975) 1207. 33 C. G a l e f f i , P. S c a r p e t t i and G.B. M a r i n i B e t t o l o , Farmaco ed S c i . 32 (1977) 665. 34 C. G a l e f f i , P. S c a r p e t t i and G.B. M a r i n i B e t t o l o , Farmaco ed S c i . 32 (1977) 853. 35 C. G a l e f f i , R. La Bua, I. Messana, R. Zapata A l c a z a r and G.B. M a r i n i Bett o l o , Gazz. Chim. I t a l . 108 (1978) 97.

303

36 C. G a l e f f i , I . Messana and G.B. M a r i n i B e t t o l o , J . Nat. Prod. ( L l o y d i a ) 46 (1983) 586. 37 J.U. Oguakwa, C. G a l e f f i , I. Messana, M. Patamia, M. N i c o l e t t i and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . 113 (1983) 533. 38 I. Messana, R. La Bua and C. G a l e f f i , Gazz. Chim. I t a l . 110 (1980) 539. 39 M.L. Bruzzone, C.G. Casinovi, C. G a l e f f i , A. Tonolo and A. T r i l l i , A t t i Accad. Naz. L i n c e i . C1. Sci. F i s . Mat. Nat. Rend. 57 (1974) 662. 40 S. Erazo G i u f f r a , C. G a l e f f i , M.A. Ciasca-Rendina & E. Miranda D e l l e Monache, Ann. 1st. Super. Sanith, 7 (1971) 23. 41 G.B. M a r i n i B e t t o l o , M. Patamia, M. N i c o l e t t i , C. G a l e f f i and I. Messana, Tetrahedron 38 (1982) 1683. 42 A. V i l l a r , M.C. Zafra-Polo, M. N i c o l e t t i and C. G a l e f f i , Phytochemistry, 22 (1983) 777. 43 J.O. Kokwaro, I . Messana, C. G a l e f f i , M. Patamia and G.B. M a r i n i B e t t o l o , Planta Med. 47 (1983) 251. 44 M. N i c o l e t t i , W.A. Chapya, I. Messana, C. G a l e f f i , M. Sperandei and G.B. M a r i n i B e t t o l o , Gazz. Chim. I t a l . 114 (1984) 49. 45 I. Messana, M. Sperandei, G . M u l t a r i , C . G a l e f f i and G.B. M a r i n i B e t t o l o , Phytochemistry, 23 (1984) 2617.

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305

MINIATURIZED SEPARATION SYSTEMS MILOS NOVOTNY Department of Chemistry, Indiana University, Bloomington, Indiana 47405, U.S.A. INTRODUCTION Throughout the history of modern chromatography, there has been a consistent trend to work with ever-decreasing amounts of analyzed materials and at increasing demands on detection sensitivity. Only gradually has this direction been translated into smaller chromatographic columns and corresponding instrumentation. While addressing the Symposium on Vapour Phase Chromatography held in 1956 in London, A.J.P. Martin (ref. 1 ) made a prediction that " ... w e should be able to work from the milligramme down to the microgramme scale. O f course, that will imply that we decrease the diameter of our columns correspondingly. We shall have columns only two tenths of a millimetre in diameter, and these will carry, I believe, advantages of their own This forsightful remark is now frequently associated with the beginning of an exciting era in separation science, that of capillary chromatography. Soon afterwards, Golay's investigations (refs. 2 , 3 ) led to the inception of the open tubular chromatographic column and the basic understanding of its physicochemical processes. Martin's belief in the potential of "microanalysis" with very small chromatographic columns was further expressed in his opening lecture to the Hamburg Gas Chromatography Symposium in 1962 (ref. 4), reasoning that if chemists learn proper techniques of manipulation, they should be able to pursue chemical studies with quantities of many orders of magnitude smaller than commonly practiced. In the current analytical activities, the term of "miniaturization" has been used relatively loosely. Naturally, decreasing the sizes of our analytical tools has been viewed as a desirable trend in many directions o f modern science and technology. Developing various microelectrodes and miniaturizing plasma spectroscopic media are exsmples of two areas where "miniaturization" has been employed for reasons entirely different from chromatography. Dramatic advances in microelectronics occurring during the last 10-15 years may serve as yet another example area where new miniaturization technologies have had a major impact on our capabilities to perform better analytical measurements; these rapidly developing areas together with microsensors technology are likely to be a very valuable resource for modern analytical chemistry for some time ....I'

306

to come. Various examples from chromatography may range from the development of a miniaturized GC column and a mass spectrometer placed aboard the Viking mission to Mars (refs. 5,6), to the design of a total GC instrument on a silicon electronic chip (ref. 7). Indeed, future availability of small, portable biomedical and environmental analyzers, based on chromatographic principles, is a very distinct possibility. One may effectively argue that the first developments in capillary GC during the late 1950s and early 1960s represented significant efforts toward chromatographic miniaturization. The major reasons for these developments, as exemplified by the elegant investigations of Desty and co-workers (refs. 8,9) in the area of hydrocarbon analysis, were achievements of high chromatographic efficiencies and greater speed of analysis. However, some beneficial elements of the small-column technology and related instrumentation became explicitly visible during the subsequent years, including the inherent advantages of capillary GC for trace analysis through certain specialized sampling techniques and detection principles. These benefits are widely utilized in today's wide practice of capillary GC throughout various fields of science and technology. "Miniaturization efforts" were also evident in the field of modern liquid chromatography (LC) during its early developmental stages. In a broader sense, the emphasis on using very small packing particles in LC (refs. 10-12), a major breakthrough in the field of chromatographic separations, may be viewed as a part of the overall picture. In a more restrictive view, such efforts have been primarily associated with the more recent trends toward decreasing column diameters and, correspondingly, the associated instrumental components (refs. 13-17). Overall, these directions shall have lasting influence on future developments in LC. In this author's opinion, current miniaturization efforts in the field of separation science should reinforce the overall development of modern analytical techniques, and may indeed have a certain unifying effect on the artificially divided GC and LC scientific audiences. Giddings (ref. 18) pointed out long ago that such division is both artificial and undesirable. During the further development of small-bore columns in LC, we should be able to take effective advantage of the technical knowledge accumulated over the years in capillary GC. Likewise, the recently developed (refs. 19-21) capillary supercritical fluid chromatography (SFC) borrows heavily from both capillary GC and modern LC. High-voltage capillary electrophoresis (refs. 22,23) further shares many instrumental features with microcolumn LC. Further exchanges of ideas from one area into the other will be needed in future developments of microcolumn separation techniques.

307 I t i s becoming i n c r e a s i n g l y e v i d e n t t h a t t h e r e a r e s e v e r a l d i s t i n c t

advantages a s s o c i a t e d w i t h t h e m i n i a t u r i z e d s e p a r a t i o n systems. M o s t importantly, these include (a.) high separation e f f i c i e n c i e s ; mass s e n s i t i v i t i e s o f t h e c o n c e n t r a t i o n - s e n s i t i v e d e t e c t o r s ;

(b.) (c.)

increased decreased

c o n s u m p t i o n o f e x p e n s i v e m o b i l e phases, column m a t e r i a l s , r e a g e n t s , e t c . ; (d.)

t h e p o s s i b i l i t y o f u s i n g " e x o t i c " m o b i l e phases; and, ( e . ) t h e d e v e l o p -

ment o f u n c o n v e n t i o n a l d e t e c t i o n t e c h n i q u e s t h a t b e n e f i t f r o m t h e d r a s t i c a l l y r e d u c e d f l o w o f t h e m o b i l e phase a s s o c i a t e d w i t h s u c h m i c r o c o l u m n s . The l a s t area, v i e w e d b y a number o f r e s e a r c h e r s i n t h e f i e l d as b e i n g p e r h a p s t h e most i m p o r t a n t , b r i n g s c h r o m a t o g r a p h e r s i n t o new, e x c i t i n g d e t e c t i o n t e c h nologies (microelectrodes,

l a s e r s , plasma sources, e t c . ) .

While formidable

t e c h n i c a l d i f f i c u l t i e s a r e sometimes e n c o u n t e r e d i n t h e d e v e l o p m e n t o f m i n i a t u r i z e d a n a l y t i c a l systems, s i g n i f i c a n t p r o g r e s s has been made i n t h e area. The p r e s e n t a r t i c l e w i l l b r i e f l y summarize t h e m o s t i m p o r t a n t r e c e n t d e v e l o p m e n t s i n t h e f i e l d o f m i n i a t u r i z e d s e p a r a t i o n t e c h n i q u e s and s p e c u l a t e on some f u t u r e t r e n d s . The d i s c u s s i o n w i l l i n c l u d e s m a l l - b o r e c a p i l l a r y GC,

m i c r o c o l u m n LC, c a p i l l a r y SFC and c a p i l l a r y e l e c t r o p h o r e s i s . CAPILLARY GAS CHROMATOGRAPHY As r e v i e w e d i n s e v e r a l r e c e n t books ( r e f s . 24-26), c a p i l l a r y GC i s seen t o d a y as a v e r y m a t u r e a n a l y t i c a l t e c h n i q u e . Numerous a p p l i c a t i o n s i n d i v e r s e f i e l d s o f s c i e n c e and t e c h n o l o g y a t t e s t t o i t s g r e a t scope. S i n c e v a r i o u s a s p e c t s o f c a p i l l a r y GC, i n c l u d i n g t h e q u e s t i o n s o f column t e c h n o l o g y , s a m p l i n g t e c h n i q u e s , and i n s t r u m e n t a t i o n , h a v e been a d e q u a t e l y r e v i e w e d , o n l y r e c e n t a t t e m p t s t o f u r t h e r d e c r e a s e t h e column d i a m e t e r w i l l b r i e f l y b e m e n t i o n e d h e r e f o r t h e sake o f c o m p l e t e n e s s and i n o r d e r t o emphasize a l o g i c a l t r a n s i t i o n o f i d e a s f r o m one a r e a o f s e p a r a t i o n s c i e n c e t o a n o t h e r . By t h e way o f c o m p a r i s o n between t h e s e p a r a t i o n g e o m e t r i e s o f GC and LC systems, m i n i a t u r i z a t i o n i n GC had a l r e a d y s t a r t e d w i t h G o l a y ' s work ( r e f . 2). The t e c h n i c a l competence o f D e s t y ' s e a r l y work ( 8 ) o n f a s t s e p a r a t i o n s , u s i n g s m a l l - b o r e g l a s s c a p i l l a r y columns, i s r e m a r k a b l e e v e n b y t o d a y ' s s t a n d a r d s . I n o r d e r t o f u r t h e r i n c r e a s e column e f f i c i e n c i e s , c a p i l l a r y i n n e r d i a m e t e r s a r e now b e i n g r e d u c e d down t o 50 pm t o g e t h e r w i t h d e s i g n i n g a d e q u a t e i n s t r u mentation. The r e a s o n s f o r d e c r e a s i n g t h e column d i a m e t e r f r o m t h e w i d e l y employed 200-300 vm down t o 50 pm a r e s t r a i g h t f o r w a r d consequences o f t h e G o l a y equation:

(a.)

i n c r e a s i n g t h e t h e o r e t i c a l p l a t e numbers f r o m l o 5 t o lo6;

5

and, ( b . ) a c h i e v i n g c o n s i d e r a b l y f a s t e r s e p a r a t i o n s i n t h e r a n g e o f 10 t o 5 10 t h e o r e t i c a l p l a t e s . I n an o p t i m i z a t i o n s t u d y , Guiochon ( r e f . 27) e x p l i c i t l y

308 d i s c u s s e d v a r i o u s c i r c u m s t a n c e s p e r t a i n i n g t o t h e speed and e f f i c i e n c y o f a n a l y s i s w i t h s m a l l - d i a m e t e r columns. Considering v a r i o u s t e c h n o l o g i c a l problems, t h e r e i s l i k e l y t o be o n l y a g r a d u a l a c c e p t a n c e o f s m a l l - b o r e GC columns. N e v e r t h e l e s s , a n a l y t i c a l a d v a n t a g e s o f I O O p m , i.d.,

significant

columns have a l r e a d y been d e m o n s t r a t e d .

Current d i f f i c u l t i e s l i e p r i m a r i l y i n t h r e e areas:

(a.) adequate d e a c t i v a t i o n

o f t h e c o l u m n ' s i n n e r w a l l ; ( b . ) i m p r o v e d ways o f sample i n t r o d u c t i o n ; and, ( c . ) development o f a v a r i e t y o f s u f f i c i e n t l y f a s t d e t e c t o r s and a s s o c i a t e d r e c o r d i n g d e v i c e s . The needs f o r b e t t e r d e a c t i v a t i o n t e c h n i q u e s a r e e v i d e n t because d e c r e a s i n g sample amounts e n c o u n t e r an e n l a r g e d s u r f a c e a r e a w i t h such s m a l l columns. These u n i q u e needs have been s t r e s s e d ( r e f . 28). A t t h i s p o i n t o f development, o n l y i n d i r e c t s a m p l i n g a p p r o a c h e s such as h i g h - r a t i o s p l i t t i n g o r f l u i d i c l o g i c d e v i c e s ( r e f . 29) a r e a v a i l a b l e .

I n s p i t e o f numerous t e c h n o l o g i c a l d i f f i c u l t i e s , f u r t h e r m i n i a t u r i z a t i o n o f c a p i l l a r y GC i s l i k e l y t o b r i n g o u r s e p a r a t i o n c a p a b i l i t i e s down t o t h e m i l l i s e c o n d time scale. T h i s t r e n d i s evident from F i g u r e 1 ( r e f . 29). I n t h e a r e a o f h i g h - r e s o l u t i o n a n a l y s i s o f complex v o l a t i l e m i x t u r e s , a p o t e n t i a l i m p a c t o f 50 pm columns i s l e s s c l e a r . S o p h i s t ' i c a t e d a n a l y t i c a l p r o b l e m s may b e t t e r be s e r v e d t h r o u g h t h e u s e of m u l t i d i m e n s i o n a l c a p i l l a r y GC t e c h n i q u e s . MICROCOLUMN L I Q U I D CHROMATOGRAPHY S t u d i e s on t h e u s e o f s m a l l e r - t h a n - u s u a l

inner diameters i n high-

p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) were i n i t i a t e d d u r i n g t h e l a t e

1970s. F o l l o w i n g t h e i n i t i a l work c a r r i e d o u t w i t h t h e s o - c a l l e d m i c r o b o r e

0.2

0.4

rec

F i g . 1 . Pentane peak w i t h u = 4.5 msec, o b t a i n e d w i t h t h e f l u i d i c i n j e c t o r a t 20.3 b a r , on a 60 cm x 5 0 p m , i . d . , f u s e d s i l i c a column. Reproduced w i t h p e r m i s s i o n f r o m r e f . 29.

309

columns (refs. 13,14,30), open tubular (capillary) columns (refs. 16,17) and packed capillaries (ref. 1 5 ) , a number of laboratories began to investigate both the column technology and unique detection aspects of microcolumn LC. At this stage of development, a significant number of applications have already been published, while further developments are likely to continue at an increasing pace. At this date, three books have already been edited (refs. 31-33) that deal with various aspects of LC microcolumn techniques. Developments on the three major microcolumn types, as depicted in Figure 2 (ref. 34), have recently provided a clearer assessment of their potential: totally packed microcolumns, such as 1 mm, i.d., microbore columns (refs. 13, 30) and their further miniaturized version, the so-called slurry-packed capillaries (refs. 35-38), are currently at the stage of a straightforward analytical application, while further extensive research is needed to reap the theoretical advantages of open tubular and semipermeable packed capillaries. The column developments are intimately linked with concurrent advances in the instrumental design, as the small column dimensions and correspondingly low flow-rates dictate the minimum volume of detection cells, sampling units, and all interconnecting lines. In addition, time constants within a miniaturized LC system must also be considered for the cases of very fast separations. Thus far, advances in the system designs and their commercial availability lag far behind the current capabilities of microcolumns. The situation with the individual column types will now briefly be reviewed.

Fig. 2. Types o f HPLC microcolumns. Reproduced with permission from ref. 34.

310 I t should be p o i n t e d o u t t h a t 1 mm, i.d.,

columns were used w i t h p e l l i c u a r

packings i n a p i o n e e r i n g s t u d y on HPLC ( r e f . 1 0 ) . However, w i t h t h e advent o f s m a l l e r p a r t i c l e s i n t h e e a r l y 1970s, w i d e r column d i a m e t e r s ( t y p i c a l l y , 4.6 mm) were g e n e r a l l y p r e f e r r e d due t o e x p e r i m e n t a l d i f f i c u l t i e s i n p a c k i n s m a l l e r columns. A s t u d y by S c o t t and Kucera ( r e f . 13) on p a c k i n g 1 mm, i.d., s t a i n l e s s - s t e e l columns up t o 1 meter i n l e n g t h under v e r y h i g h p r e s s u r e , was a m a j o r d e p a r t u r e f r o m common p r a c t i c e . A l t h o u g h t h e s e s o - c a l l e d " m i c r o bore columns,"

as seen f r o m t h e chromatogram reproduced i n F i g u r e 3, were

n o t up t o t h e i r t h e o r e t i c a l p o t e n t i a l [ a 14-meter l o n g (segmented) column i n F i g u r e 3 y i e l d e d o n l y 510,000 t h e o r e t i c a l p l a t e s i n a r e l a t i v e l y l o n g time o f analysis],

t h e o b t a i n e d e f f i c i e n c i e s were a s t o n i s h i n g as viewed by

t h e standards o f c o n v e n t i o n a l HPLC. However, a c c o r d i n g t o Kucera ( r e f . 39), t h e s t a i n l e s s - s t e e l c a p i l l a r i e s ' w i t h i n t e r n a l d i a m e t e r s o f l e s s t h a n 1 mm a r e d i f f i c u l t t o pack, whereas u s i n g p a r t i c l e s s m a l l e r t h a n 10 p m f u r t h e r c o m p l i c a t e s t h e t a s k o f column p r e p a r a t i o n . N e v e r t h e l e s s , segmented columns were r e c e n t l y used ( r e f . 40) i n a p e t r o c h e m i c a l a p p l i c a t i o n where o v e r 1,000,000

t h e o r e t i c a l p l a t e s were achieved, p e r m i t t i n g s e p a r a t i o n o f compound

p a i r s w i t h U-values around 1.011.

130.0 480.0 dB0.0 920.0 1150.0 1380.0 1610.0 1840.0 2070.0 nllw (mln)

F i g . 3. Chromatogram o f an e s s e n t i a l o i l o b t a i n e d w i t h a 14-in x 1 mm, i.d., m i c r o b o r e s i l i c a column (510,000 t h e o r e t i c a l p l a t e s ) ; n - h e x a n e l e t h y l a c e t a t e (95:5) s o l v e n t . Reproduced f r o m r e f . 13.

311

Column materials alternative to stainless steel were also explored in microcolumn LC. Plastic tubes (ref. 14) permit only low-pressure applications. However, the exploration of fused-silica and glass columns, with a typical inner diameter range of 150-300 pm, has been considerably more successful (refs. 35-38). Such columns, also referred to as "slurry-packed capillary columns," have now been employed in a number of applications, demonstrating both excellent efficiencies and good permeabilities. A systematic study of their preparation and analytical performance has been reported by our group (ref. 38). The initial attempts to use open tubular columns in LC (refs. 10,41) may long be forgotten by most researchers in the field. The authors performing these studies used the capillary tubes of roughly the same dimensions as those in conventional capillary GC; in a rough agreement with the Golay theory, the column inner diameter must be reduced at least ten times (and, for optimum results, more like fifty-fold) to counterbalance considerably slower mobile-phase diffusion processes in liquids. Tsuda and Novotny (ref. 16), who showed that thick-walled stock glass tubing can be drawn into capillary tubes of a variety of lengths and diameters for open tubular LC, found the experimental agreement with the Taylor equation (ref. 42) down to 50 pm, i.d., tubes. However, the more explicit subsequent optimization studies by Knox and Gilbert (ref. 43) and Jorgenson and Guthrie (ref. 44) point out that the inner diameters of 10 pm and below are needed to provide a qualitatively new step, i.e., the numbers o f theoretical plates above one million in reasonable analysis times, through the open tubular approach. While encouraging results have recently been achieved with LC open tubular columns (refs. 44-48), technological difficulties associated with both the column preparation and optimum instrumental design remain formidable. Following the fabrication of a capillary tube with suitable dimensions, a uniform deposition or in situ formation of a stationary phase is essential. Much o f the surface chemistry developed in the capillary GC field (ref. 4 9 ) , including both geometrical and chemical modification, can hopefully be translated into the specific conditions of capillary LC. Some work along these lines has already been initiated in several groups (refs. 44-48). In the area of open tubular LC instrumentation, the need of resorting to indirect sampling procedures (refs. 16,44,50,51) is viewed as the technique's weakest point. In spite of the general difficulties encountered with this "infant technique," a number of workers in the field still view it as a potentially rewarding and unique route to extremely large plate numbers that "will come, some day" (ref. 5 2 ) . In completing the discussion on different LC microcolumns, semipermeable

312

packed capillaries should also be mentioned. In the initial work on these columns (ref. 1 5 ) , alumina particles were drawn inside the glass columns, yielding a relatively low density of particles imbedded into the column's wall. In the following studies (refs. 5 3 - 5 5 ) , both additional column selectivities and smaller particles were explored. The effects of the column diameter, particle size, particle shape and porosity were systematically evaluated for these packed capillaries by McGuffin and Novotny (ref. 55); smaller diameters and particles yielded more efficient columns, while irregular particles seemed to provide somewhat superior results over the spherical particles. Thus far, it has been very difficult to draw inside the glass capillaries particles smaller than 10 pm. The state-of-the-art situation with packed capillary (ref. 56) and columns is exemplified by the recent results of Tsuda et. Figure 4, showing separation o model phthalates. Interestingly, the column permeabilities were quite high considering the relatively high density of the packing beads (Figure 5 ) . Admittedly, even HPLC exper s cannot readily agree on the relative merits of the individual microcolumns The efficiency of various packing procedures is reflected in the reduced plate-height values, but this criterion is difficult to apply to all microcolumn types. The numbers of theoretical plates alone, although necessary in resolving various hard-to-separate components, are not entirely meaningful, if the major "technological price" (high inlet pressure) and time of analysis are not specified. A useful

Fig. 4. Separation of dialkyl phthalates: column, 10.3 m x 47 pin; inlet 2 pressure, 500 kg/cm ; sample, (1) didecyl, (2) dinonyl and dioctyl, (3) diheptyl, (4) dicyclohexyl, ( 5 ) dibutyl, ( 6 ) dipropyl, (7) diethyl, and (8)dimethyl phthalate; linear velocity, 4.2 cm/s. Reproduced with permission from ref. 56. Copyright 1984 American Chemical Society.

313

Fig. 5. Electron micrographs of a cross section of a packed microcapillary column. B was part o f A. Magnifications o f A and B were 1500x and 7500x, respectively. Reproduced with permission from ref. 56. Copyright 1984 American Chemical Society. criterion that incorporates all these variables into one equation is the socalled separat on impedance suggested by Bristow and Knox (ref. 57):

314 The v a l u e o f s e p a r a t i o n impedance (E) e v a l u a t e s column e f f i c i e n c y a l s o c o n s i d e r s t h e t i m e o f a n a l y s i s (t,)

(N), b u t

and t h e p r e s s u r e g r a d i e n t (Ap)

r e q u i r e d t o achieve t h e s e p a r a t i o n . The s o l v e n t v i s c o s i t y

(n)

and c a p a c i t y

r a t i o ( k ) a r e a l s o taken i n t o account. I t i s f u r t h e r shown t h a t t h e s e p a r a t i o n impedance i s equal t o t h e square o f t h e p l a t e h e i g h t ( H ) d i v i d e d b y t h e column o r , a l t e r n a t i v e l y , i s equal t o t h e square o f reduced p l a t e 2 h e i g h t ( h ) m u l t i p l i e d by a column r e s i s t a n c e parameter (+I), where + ’ = d / K O ,

permeability

(KO),

and d i s t h e c h a r a c t e r i s t i c column dimension. The e x p e r i m e n t a l l y observed q u a n t i t i e s can be compared t o t h e t h e o r e t i c a l e s t i m a t e s made b y Knox ( r e f . 58) and shown i n Table I . Since s m a l l e r E-values correspond t o b e t t e r columns, open t u b u l a r , columns should e a s i l y w i n t h e c o n t e s t (however, o n l y i f t h e necessary l o w - d i s p e r s i o n i n s t r u m e n t a t i o n becomes a v a i l a b l e ) . Whereas, i n t h e o r y , t h e o t h e r column t y p e s do n o t appear p a r t i c u l a r l y a t t r a c t i v e as compared t o c o n v e n t i o n a l packed columns, some e x p e r i m e n t a l o b s e r v a t i o n s make notable d i f f e r e n c e i n practice:

s e p a r a t i o n impedances much l a r g e r t h a n

2,000 a r e commonly observed w i t h c o n v e n t i o n a l packed columns ( c h i e f l y due t o l a r g e r - t h a n - e x p e c t e d i n l e t p r e s s u r e s ) , w h i l e E-values c o n s i d e r a b l y lower than t h e t h e o r e t i c a l can be measured ( r e f . 59) w i t h s l u r r y - p a c k e d f u s e d s i l i c a columns. S i m i l a r d i s c r e p a n c i e s a r e o c c a s i o n a l l y observed ( r e f . 5 5 ) w i t h t h e semipermeable packed c a p i l l a r i e s . These p o t e n t i a l l y i m p o r t a n t o b s e r v a t i o n s c l e a r l y p o i n t o u t t h a t much remains t o be l e a r n e d about t h e r o l e o f column i n n e r diameter and i t s r e l a t i o n t o t h e p a r t i c l e s i z e .

TABLE I Comparison o f t h e T h e o r e t i c a l Performance o f Conventional and M i n i a t u r i z e d Columns i n L i q u i d Chromatography ( r e f . 5 8 ) . Column Type

hmi n

Conventional o r Small-Bore Packed Column

2

Packed C a p i l l a r y Column

2

Open-Tubular Capi 11a r y

0.8

+‘ 500- 1000

150 32

Emin

2000 600 20

The a n a l y t i c a l advantages o f v a r i o u s microcolumns may be m a n i f e s t e d i n s e v e r a l ways, depending on our g o a l s . I f t h e t i m e s o f a n a l y s i s on t h e o r d e r o f hours a r e acceptable, e x t r e m e l y h i g h e f f i c i e n c i e s can be achieved:

an

315 I

L1

I"

,,

I" 1.

.,

T

Fig. 6. High-resolution chromatogram of large polyaromatic compounds extracted from carbon black. Column and detection conditions: 1.8 meter x 250 um fused silica capillary packed with 3 um CI8 spherical beads; fluorescence detection; stepwise gradient. Reproduced with permission from ref. 60. Copyright 1984 Pergamon Press. example is shown in Figure 6 (ref. 60) where polyaromatic components up to nine-ring structuresfromthe carbon black extract are resolved on a slurrypacked capillary column, exhibiting 240,000 theoretical plates. Using similar techniques, complex nonvolatile mixtures originated from fossil fuels (refs. 61,621 and biological materials (refs. 63-65) were adequately resolved. It is quite important that such columns require only a moderate degree of instrumental miniaturization (volumes around 100 nL are quite adequate for both sampling and detection), while up to microgram sample amounts are tolerated without column overloading (ref. 38). Consequently, a variety of ancillary techniques can be utilized for the solute identification. Alternatively, if moderate chromatographic efficiencies are acceptable, it is feasible to perform some very fast separations. Several years ago, Scott Gal. (ref. 66) demonstrated the resolution of a seven-component mixture, on a microbore column, in as little as 30 sec. Yet another example of a fast separation, with a small-bore packed column, published by Ishii and co-workers (ref. 67), is shown in Figure 7. Here, microcolumns have a very distinct advantage over the conventional columns as far as the mobile-

316

L

-

0

1

T I ME (rn 1n 1

Fig. 7. Rapid separation of PAHs on a 5 y m ODS column. Column: 100 x 0.34 mm, i.d., packed with Silica ODs SC-01. Mobile phase: acetonitrile/water = 7/3. Inlet pressure: 160 kg/cm2. Sample: 1) benzene; 2) naphthalene; 3) biphenyl; 4) fluorene; 5) phenanthrene; 6) anthracene; 7) fluoranthene; 8) pyrene. Wavelength of UV detection: 254 nm. Reproduced with permission from ref. 67. phase consumption is concerned. While several detector types, based on the conventional detection principles such as U V absorption, spectrofluorimetric, or electrochemical devices, have been adequately miniaturized for microcolumn LC work, some unique detection opportunities exist. Since the remaining microcolumn techniques reviewed below bear resemblance instrumentally to each other, these unique detection possibilities will briefly be reviewed in the last section of this article.

317 CAPILLARY SUPERCRITICAL FLUID CHROMATOGRAPHY The u s e o f s u p e r c r i t i c a l f l u i d s as t h e m o b i l e s phases i n c h r o m a t o g r a p h y was f i r s t r e p o r t e d i n 1962 ( r e f . 6 8 ) , however, t h e t e c h n o l o g i c a l p r o b l e m s of t h i s a p p r o a c h t o g e t h e r w i t h an i n c r e a s i n g p o p u l a r i t y o f HPLC i n t h e f o l l o w i n g decade l i m i t e d i t s a c c e p t a n c e . S u p e r c r i t i c a l f l u i d c h r o m a t o g r a p h y (SFC) has r e c e n t l y been r e v i t a l i z e d due t o a number o f t e c h n o l o g i c a l i m p r o v e ments i n t h e a r e a as w e l l as a v e r y a c t i v e c u r r e n t i n t e r e s t i n t h e s c i e n c e and t e c h n o l o g y o f s u p e r c r i t i c a l media. I n p a r t i c u l a r ,

t h e i d e a o f u s i n g open

t u b u l a r columns i n SFC ( r e f . 1 9 ) i s l i k e l y t o have a m a j o r i m p a c t o n t h e development o f new a n a l y t i c a l c a p a b i l i t i e s . The p r i n c i p a l i n t e r e s t i n SFC stems f r o m t h e f l u i d d e n s i t i e s a p p r o a c h i n g those o f l i q u i d s ;

increasing i n t e r a c t i o n s o f t h e mobile-phase molecules with

t h e s o l u t e s c a u s e s o l v a t i o n and f o r m t h e b a s i s o f p r e s s u r e ( d e n s i t y ) p r o g r a m m i n g i n SFC so t h a t t h e m o l e c u l e s o f i n c r e a s i n g m o l e c u l a r w e i g h t s and p o l a r i t i e s c a n b e s u c c e s s f u l l y chromatographed. S i m u l t a n e o u s l y ,

greater solute

d i f f u s i v i t i e s and l o w e r m o b i l e - p h a s e v i s c o s i t i e s make s u p e r c r i t i c a l f l u i d s c h r o m a t o g r a p h i c a l l y s u p e r i o r t o t h e c o n v e n t i o n a l l i q u i d s . P r i n c i p a l l y , SFC can p r o v i d e g r e a t e r c h r o m a t o g r a p h i c e f f i c i e n c i e s , u n d e r c e r t a i n c i r c u m s t a n c e s , t h a n LC, w h i l e t h e r e a r e a l s o c e r t a i n u n i q u e a n a l y t i c a l a d v a n t a g e s a s s o c i a t e d w i t h t h i s method ( e . g . ,

i t s use w i t h flame detectors,

I R s p e c t r o s c o p y , and

mass s p e c t r o m e t r y ) . C o n s e q u e n t l y , SFC i s l i k e l y t o o c c u p y a s p e c i a l p o s i t i o n between GC and LC, b e i n g b o t h complementary and c o m p e t i t i v e t o them i n future applications. Open t u b u l a r columns a r e a t t r a c t i v e t o SFC because o f t h e i r v e r y l o w p n e u m a t i c r e s i s t a n c e and, c o n s e q u e n t l y ,

an e a s y c o n t r o l o f r e t e n t i o n t h r o u g h

p r e s s u r e c o n t r o l . J u s t as i n c a p i l l a r y GC, open t u b u l a r columns i n SFC c a n p r o v i d e h i g h e f f i c i e n c i e s needed i n t h e r e s o l u t i o n o f complex n o n v o l a t i l e m i x t u r e s . As p r e d i c t e d b y an o p t i m i z a t i o n s t u d y ( r e f . 6 9 ) , e f f i c i e n c i e s i n 5 6 t h e r a n g e o f 1 0 t o 10 t h e o r e t i c a l p l a t e s a r e now f e a s i b l e w i t h c a p i l l a r y i n n e r d i a m e t e r s between 50-100

m. I n

c o n t r a s t t o t h e above-mentioned open

t u b u l a r LC where c o n s i d e r a b l y s m a l l e r column d i a m e t e r s a r e e s s e n t i a l t o success, t h e columns used i n c a p i l l a r y SFC a r e w e l l w i t h i n o u r t e c h n o l o g i c a l means. T h i s n e c e s s a r y column r e d u c t i o n ( f r o m GC t o SFC and LC i n t h e i r c a p i l l a r y modes) r o u g h l y c o r r e s p o n d s t o t h e v a l u e s o f m o b i l e - p h a s e s o l u t e d i f f u s i v i t i e s ( r e f . 70) i n t h e i r r e s p e c t i v e phases. As shown i n F i g u r e 8 ( r e f . 2 1 ) , p r a c t i c a l c h r o m a t o g r a p h i c e f f i c i e n c i e s o f GC and SFC a r e e x t r e m e l y s i m i l a r , t a k i n g t h e d i f f e r e n c e i n column d i a m e t e r i n t o a c c o u n t .

A typical capillary

SFC s y s t e m i s shown i n F i g u r e 9. A h i g h - p r e s s u r e pump

c o n t a i n s an a p p r o p r i a t e f l u i d w h i c h i s s u b s e q u e n t l y t r a n s f o r m e d i n t o a s u p e r c r i t i c a l medium i n a h e a t e d oven. A p r e s s u r e c o n t r o l l e r a d j u s t s t h i s f l u i d t o

318

P ,

40

50

11, Time (min)

yl

20

30

150 Tempfalure ('C)

100

en

50

40

m

250

FC

0

Time (mm) 20

22502150'b

W

40

050 Densily IglmL)

Oh0

en

(XI

lpo

OW

070

F i g . 8. GC and SFC chromatograms o f c o a l t a r . C o n d i t i o n s : Flame i o n i z a t i o n d e t e c t o r ( F I D ) . GC chromatogram: 20 m x 300 pm, i.d., f u s e d - s i l i c a column; SE-54 s t a t i o n a r y phase (0.25 urn f i l m t h i c k n e s s ) ; temperature program f r o m 40°C t o 265°C a t 4°C min-' a f t e r an i n i - t i a l 4 min i s o t h e r m a l p e r i o d ; hydrogen c a r r i e r gas. SFC chromatogram: 24 m x 50 pm, i.d., f u s e d - s i l i c a column; SE-54 s t a t i o n a r y phase (0.25 Um f i l m t h i c k n e s s ; Con m o b i l e ehase a t 40°C; a t 0.005 g mL-' rnin a f t e r an i n i t i a l d e n s i t y program f r o m 0.225 t o 0.70 mL 15 min i s o c o n f e r t i c ( c o n s t a n t d e n s i t y ) p e r i o d . Reproduced w i t h p e r m i s s i o n f r o m r e f . 21. C o p y r i g h t 1984 American Chemical S o c i e t y .

'

319

F i g . 9. Block diagram showing t h e components o f a c a p i l l a r y s u p e r c r i t i c a l f l u i d chromatograph. Reproduced w i t h p e r m i s s i o n f r o m t h e American Chemical S o c i e t y . C o p y r i g h t 1981.

an a p p r o p r i a t e d e n s i t y w h i l e t h e sample i s i n t r o d u c e d t h r o u g h a h i g h - p r e s s u r e v a l v e . A f t e r t h e sample has separated i n t o i t s components d u r i n g t h e passage t h r o u g h a c a p i l l a r y column, d e t e c t i o n i s u s u a l l y accomplished i n a h i g h p r e s s u r e c e l l , f o l l o w e d by decompression a t t h e c a p i l l a r y r e s t r i c t o r end. While n o n - e x t r a c t a b l e s t a t i o n a r y phases a r e a t t a c h e d t o t h e c o l u m n ' s i n n e r s u r f a t e t h r o u g h processes known f r o m t h e contemporary c a p i l l a r y GC, t h e o v e r a l l sy_stem i s a " h y b r i d " between GC and LC i n s t r u m e n t a t i o n . F u s e d - s i l i c a c a p i l l a r i e s a r e easy t o use i n t h i s t y p e o f chromatography.

320 W h i l e many s u b s t a n c e s can be c o n v e r t e d i n t o s u p e r c r i t i c a l f l u i d s when g i v e n a p p r o p r i a t e v a l u e s o f t e m p e r a t u r e and p r e s s u r e , p r a c t i c a l c o n s i d e r a t i o n s

l i m i t us t o a s m a l l number o f systems:

c a r b o n d i o x i d e and n i t r o u s o x i d e a r e

c o n v e n i e n t t o u s e because o f t h e i r m o d e r a t e c r i t i c a l t e m p e r a t u r e s , w h i l e

C2 - C 5 h y d r o c a r b o n s a r e a t t r a c t i v e , o w i n g t o l a r g e s o l u t e d i f f u s i v i t i e s ( r e f . 7 0 ) , f o r s e p a r a t i o n s o f n o n p o l a r m o l e c u l e s . The c h o i c e o f o t h e r s u p e r c r i t i c a l m e d i a c a n be made a p p r o x i m a t e l y a c c o r d i n g t o t h e " p o l a r i t y " s c a l e suggested by Giddings

e. ( r e f . 71),

based on v a l u e s o f t h e H i l d e b r a n d

s o l u b i l i t y p a r a m e t e r . As c a p i l l a r y columns t y p i c a l l y pass a f e w m i c r o l i t e r s o f a f l u i d p e r m i n u t e , i t i s f e a s i b l e t o employ " e x o t i c " m o b i l e phases, such as s u p e r c r i t i c a l xenon [ f o r t h e sake o f d e t e c t i o n ( r e f . 7 2 ) ] .

The s o l u t e

r e t e n t i o n i n SFC i s p r i m a r i l y c o n t r o l l e d b y t h e d e n s i t y o f s o l v a t i n g m o l e c u l e s . C o n s e q u e n t l y , d e n s i t y programming i s p r i m a r i l y employed ( r e f . 7 3 ) , a l t h o u g h some s e l e c t i v i t y a d j u s t m e n t s a r e a l s o f e a s i b l e w i t h t h e a p p l i c a t i o n o f p o l a r m o d i f i e r s ( r e f s . 74,75). W i t h an i m p r o v e d u n d e r s t a n d i n g o f t h e p h y s i c o c h e m i c a l p r o c e s s e s i n c a p i l l a r y SFC, t h e method i s l i k e l y t o f i n d an i n c r e a s i n g number o f a p p l i c a t i o n s . Once a g a i n , u n i q u e d e t e c t i o n t e c h n i q u e s e x i s t i n t h i s area; t h e y w i l l b e d i s c u s s e d i n t h e l a s t s e c t i o n ,if t h i s a r t i c l e . CAPILLARY ELECTROPHORESIS D u r i n g t h e many y e a r s o f t h e i r e x i s t e n c e , v a r i o u s e l e c t r o p h o r e t i c methods have f o u n d a g r e a t u t i l i z a t i o n i n t h e s e p a r a t i o n o f i o n i c m a c r o m o l e c u l e s . F l a t bed t e c h n i q u e s a r e c o n s i d e r a b l y more common t h a n t h e column t e c h n i q u e s , w i t h t h e t w o - d i m e n s i o n a l method o f O ' F a r e l l ( r e f . 7 6 ) r e p r e s e n t i n g t h e h i g h e s t a v a i l a b l e r e s o l u t i o n . A f e w e l e c t r o p h o r e t i c t e c h n i q u e s have been m i n i a t u r i z e d ( r e f . 7 7 ) f o r w o r k i n g w i t h s m a l l e r amounts o f m a t e r i a l s . A r e p r e s e n t a t i v e example o f such e f f o r t s i s t h e r e p o r t o f Hyden

e.( r e f .

7 8 ) who i n 1966

described a micro-disc electrophoresis o f p r o t e i n s a t a very small scale. As seen i n F i g u r e 1 0 ( r e f . 7 8 ) , s t a i n i n g o f n e r v e c e l l p r o t e i n s ( c o r r e s p o n d i n g t o s i x t y n e u r o n s ) has been a c c o m p l i s h e d on a 215 pm g e l . I n s t r u m e n t a l d e v e l o p m e n t s i n e l e c t r o p h o r e s i s have been c o n s i d e r a b l y l e s s common t h a n t h o s e i n c h r o m a t o g r a p h y . Among t h e n o t a b l e e x c e p t i o n s i s i s o tachophoresis,

a method c o n c e i v e d b y A.J.P.

M a r t i n ( r e f . 7 9 ) and f u r t h e r

d e v e l o p e d b y E v e r a e r t s and c o - w o r k e r s [ f o r a r e v i e w , see ( r e f . 8 0 ) ] . T h i s method, o c c u r r i n g i n c a p i l l a r y t u b e s o r s m a l l c h a n n e l s , i s f i n d i n g a number o f a p p l i c a t i o n s i n c e r t a i n f i e l d s . I n a d d i t i o n , zone e l e c t r o p h o r e s i s i n n a r r o w p l a s t i c t u b e s was i n v e s t i g a t e d b y M i k k e r s

9. (ref.

81) i n order t o reduce

t h e zone d i s p e r s i o n o r i g i n a t i n g f r o m t h e c o n v e c t i o n c u r r e n t s .

A more r e c e n t v e r s i o n o f c a p i l l a r y e l e c t r o p h o r e t i c t e c h n i q u e s i n t h e z o n a l

3 21

F i g . 10. P h o t o m i c r o g r a p h o f a 215 PI$ g e l on w h i c h s e p a r a t i o n o f n e r v e c e l l p r o t e i n has been p e r f o r m e d . S t a i n e d b y amido b l a c k . Reproduced w i t h p e r m i s s i o n f r o m r e f . 78.

mode o f o p e r a t i o n has been d e s c r i b e d b y J o r g e n s o n and L u k a c s ( r e f s . 2 2 , 2 3 ) . T h e i r e x p e r i m e n t a l s e t u p i s q u i t e s i m p l e ( F i g u r e I I ) , w i t h t h e on-column d e t e c t i o n somewhat r e m i n i s c e n t o f c a p i l l a r y LC. A r e l a t i v e l y s h o r t c a p i l l a r y , attached t o t h e r e s p e c t i v e r e s e r v o i r s , i s s u b j e c t e d t o h i g h v o l t a g e s i n excess o f 30 kV. F o l l o w i n g a t h e o r e t i c a l p r e d i c t i o n b y G i d d i n g s ( r e f . 82) t h a t t h e zone b r e a d t h i s i n v e r s e l y p r o p o r t i o n a l t o t h e a p p l i e d v o l t a g e , J o r g e n s o n and L u k a c s ( r e f . 2 2 ) u t i l i z e d n a r r o w c a p i l l a r y t u b e s ( 7 5 p m o r l e s s ) w h i c h

~-gJ.----=* Plexiglas

Capi 11ary

Buffer Reservoirs

F i g . 1 1 . S c h e m a t i c o f a c a p i l l a r y e l e c t r o p h o r e s i s system.

322

allow an easy dissipation of generated heat through the wall. A sample is drawn inside the capillary by a short exposure at high voltage. Except for employing relatively high voltages, the method is relatively simple. Extremely efficient separations have been reported (ref. 23) using capillary zone electrophoresis, as exemplified (Figure 12) by a separation of fluorescamine-labeled peptides from a tryptic digest of egg white lysozyme (ref. 83). However, further research appears necessary to achieve equally impressive results with larger biomolecules (ref. 23).

is

20

Fig. 12. Separation of fluorescamine-labeled peptides from a tryptic digest of egg white lysozyme. Glass capillary, 75 pm, i.d., by 100 cm long, filled with 0.05 phosphate buffer at pH 7; potential, 30 k V . Reproduced with permission from ref. 23. Further developments in microcolumn electrophoresis are highly desirable from the theoretical as well as practical point of view. The rapidly developing fields of biology and medicine will necessitate both high-resolution techniques and improved capabilities to analyze charged biological macromolecules present in ever-decreasing amounts o f sample.

323

UNIQUE DETECTION OPPORTUNITIES The scope of chromatographic separations and measurements has significantly expanded with the availability of an increasing number of highly sensitive and selective detectors. The column-detector relationship has been emphasized by both the realization that highly efficient separations are feasible primarily when working with minute sample quantities and by the recognition that detection conditions should not compromise optimum column performance. In gas chromatography, capillary columns and highly sensitive ionization detectors could hardly exist without each other. I n most instances, column miniaturization efforts further expand rather than limit the scope of detection. In the field of gas chromatography, the transition from packed to capillary columns already improved numerous detection possibilities: while there is little improvement with mass-flow-sensitive detectors, concentration-sensitive devices greatly benefit from miniaturization. These advantages were recently translated into viable.smal1-volume GC detectors based on photoionization (ref. 84), thermal conductivity (ref. 8 5 ) , optical absorption (ref. 86), and the electron capture phenomena (refs. 87,88). In addition, even the mass spectrometer operates more reliably and sensitively with low-bleed capillary columns, as compared to packed columns; sensitivities in the picogram range were recently indicated (ref. 89) in GC/MS employing small-bore capillary columns. An increased mass sensitivity of miniaturized LC concentration-sensitive detectors (such as those based on UV absorption, fluorescence or electrochemical processes) is becoming recognized as a valuable asset of microcolumn techniques in sample-limited situations. In spite of their decreased concentration sensitivity (due to a reduced optical path), miniaturized UV absorbance detectors have been shown to work adequately for a number o f applications (refs. 14,31,63). Oependingon the measurement principle involved, miniaturization of electrochemical detectors also results in dramatic improvements of detection sensitivity. While the earlier versions of miniaturized amperometric detectors (refs. 90-92) indicated satisfactory performance, more recent investigations show even greater promise. Manz and Simon (ref. 47) constructed a miniature ion-selective electrode sensing changes in the effluent concentration at the end of an open tubular column (see Figure 13). The detector demonstrated detection limits for K+ on the order of M. Yet another example of a highly sensitive miniature electrochemical detector .was recently demonstrated (ref. 93) who inserted a single carbon fiber electrode into by Knecht the outlet of a 15 pm, i.d., open tubular column, achieving nearly 100%

u.

324

ION-SELECTIVE MlcRoELECTROoE

SEPU?ATON

/

GLASS KATE

DROP

ELECTROOE

CONVERTER

Fig. 13. Schematic diagram of the ion-selective detection system. Reproduced with permission from ref. 47.

utilization of electrochemically active substances and, consequently, femtogram-level detection limits. Considering the overall general importance of microsensors, the area is likely to grow in importance. Although certain techniques employed in the above investigations may appear "tricky" to an average user of HPLC, electrophysiologists have known for some time how to manipulate small volumes and electrodes. Exciting opportunities also exist in the area of miniaturized spectrofluorimetric detection. Besides the mentioned increases in mass sensitivity with concentration-sensitive detector (including fluorescence), the current and future availability of laser light sources will undoubtedly be a major resource for ultrahigh-sensitivity measurements. Lasers are inherently well-suited for various uses in microcolumn separation techniques because of their highly collimated beams containing a large radiant power within an extremely small cross-sectional area. Miniaturized laser-based fluorescence detectors have been described (refs. 94-96) that exhibit predictably high sensitivities. A recent report on the detection of 22,000 molecules i n a flowing stream (ref. 97) provides further incentive for improvements. While a variety of biological and environmentally important molecules exhibiting native fluorescence may eventually require a wider availability of tunable, powerful lasers for their detection, in some instances it appears eminently worthwhile to "tune" our chemistry (refs. 64,65) to the wavelengths of relatively inexpensive lasers. An example of this is shown in Figure 14,

325

Fig. 14. Chromatogram of solvolyzed plasma steroids: column, 220 wm, i.d., x 2.25 m length, packed with 5 Spherisorb 0 0 s ; mobile phase: continuous gradient 75-100% aqueous acetonitrile (1.5 wl/min); tentatively identified components: ( 1 ) 5a-androstan-3a,ll~-diol-l7-one, (2) 5B-androstan-3a,llBdiol-17-one, (3) 5~-pregnane-3a,ll~,17a,2l-tetrol-2O-one, (4) 5B-pregnane3a,17a ,20B,21-tetrol-ll-one, (5) 5B-pregnane-3a,IlB,17a-20!3,21-pentol, (6) 5~-pregnane-3a,17a,2Oa,2l-tetrol-ll-one, (7) 58-pregnane-3a 118.17~. . . 20a, 21-pentol , (8) 5a-androstan-3a-ol-l7-one, (9) 5-androstene-3B-o -1 7-one, (10) 5B-pregnane-3a,20a,2l-triol, (11) 5B-androstan-3a,l7B-diol Approxima 50 pg of each steroid was injected. Reproduced from ref. 96. where femtomole amounts of blood steroids are recorded with a f uorescence detector based on a helium-cadmium laser (ref. 96) after their precolumn derivatization with a novel coumarin-based reagent (ref. 65). Other uses of lasers in conjunction with miniaturized chromatographic systems have recently been reviewed by Yeung (ref. 98). Optical imaging detectors are becoming popular as ancillary techniques for modern LC. Based on different technologies, the detectors consist o f multichannel systems that can yield a vastly increased volume of information (conplete spectra o f substances) as compared to that provided by singlewavelength detection. These types of detectors were recently decreased in (ref. 99) have miniaturized a commercial UV volume: while Ishii Gal.

326

absorbance photodiode array detector, our group designed (ref. 100) a highly sensitive imaging fluorescence detection system based on an intensified photodiode array. Detection possibilities in the infrared region have recently been revolutionized by the availability of Fourier-transform infrared (FTIR) techniques. While capillary GC columns were successfully developed in coupling with FTIR (refs. 86,101), an intensive search for similar capabilities in LC will continue for some time. Once again, the system miniaturization appears beneficial, as shown by both the design of solute transport devices (ref. 102) and the use of "exotic" (e.g., deuterated) mobile phases (ref. 103) allowed by very low flow-rates of microcolumns. However, a potentially more fruitful area appears to be coupling of capillary SFC with FTIR. It has already been demonstrated (refs. 104,105) that carbon dioxide is sufficiently transparent in the mid-IR region to allow the sensitive detection and identification of numerous organic compounds. A s illustrated by a representative chromatogram (Figure 15), submicrogram sensitivities are achievable in capillary SFC/FTIR (ref. 105). Further interesting possibilities lie in the use of supercritical xenon (ref. 72) for similar types of measurements.

I1.0

c L

i

A r

11.1

0

o

10

to

m

w

10

YIMUTLS

Fig. 15. Gram-Schmidt real-time chromatogram of test mixture: ( 1 ) benzaldehyde, (2) o-chlorobenzaldehyde, (3) 2,6-di-t-butylphenol, (4) 2-naphthol, (5) benzophenone, 2 ~g of each injected. Reproduced from ref. 105 with permission of Pergamon Press. Copyright 1984.

327 Mass s p e c t r o m e t r y o c c u p i e s a p r o m i n e n t p l a c e among t h e a n c i l l a r y t e c h n i q u e s o f modern c h r o m a t o g r a p h y . Whereas GC/MS (and, d u r i n g r e c e n t y e a r s , i n m a j o r p a r t c a p i l l a r y GC/MS) has now been w e l l e s t a b l i s h e d , t h e LC/MS c o m b i n a t i o n h a s been r e l a t i v e l y s l o w t o d e v e l o p due t o t h e o b v i o u s t e c h n o l o g i c a l d i f f i c u l t i e s o f t h e s o l u t e t r a n s f e r and i o n i z a t i o n . T h e r e a r e some i n d i c a t i o n s t h a t m i n i a t u r i z e d LC columns and t h e i r c o r r e s p o n d i n g l y l o w e l u e n t f l o w - r a t e s w i l l p l a y a m a j o r r o l e i n f u t u r e t r e n d s . Recent r e v i e w a r t i c l e s b y Tsuge ( r e f . 1 0 6 ) and H e n i o n ( r e f . 1 0 7 ) h i g h l i g h t t h e u n i q u e a d v a n t a g e s o f m i c r o c o l u m n s i n LC/MS. On t h e o t h e r hand, t h e r e c e n t l y d e v e l o p e d t h e r m o s p r a y a p p r o a c h ( r e f . 1 0 8 ) appears t o work b e s t w i t h h i g h f l o w - r a t e s o f p o l a r m o b i l e phases. I n t h e a r e a o f s u p e r c r i t i c a l f l u i d c h r o m a t o g r a p h y , a c o m b i n a t i o n o f c a p i l l a r y columns w i t h mass s p e c t r o m e t r y has a l s o been s u c c e s s f u l ( r e f s . 109,110). F i n a l l y , t h e u s e o f f l a m e - b a s e d d e t e c t o r s f o r m i c r o c o l u m n LC and SFC s h o u l d a l s o b e m e n t i o n e d . The m o b i l e - p h a s e f l o w s o f a f e w u L / m i n p r e s e n t h e r e a u n i q u e p o s s i b i l i t y t o n e b u l i z e t h e e n t i r e column e l u e n t i n t o a f l a m e d e t e c t o r . P r e v i o u s a t t e m p t s t o c o u p l e c o n v e n t i o n a l f l a m e s p e c t r o m e t e r s w i t h HPLC were o n l y m a r g i n a l l y s u c c e s s f u l . M i n i a t u r i z e d LC f l a m e d e t e c t o r s can work on t h e p r i n c i p l e o f flame emission ( r e f . I l l ) , thermionic e f f e c t ( r e f s . 112-114), o r even f l a m e i o n i z a t i o n ( r e f . 4 8 ) . A d d i t i o n a l modes o f o p e r a t i o n a r e p o t e n t i a l l y f e a s i b l e . S i n g l e - and d o u b l e - f l a m e d e t e c t o r v e r s i o n s o r i g i n a t e d i n o u r l a b o r a t o r y ( r e f s . 111-114) p r o v i d e s e l e c t i v e d e t e c t i o n o f p h o s p h o r u s - and n i t r o g e n - c o n t a i n i n g compounds. An example o f such d e t e c t i o n d e v i c e s i s shown i n F i g u r e 16. T h i s d e t e c t o r , used i n a t h e r m i o n i c d e t e c t i o n mode, c o n s i s t s o f two f l a m e s :

t h e p r i m a r y flame t h a t causes combustion, s o l u t e v o l a t i l i z a -

t i o n and breakdown; and t h e a n a l y t i c a l f l a m e , w h i c h c a n be o p t i m i z e d f o r a maximum r e s p o n s e . A n a l y t i c a l p e r f o r m a n c e o f t h e m i n i a t u r i z e d LC f l a m e d e t e c t o r s has been d e s c r i b e d ( r e f s . 1 1 1 - 1 1 4 ) . The f l a m e - b a s e d d e t e c t o r s may become i n c r e a s i n g l y p o p u l a r i n c a p i l l a r y SFC. I m p o r t a n t l y , such m o b i l e phases as c a r b o n d i o x i d e and n i t r o u s o x i d e y i e l d l i t t l e b a c k g r o u n d s i g n a l i n t h e f l a m e i o n i z a t i o n d e t e c t o r and, t h u s , a l l o w d e t e c t i o n o f many s o l u t e s . An example o f t h i s t y p e o f d e t e c t i o n i s d e m o n s t r a t e d i n F i g u r e 17 ( r e f . Z I ) , where t h e s e r i e s o f s i l i c o n e o l i g o m e r s a r e d i s p l a y e d . I n a d d i t i o n , i t i s e q u a l l y f e a s i b l e t o employ t h e s e l e c t i v e d e t e c t i o n p r i n c i p l e s i n c a p i l l a r y SFC ( r e f . 1 1 5 ) .

328

i

@ *

COLLECTOR ELECTRODE RUBIDIUM BEAD ANALYTICAL FLAME

HYDROGEN AND AIR I N L E T S

PRIMARY FLAME

AIR I N L E T CAPILLARY HYDROGEN AND NITROGEN I N L E T S

Fig. 16. Dual-flame thermionic detector for microcolumn LC. Reproduced with permission from ref. 112. Copyright 1983 American Chemical Society.

329

0

0.3

30 I

0.3

I

0.4

lime (min) I

0.5

80 I

0.6

80 I

0.7

I

0.8

’

Density (g/mL)

F i g . 17. SFC chromatogram o f a m e t h y l p o l y s i l o x a n e o l i g o m e r m i x t u r e (Dow C o r n i n g DC 200 f l u i d , 20 c s ) . C o n d i t i o n s : 27 m x 50 urn, i . d . , f u s e d - s i l i c a column; SE-54 s t a t i o n a r y phase (0.25 p m f i l m t h i c k n e s s ) ; C02 m o b i l e phase a t 40°C; d e n s i t y p r o g r a m f r o m 0.3 t o 0 . 8 g i n i t i a l 15 m i n i s o c o n f e r t i c p e r i o d ; F I D r e p r e s e n t t h e number o f r e p e a t i n g u n i t s p e r m i s s i o n f r o m r e f . 21. C o p y r i g h t 1984

mL-’ a t 0.0075 g mL-’ m i n - ’ a f t e r an d e t e c t o r . Numbers on l a b e l e d peaks i n t h e o l i g o m e r c h a i n . Reproduced w i t h A m e r i c a n Chemical S o c i e t y .

CONCLUSIONS M i n i a t u r i z a t i o n o f a n a l y t i c a l s e p a r a t i o n t e c h n i q u e s most v i s i b l y s t a r t e d w i t h t h e i n v e n t i o n o f c a p i l l a r y GC columns and has c o n t i n u e d t h r o u g h o u t t h e y e a r s t o w a r d t h e uses o f s m a l l e r column d i a m e t e r s i n b o t h c a p i l l a r y GC and LC. The d e v e l o p m e n t s i n m i c r o c o l u m n LC d u r i n g t h e l a t e 1970s have s t i m u l a t e d renewed i n t e r e s t i n s u p e r c r i t i c a l f l u i d c h r o m a t o g r a p h y and c a p i l l a r y e l e c t r o p h o r e s i s . W h i l e d e c r e a s i n g column d i m e n s i o n s and t h e n e c e s s a r y a d j u s t m e n t s i n r e l a t e d i n s t r u m e n t a t i o n h a v e been r e m a r k a b l e , f u r t h e r d e v e l o p m e n t s a r e i n o r d e r . M i n i a t u r i z e d s e p a r a t i o n t e c h n i q u e s c l e a r l y i n v i t e new d e t e c t i o n t e c h n o l o g i e s . I t i s v e r y l i k e l y t h a t combined e f f o r t s i n t h i s a r e a w i l l r e s u l t i n a f u t u r e g e n e r a t i o n o f a n a l y t i c a l i n s t r u m e n t s w h i c h c a n meet i n c r e a s i n g demands o f modern s c i e n c e and t e c h n o l o g y .

330 ACKNOWLEDGEMENT I n v e s t i g a t i o n s on t h e fundamental and a p p l i e d aspects o f microcolumn l i q u i d chromatography and c a p i l l a r y s u p e r c r i t i c a l f l u i d chromatography conducted i n t h e a u t h o r ' s l a b o r a t o r y over t h e l a s t s e v e r a l y e a r s have been supported by g r a n t s f r o m t h e N a t i o n a l I n s t i t u t e o f H e a l t h (GM 24349), N a t i o n a l Science Foundation (NSF CHE 82-00034),

U.S.

Department o f Energy (DE-FG2-84

ER 60215), and t h e O f f i c e o f Naval Research (N14-82-K-0561).

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3 33

CHROMATOGRAPHY BEYOND ANALYSIS

C.S.G. PHILLIPS I n o r g a n i c Chemistry L a b o r a t o r y , Oxford U n i v e r s i t y , South Parks Road, O x f o r d OX1 3QR ( U . K . )

SUMMARY The a p p l i c a t i o n o f chromatography t o n o n - a n a l y t i c a l problems i s i l l u s t r a t e d by p a r t i c u l a r r e f e r e n c e t o s t u d i e s o f heterogeneous c a t a l y s i s . Emphasis i s l a i d on t h e unusual v a r i a t i o n s o f t h e chromatographic method which may be usef u l l y employed and t h e advantages which can a r i s e f r o m t h e a b i l i t y t o probe s u r f a c e s w i t h a v a r i e t y o f molecules. Some p o s s i b l e p r e p a r a t i v e , s y n t h e t i c and 1.

pedagogic a p p l i c a t i o n s o f chromatography a r e a l s o discussed. INTRODUCTION Chromatography i s now v e r y f i r m l y e s t a b l i s h e d as t h e most p o w e r f u l o f ana-

l y t i c a l methods. I t i s t h u s t h e v a l u e d s e r v a n t of a l m o s t e v e r y s c i e n c e . Each y e a r we see t h e p u b l i c a t i o n o f a h o s t o f papers which i l l u s t r a t e i t s use t o s o l v e new and even more v a r i e d a n a l y t i c a l problems. I n many l a b o r a t o i r e s i t has become as commonplace as t h e t e s t tube, t h e b u r e t t e and t h e balance. O t h e r papers i n t h i s Symposium w i l l i l l u s t r a t e t h i s a n a l y t i c a l d i v e r s i t y , and show how even f u r t h e r improvements and e x t e n s i o n s o f t h e a n a l y t i c a l power o f c h r o matography may be expected. I w i l l be concerned, however, w i t h what I see as t h e w i d e r and p o s s i b l y a more dominant r o l e f o r chromatography i n s c i e n c e . My prime i n t e r e s t w i l l be t h e use o f chromatographic methods t o i n v e s t i g a t e a whole range o f p h y s i c o chemical phenomena. I propose t o i l l u s t r a t e t h i s by s p e c i f i c r e f e r e n c e t o an area i n which I have been p e r s o n a l l y most i n v o l v e d i n r e c e n t y e a r s , and y e t i n which, so i t seems t o me, we have o n l y begun as i t were " t o s c r a t c h t h e s u r f a ce". T h i s i s t h e s t u d y of heterogeneous c a t a l y s i s by gas-chromatographic methods. However t h e general p r i n c i p l e s a r e o f much w i d e r a p p l i c a t i o n , and o f course embrace b o t h gas and l i q u i d chromatography.

334

I n such a s t u d y we a r e e s s e n t i a l l y s e t t i n g o u t t o f e e l o r probe c a t a l y s t s u r f a c e s w i t h molecules r a t h e r t h a n say w i t h e l e c t r o n s or photons, and t h u s under c o n d i t i o n s much c l o s e r t o t h o s e met i n t h e i r p r a c t i c a l a p p l i c a t i o n . A p a r t i c u l a r f e a t u r e o f such chromatographic i n v e s t i g a t i o n s i s t h e i n f o r m a t i o n which i s p r o v i d e d by t h e r e a d y exchange o f one m o l e c u l a r probe f o r a n o t h e r : t h i s w i l l be a r e c u r r i n g theme t h r o u g h o u t t h i s paper. I t i s a l s o p o s s i b l e t o f o l l o w t h e r e a c t i o n s themselves w i t h i n v a r i o u s chromatographic systems, and t o make use o f s u b t l e combinations o f r e a c t i o n and s e p a r a t i o n t o p r o v i d e an e x t r a dimension t o t h e s t u d y o f c a t a l y t i c processes. I n such work, v a r i a t i o n s o f chromatcgraphy a r e commonly employed which a r e n o t u s u a l l y f a m i l i a r t o those concerned e s s e n t i a l l y w i t h i t s a n a l y t i c a l a p p l i c a t i o n s . Some o f these v a r i a t i o n s w i l l be r e f e r r e d t o i n v a r i o u s p a r t s o f t h i s paper. These a r e l i s t e d i n Table I t o g e t h e r w i t h r e f e r e n c e s i n which t h e y a r e d e s c r i b e d i n more d e t a i l . Table 1 Some v a r i a t i o n s o f Chromatographic Method Dusted Column E l u t i o n on a P l a t e a u F r o n t a l A n a l y s i s and E l ut ion by C'harac t e r is t ic P o i n t Heater Displacement I s o t o p e Exchange ( T r a c e r Pul se) Peak Shape A n a l y s i s Pulse T i t r a t i o n Reversed Flow Sample Vacancy Stopped Flow Thermal D e s o r p t i o n

1,2 2 293 495

6,7,8 2Y9

l o y l l ,12

13 14 2,15 16,17

I n t h e l a s t p a r t o f my paper, I wish a l s o t o draw a t t e n t i o n t o some p o s s i b l e i m p l i c a t i o n s f o r Chromatography i n p r e p a r a t i v e and s y n t h e t i c chemist r y , and t o suggest f u r t h e r t h a t chromatography c o u l d p l a y a q u i t e fundamental r o l e i n t h e t e a c h i n g o f c h e m i s t r y and p a r t i c u l a r l y i n i t s most f o r m a t i v e stages. 2.

THERMODYNAMIC ASPECTS OF ADSORPTION Chromatographic d a t a p r o v i d e v e r y p r e c i s e and d e t a i l e d i n f o r m a t i o n on t h e

335 d i s t r i b u t i o n o f molecules between a gas and a s t a t i o n a r y phase. I n t h e case o f s u r f a c e s t h e y l e a d t h u s d i r e c t l y t o f r e e e n e r g i e s , heats and e n t r o p i e s of a d s o r p t i o n a t a v a r i e t y o f s u r f a c e coverages, t o a d s o r p t i o n i s o t h e r m s and t o s u r f a c e areas ( r e f . 2 ) . I t i s w o r t h s t r e s s i n g moreover t h a t q u i t e s m a l l e n e r g i e s o f i n t e r a c t i o n a r e c h r o m a t o g r a p h i c a l l y s i g n i f i c a n t . Thus i n t h e s t u d y o f complex f o r m a t i o n (e.g.,

between a vapour l i g a n d and a metal s u r f a c e atom)

t e n s o f j o u l e s have r e a l chromatographic impact, w h i l e i n t h e normal preparat i o n o f s t a b l e complexes one would be concerned w i t h t e n s o f k i l o j o u l e s . I t i s t h u s p o s s i b l e t o i n v e s t i g a t e complexec, such as t h o s e f o r example between o l e f i n s and Cd2+ o r Zn2+ i o n s , which have never been i s o l a t e d by t h e p r e p a r a t i v e chemist(ref.18,

1 9 ) . Furthermore t h e s e i n t e r a c t i o n s a r e o f t e n enhanced

when t h e metal i o n i s exposed on a s u r f a c e r a t n t r t h a n embedded i n a s o l u t i o n . Thus

t h e use o f s o l u t i o n s o f AgNO,

f o r g a s - l i q u i d chromatography can g i v e a

s e l e c t i v e r e t a r d a t i o n o f o l e f i n s o v e r p a r a f f i n s c o r r e s p o n d i n g t o a few carbon atoms, b u t w i t h AgNO,

adsorbed on A120,

t h i s s e l e c t i v i t y i s extended t o some

f i f t y carbon atoms.

Two examples w i l l i l l u s t r a t e t h e use o f s i m p l e e l u t i o n chromatography t o i l l u m i n a t e c a t a l y t i c studies.

I n t h e f i r s t ( r e f . 20) i t was observed t h a t

b o t h t h e a c t i v i t y and t h e s e l e c t i v i t y (e.g.,

b u t a d i e n e from butene, o r acro-

l e i n f r o m propene) o f a Bi2MoO6 s e l e c t i v e - o x i d a t i o n c a t a l y s t were d r a m a t i c a l l y improved as t h e b u l k c a t a l y s t c o m p o s i t i o n changed from one t h a t was s l i -

g h t l y B i - r i c h t o one t h a t was s l i g h t l y M o - r i c h . A t t h e same t i m e t h e chromat o g r a p h i c p r o p e r t i e s o f t h e s u r f a c e a l s o changed c o m p l e t e l y ( f o r example t h e r e l a t i v e r e t e n t i o n o f o f benzene as a g a i n s t cyclohexane i n c r e a s e d b y a f a c t o r o f 1 6 ) , and i n a manner v e r y s i m i l a r t o t h a t found on p a s s i n g f r o m a B i 2 0 3 t o an Moo3 s u r f a c e . P h o t o e l e c t r o n spectroscopy t h e n c o n f i r m e d t h a t s m a l l b u l k d e v i a t i o n s f r o m s t o i c h i o m e t r y wert accompanied by d r a s t i c changes i n t h e comp o s i t i o n o f t h e s u r f a c e . The small excess o f Bi20, or Moo3 was t h u s c o n c e n t r a t e d on t h e r e l a t i v e l y small area (2m2g-l) o f t h e s u r f a c e and gave r i s e t o t h e p r e c i p i t a t e change i n c a t a l y t i c p r o p e r t i e s . The second example ( r e f . 15) concerns t h e mechanism o f w a t e r e l i m i n a t i o n f r o m a l c o h o l s on t h e s u r f a c e s o f m o d i f i e d aluminas. I n t h i s case m o l e c u l a r models o f t h e a l t e r n a t i v e cis- and t m n s - e l i m i n a t i o n mechanisms showed t h a t t h e r e should be d i f f e r e n c e s i n t h e number o f CH2-groups which m i g h t be expe-

336

c t e d t o be a t t a c h e d t o t h e c a t a l y s t s u r f a c e i n t h e t r a n s i t i o n s t a t e s . From t h e d i f f e r e n c e i n t h e r e t e n t i o n t i m e s o f two a d j a c e n t alkanes when chromatographed on t h e c a t a l y s t s u r f a c e i t was t h e n s i m p l e t o compute t h e d i f f e r e n c e s i n t h e f r e e e n e r g i e s of these t r a n s i t i o n s t a t e s and hence t h e expected r a t i o s o f t h e v a r i o u s p r o d u c t o l e f i n s . The e x p e r i m e n t a l r e s u l t s , o f which examples a r e g i v e n i n Table 2, f i t t e d q u i t e remarkably w e l l w i t h t h e t r a n s - e l i m i n a t i o n mechanism and n o t t h e cis. Table 2 P r e d i c t e d and Experimental R a t i o s o f P r o d u c t O l e f i n s Predicted product r a t i o s cis-Elimination traans-El i m i n a t i o n

Alcohol n-Heptan-4-01

trans ____

trans ___

0.6

cis

cis

1.8

Products observed

trans cis

__

1.86

Large ( 5 0 ) % n-but-1-ene

n- But an- 2- o 1 trans _ _

cis 1-enettruns

i.e.,

trans __

1.8

CiS

0.6

cis

3.2

+-ene+truns cis 1.4

1- e n e + t r m s cis 1.20

trans __

Small % n-pent-1-ene traans 1 .8 cis 0.6

lrans c i s + l - e n e 0.83

n-Pentan-2-01 cis n- Hexan- 2- o 1

No n-hex-1 -ene trans -

trans -

c1.s

1.8

trans __

No n-oct-1 -ene trans 1.8 cis

n-Octan-2-01 cis

2- Methyl-n-pentan-2-01

cis

~

a1 k-1 -ene a1 k-2-ene

1.5

0.6

trans c i s + l -ene 0.58

No n-oct-1-ene 0.6

trans cis

11 k-1 -ene a1 k - l - e n e

0.60

1.53

By employing d i f f e r e n t sample molecules i t i s o f course p o s s i b l e t o probe s e l e c t i v e l y d i f f e r e n t p a r t s o f t h e s u r f a c e . Thus, f o r example, a z e o l i t e - s u p p o r t e d metal c o u l d be probed w i t h N,

t o determine i t s t o t a l s u r f a c e area, w i t h

molecules o f d i f f e r e n t m o l e c u l a r geometries t o i n v e s t i g a t e i t s p o r e s t r u c t u r e , and w i t h H,

and CO t o d e t e r m i n e t h e n a t u r e and amount o f f r e e m e t a l exposed

( r e f . 1 0 ) . A f u r t h e r e x t e n s i o n o f t h i s concept i s p r o v i d e d by a s t u d y o f a Ni/SiO,

h y d r o c r a c k i n g c a t a l y s t u s i n g isotope-exchange chromatography ( r e f . 7 ) .

The c a t a l y s t was packed i n t o a chromatographic column and a s t r e a m o f hy-

331 drogen used as c a r r i e r gas w i t h a 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 . Deuterium gas was now i n j e c t e d a t t h e column i n l e t . I t s c o r r e c t e d r e t e n t i o n t i m e ( i . e . ,

after

t h e dead t i m e of t h e column) p r o v i d e d t h e n a d i r e c t measure o f t h e exchangeab l e hydrogen on t h e s u r f a c e o f t h e c a t a l y s t . A t temperatures below about 120°C t h i s corresponded t o hydrogen chemisorbed on t h e N i - s u r f a c e : t h i s was measured i n d e p e n d e n t l y by p u l s e - t i t r a t i o n chromatography. Above 120°C k i n e t i c a l l y - c o n t r o l l e d exchange w i t h adsorbed w a t e r began t o be s i g n i f i c a n t r e a c h i n g a thermodynamically-controlled

p l a t e a u a t about 260°C: t h e s e o b s e r v a t i o n s were checked

q u a n t i t a t i v e l y by i n j e c t i o n s o f known amounts o f w a t e r . F i n a l l y , a t s t i l l h i g h e r temperatures, f u r t h e r k i n e t i c a l l y - c o n t r o l l e d exchange t o o k p l a c e which appeared t o i n v o l v e t h e H i n S i O H groups: t h i s was supported by i n f r a - r e d spectroscopy. I n t h e case o f t h e k i n e t i c a l l y - c o n t r o l l e d exchanges t h e k i n e t i c s were i n v e s t i gated i n more d e t a i l by s t o p p e d - f l o w chromatography. I n j e c t i o n o f hydrocarbons i n t o t h e system t h e n made p o s s i b l e t h e i n v e s t i g a t i o n o f t h e f u r t h e r exchange o f hydrogen w i t h adsorbed hydrocarbon. C l e a r l y as w e l l as p r o b i n g d i f f e r e n t t y p e s o f s u r f a c e s i t e , e.g.,

acidic

and b a s i c s i t e s w i t h b a s i c and a c i d i c vapours r e s p e c t i v e l y , s t r o n g l y adsorbed molecules may a l s o be used t o s e l e c t i v e l y p o i s o n such s i t e s . T h i s i s o f t e n i m p o r t a n t and r e v e a l i n g i n t h e general s t u d y o f a r e a c t i o n mechanism. I t can however become a v i t a l t e s t when t h e r e i s a r i s k t h a t t h e e x p e r i m e n t a l r e s u l t s observed c o u l d be l a r g e l y caused by r e l a t i v e l y small t r a c e s o f i m p u r i t y i n t h e c a t a l y s t , e.g.,

small amounts o f f i n e l y - d i v i d e d metal i n a supposedly a c t i v e

organometallic c a t a l y s t .

3.

ADSORPTION K I N E T I C S I n t h e p r e c e d i n g s e c t i o n we c o n s i d e r e d t h e way i n which s u r f a c e s may be

probed thermodynamically by gas-chromatographic methods. I t i s , o f course, a l s o possible t o explore chromatographically t h e k i n e t i c s o f adsorption ( r e f . 2 ) . I n t h e s i m p l e s t cases, w i t h a l l a d s o r p t i o n s i t e s h a v i n g s i m i l a r a d s o r p t i o n k i n e t i c s , i n c r e a s i n g slowness o f t h e a d s o r p t i o n - d e s o r p t i o n process i s m a n i f e s t e d i n t h e broadening o f t h e e l u t i o n peak, f o l l o w i n g f o r example t h e c l a s s i c a l t r e a t ment o f t h e van Deemter e q u a t i o n ( r e f . 2 1 ) . Thus w i t h m o l e c u l a r s i e v e 5A as s t a t i o n a r y phase t h e C t e r m o f t h i s e q u a t i o n i s markedly i n c r e a s e d on p a s s i n g f r o m t h e n o n - p e n e t r a t i n g branched t o t h e s t r a i g h t c h a i n hydrocarbons which a r e a b l e to

p e n e t r a t e i n t o t h e narrow pores o f t h e z e o l i t e s t r u c t u r e .

338

As Giddings ( r e f . 22) has shown (see a l s o r e f . 23) a d s o r p t i o n on t o two o r more k i n e t i c a l l y - d i s t i n g u i s h a b l e s i t e s w i l l l e a d t o skewed peaks. These peaks may be regarded as t h e s u p e r p o s i t i o n o f a symmetrical peak (produced by t h o s e molecules which adsorb o n l y on t h e f a s t s i t e s d u r i n g t h e i r passage t h r o u g h t h e chromatographic column) and a broad t r a i l i n g peak (produced by t h o s e molecules which have been absorbed a t l e a s t once on s l o w e r s i t e s ) . We have come across what appears t o be a good example ( r e f . 24) o f t h i s phenomenon i n s t u d i e s made w i t h a s t a t i o n a r y phase c o n s i s t i n g o f anatase c o a t e d w i t h a carbonaceous mater i a l (which had been produced by t h e d i s p r o p o r t i o n a t i o n r e a c t i o n o f 1,3-butad i e n e ) . The phenomenon i's h e r e b r o u g h t o u t p a r t i c u l a r l y c l e a r l y as i t s e f f e c t s a r e o n l y apparent w i t h c e r t a i n molecules. Thus n-hexane produces normal symmet r i c a l peaks a t a l l temperatures i n v e s t i g a t e d , w h i l e around 200°C, f o r example, i t s isomer 2,2-dimethyl

butane produces a peak which i s much b r o a d e r t h a n t h a t

o f n-hexane and which t a i l s s i g n i f i c a n t l y . The skewed n a t u r e o f t h i s peak var i e s w i t h b o t h temperature and w i t h f l o w - r a t e o f c a r r i e r gas. I t can be d e s c r i bed v e r y adequatedly i n terms o f t h r e e t y p e s o f s i t e i n v o l v i n g f a s t , medium and slow a d s o r p t i o n - d e s o r p t i o n k i n e t i c s , t h e l a s t b e i n g o f r e a l s i g n i f i c a n c e o n l y a t t h e s l o w e s t gas f l o w - r a t e s employed. I n p a r t i c u l a r t h e p o s i t i o n o f t h e peak maximum ( r e l a t i v e t o t h a t o f n-hexane)

i n c r e a s e s w i t h d e c r e a s i n g f l o w - r a t e as

a d s o r p t i o n on t h e slower s i t e s p l a y s an i n c r e a s i n g l y i m p o r t a n t r o l e . I n s t u d y i n g a wide range o f molecules t h e skewing and broadening phenomenon was found t o i n c r e a s e i n general w i t h i n c r e a s e i n c h a i n b r a n c h i n g and w i t h h y d r o g e n a t i o n o f a benzene r i n g . I n many circumstances t h e d i s t i n c t i o n between f a s t and s l o w a d s o r p t i o n - d e s o r p t i o n k i n e t i c s i n c r e a s e s even f u r t h e r so t h a t t h e chromatographic e l u t i o n peak c o n s i s t s o f an a p p a r e n t l y normal symmetrical peak t o g e t h e r w i t h a l o n g and v e r y low t a i l . T h i s t a i l i s produced by t h o s e molecules which have been i n v o l v e d i n a d s o r p t i o n - d e s o r p t i o n processes which a r e now v e r y s l o w

i n compa-

r i s o n w i t h t h o s e i n which t h e symmetrical peak molecules have been i n v o l v e d . I t i s a l l t o o easy t h e n t o be unaware o f t h e v e r y e x i s t e n c e o f such a t a i l

, altho-

ugh i t should be suspected i n view o f t h e l a c k o f q u a n t i t a t i v e sample r e c o v e r y . Indeed such a l o s s would appear t o o c c u r a l l t o o f r e q u e n t l y i n some o f t h e n o r mal a n a l y t i c a l a p p l i c a t i o n s o f gas chromatography. T h i s l o n g t a i l may be reveal e d by t h e use o f s t o p p e d - f l o w chromatography i n which molecules s l o w l y desorb i n g f r o m t h e surface a r e a c c u m i l a t e d by s t o p p i n g t h e gas f l o w f o r a p e r i o d o f

339

t i m e . An example i s p r o v i d e d i n some s t u d i e s w i t h ion-exchange r e s i n s as gas-chromatographic s t a t i o n a r y p h a s e s . ( r e f . 4.

25).

CATALYTIC REACTIONS The s t u d y o f c a t a l y s i s i s , o f course, commonly a s s i s t e d by t h e use o f gas

chromatography as a p u r e l y a n a l y t i c a l d e v i c e . Such s t u d i e s may o f t e n be simp l i f i e d c o n s i d e r a b l y by a more i n t i m a t e c o n n e c t i o n between t h e c a t a l y t i c r e a c t o r and t h e a n a l y t i c a l chromatographic column as i n t h e now c l a s s i c a l m i c r o r e a c t o r t e c h n i q u e ( r e f . 2 6 ) . The i n j e c t i o n system o f t h e chromatographic column can sometimes be s i m p l y adapted t o f u n c t i o n as t h e m i c r o r e a c t o r , and t h e t i m e spent on t h e c a t a l y s t i n t h e m i c r o r e a c t o r v a r i e d by s t o p p i n g t h e gas f l o w i m m e d i a t e l y a f t e r i n j e c t i o n ( r e f . 27, 2 8 ) . By t h e use o f sample-vacancy chromatography ( r e f . 1 4 ) , i n which a sample o f t h e r e a c t a n t f e e d i s i n j e c t e d between t h e c o u p l e d r e a c t o r and chromatographic column w h i l e r e a c t a n t i s f e d c o n t i n u o u s l y i n t o t h e r e a c t o r , d i f f e r e n t i a l r e a c t i o n chromatograms may be generated. I n these n e g a t i v e peaks correspond t o r e a c t i o n p r o d u c t s , a p o s i t i v e peak measures t h e amount o f r e a c t a n t which has r e a c t e d , w h i l e n o n - r e a c t i n g i m p u r i t i e s a r e e l i m i n a t e d f r o m t h e chromatogram and n o n - v o l a t i l e r e a c t i o n p r o d u c t s may be e s t i mated f r o m t h e d i f f e r e n c e between p o s i t i v e and n e g a t i v e peaks. However much a l s o may be l e a r n e d by combining i n t h e same column, o r even i n t h e same p a c k i n g m a t e r i a l , b o t h a n a l y t i c a l and chromatographic c h a r a c t e r i s t i c s : see f o r example t h e r e v i e w a r t i c l e s by van Swaay ( r e f . 29) on "The Study of R e a c t i o n K i n e t i c s by t h e D i s t o r t i o n o f Chromatographic E l u t i o n Peaks" and by Langer and P a t t o n ( r e f . 30) on "Chemical Reactor A p p l i c a t i o n s o f t h e Gas Chromatographic Column", and Chapter 13 on "On-Column R e a c t i o n s " i n Conder and Young ( r e f . 2 ) .

I t i s a p i t y t h a t t h e t e r m " r e a c t i o n chromatography" c a n n o t now

be a p p l i e d t o such s t u d i e s , s i n c e i t has become t h e p r a c t i c e t o employ i t t o cover almost e x c l u s i v e l y r e a c t i o n s o c c u r r i n g b e f o r e o r a f t e r b u t n o t w i t h i n a chromatographic column (e.g.,

r e f . 31). This i s a f u r t h e r i l l u s t r a t i o n o f t h e

heavy a n a l y t i c a l b i a s o f gas chromatography. One immediate advantage o f u s i n g t h e same m a t e r i a l as b o t h c a t a l y s t and chromatographic s t a t i o n a r y phase i s t h a t thermodynamic and a d s o r p t i o n - k i n e t i c s t u d i e s o f t h e s u r f a c e , such as t h o s e o u t l i n e d i n t h e two p r e v i o u s s e c t i o n s , may be made w i t h e s s e n t i a l l y t h e same e x p e r i m e n t a l system. Moreover a l l t h e s e s t u d i e s a r e t h e n c a r r i e d o u t under c o n d i t i o n s v e r y s i m i l a r t o t h o s e i n which

340 t h e c a t a l y t i c r e a c t i o n s m i g h t be o c c u r r i n g i n say i n d u s t r i a l p r a c t i c e , r a t h e r than t h e more a b s t r a c t e d c o n d i t i o n s (e.g.,

under h i g h vacuum o r s u b j e c t t o

e l e c t r o n bombardment) o f t e n used i n modern p h y s i c a l s t u d i e s o f c a t a l y s i s and c a t a l y t i c s u r f a c e s . There a r e a l s o many o t h e r advantages i n c l u d i n g r a p i d i t y and p r e c i s i o n o f measurement, and o f course, once a g a i n , t h e ready a b i l i t y t o probe t h e s u r f a c e w i t h a whole range o f m o l e c u l a r species. The i m p o s i t i o n o f a chemical r e a c t i o n on t o t h e normal chromatographic process n a t u r a l l y leads t o a more complex chromatogram. I n p r i n c i p l e t h i s comp l e x i t y can be t r a n s f o r m e d t o g i v e t h e k i n e t i c s o f t h e r e a c t i o n ( s e e e s p e c i a l l y r e f . 3 0 ) . Thus i n t h e case o f a simple r e v e r s i b l e r e a c t i o n A

B, i n j e c t i o n

o f A w i l l l e a d t o a chromatogram spanning t h e normal e l u t i o n peaks o f A and o f B f r o m t h e a n a l y s i s o f which ( r e f . 30, 32) t h e f o r w a r d and r e v e r s e r a t e c o n s t a n t s (and hence t h e e q u i l i b r i u m c o n s t a n t ) may be determined. Experimental examples i n c l u d e t h e i s o m e r i s a t i o n o f oyn- and a n t i - a c e t a l d o x i m e ( r e f . 30), t h e enantiomerization o f l-chlor0-2,2-dimethyl r y phase c o n t a i n i n g n i c k e l ( 1 1 ) b i s

a z i n i d i n e on a r e s o l v i n g s t a t i o n a -

3-(trifluoroacetyl)-l-R-.camphorate

(ref.

33), and t h e i n t e r c o n v e r s i o n o f ortho and para hydrogen ( r e f . 3 4 ) . F o r a decomposition r e a c t i o n (producing r e a c t i o n products l e s s s t r o n g l y retarded chrom a t o g r a p h i c a l l y than t h e r e a c t a n t ) e.g.,

dicyclopentadiene-

pentadiene

( r e f . 3 0 ) , t h e chromatogram c o n s i s t s o f a normal peak o f u n r e a c t e d r e a c t a n t preceded by a l o n g t r a i l i n g peak o f p r o d u c t s . T h i s l a t t e r peak s t a r t s s t e p w i s e ( i n t h e case o f more t h a n one p r o d u c t ) a t t h e r e t e n t i o n t i m e s o f t h e normal e l u t i o n peaks o f t h e p r o d u c t s ( i . e . ,

c o r r e s p o n d i n g t o p r o d u c t s produced a t t h e

v e r y b e g i n n i n g o f b o t h t h e r e a c t i o n and t h e column) and t r a i l s r i g h t back t o t h e r e a c t a n t peak. Once a g a i n t h i s chromatogram r e f l e c t s and can be t r a n s f o r med t o g i v e r e a c t i o n - k i n e t i c i n f o r m a t i o n . However, whenever t h e r e i s a r e l a t i v e l y l a r g e chromatographic s e p a r a t i o n ( b u t see a l s o r e f . 13) between r e a c t a n t and p r o d u c t s (e.g.,

decomposition,

c r a c k i n g , e l i m i n a t i o n , Fischer-Tropsch r e a c t i o n s ) , t h e a n a l y s i s o f t h e r e a c t i o n may be c o n s i d e r a b l y extended and s i m p l i f i e d by t h e use o f s t o p p e d - f l o w chromatography ( r e f . 1 5 ) . Here t h e gas f l o w i s p e r i o d i c a l l y stopped d u r i n g t h e passage o f t h e r e a c t a n t t h r o u g h t h e column, so t h a t t h e r e a c t i o n and t h e chromatographic processes a r e e f f e c t i v e l y uncoupled. The f o r m e r c o n t i n u e s w h i l e t h e l a t t e r s t o p s d u r i n g t h e f l o w i n t e r r u p t i o n s . Each t i m e t h e gas f l o w i s

341

stopped, r e a c t i o n p r o d u c t s accumulate a t t h a t p o i n t i n t h e column where t h e r e a c t a n t was s i t t i n g d u r i n g t h e s t o p . On r e s t a r t i n g t h e f l o w , t h e s e accumulat i o n s o f p r o d u c t t h e n behave as though t h e y had been i n j e c t e d a t t h i s p a r t i c u l a r p o i n t and produce sharp chromatographic peaks superimposed on t h e broad r e a c t i o n chromatogram.

I n f a v o u r a b l e cases i t i s p o s s i b l e t o g e n e r a t e more

t h a n two hundred s e t s o f such peaks d u r i n g one passage o f r e a c t a n t t h r o u g h t h e column. F u r t h e r i n f o r m a t i o n can be o b t a i n e d e.g.,

by v a r y i n g t h e s t o p t i m e

( t o i d e n t i f y a u t o c a t a l y t i c and s u c c e s s i v e r e a c t i o n s ) , by i n t r o d u c i n g p o t e n t i a l i n h i b i t o r s and c o c a t a l y s t s i n t o t h e column ( t h e i r p o s i t i o n s i f t h e y a r e V O l a t i l e b e i n g r e a d i l y determined a t any t i m e from t h e i r chromatographic b e h a v i o u r ) , and by c a r r y i n g o u t s i m u l t a n e o u s l y more t h a n one r e a c t i o n a t d i f f e r e n t p a r t s o r a t t h e same p a r t o f t h e column. There a r e o t h e r ways i n which advantage may be t a k e n o f t h e v a r i e t y o f m o l e c u l a r probes. Thus t h e h y d r o g e n a t i o n o f an adsorbed C, c i n g C,

s p e c i e s on a Ni/SiO,

s u r f a c e ( r e f . 3 5 ) can be i n v e s t i g a t e d by produ-

s p e c i e s f r o m methane, f r o m C O Y o r v i a t h e i d r o c r a c k i n g o f p a r a f f i n

hydrocarbons. So a l s o t h e d e t a i l s o f a s e r i e s o f s u c c e s s i v e r e a c t i o n s ( r e f . 36) may be u n r a v e l l e d by i n j e c t i o n o f t h e p r o d u c t s . Even t h e r e a c t i o n s themselves may be used t o s t u d y t h e n a t u r e o f t h e s u r face. Thus on c e r t a i n s u r f a c e s some e l i m i n a t i o n r e a c t i o n s (e.g.,

water from

a l c o h o l s o r hydrogen c h l o r i d e f r o m a l k y l c h l o r i d e s t o g i v e o l e f i n s i n each case) t a k e p l a c e w i t h o u t any p e r c e p t i b l e movement o f r e a c t a n t on t h e column ( t h e r e t e n t i o n t i m e s o f t h e p r o d u c t o l e f i r l s remain c o n s t a n t f o r a whole s e r i e s o f successive s t o p p e d - f l o w chromatograms). The a n a l y s i s o f t h e k i n e t i c s t h e n l e a d s t o a c h o i c e between two mechanisms, one i n v o l v i n g f i r s t - o r d e r r e a c t i o n s on a s e r i e s o f s i t e s o f d i f f e r e n t a c t i v i t y and t h e o t h e r a h i g h e r - o r d e r r e a c t i o n ( r e f . 3 7 ) . The second mechanism can be e f f e c t i v e l y d i s p r o v e d by demons t r a t i n g t h a t t h e e l i m i n a t i o n r e a c t i o n o f one m o l e c u l a r species i s u n a f f e c t e d by t h a t o f a n o t h e r s i m i l a r m o l e c u l a r species and, more s u b t l y , by p a r t i a l l y r e a c t i n g one m o l e c u l a r species (and t h u s on t h e a l t e r n a t i v e h y p o t h e s i s f r e e i n g t h e more r e a c t i v e s i t e s ) and t h e n i n j e c t i n g and f o l l o w i n g t h e r e a c t i o n o f anot h e r . The d e t a i l e d computer a n a l y s i s o f t h e k i n e t i c s p e r m i t s a c a l c u l a t i o n o f t h e d i s t r i b u t i o n on t h e s u r f a c e o f s i t e s o f v a r y i n g c a t a l y t i c a c t i v i t y , i n ess e n t i a l l y t h e same manner as i n d i v i d u a l r a d i o a c t i v e i s o t o p e s may be i d e n t i f i e d by d e t a i l e d a n a l y s i s o f t h e decay r a t e s o f a m i x t u r e o f i s o t o p e s measured o v e r a period o f time.

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5.

PREPARATIVE AND SYNTHETIC CHEMISTRY

I t u r n now t o a d i f f e r e n t n o n - a n a l y t i c a l a s p e c t o f chromatography: i t s w i der a p p l i c a t i o n i n p r e p a r a t i v e and s y n t h e t i c c h e m i s t r y . Chromatography has o f course been used f o r a v e r y l o n g t i m e as a p r e p a r a t i v e t o o l . However i t s potent i a l i t i e s seem n o t even y e t t o have been f u l l y developed. I propose t o comment i n p a r t i c u l a r on two m a t t e r s namely (A) t h e a b i l i t y t o d r i v e a chemical r e a c t i o n beyond i t s normal thermodynamic l i m i t s and ( B ) t h e p o s s i b i l i t y o f c o n t r o l l i n g t h e n a t u r e o f t h e s y n t h e t i c i n v e s t i g a t i o n s themselves. (A) As has been i n d i c a t e d above i n t h e d i s c u s s i o n o f heterogeneous c a t a l y s i s , chemical r e a c t i o n s ( c a t a l y s e d o r u n c a t a l y s e d ) may be c a r r i e d o u t i n a c h r o matographic column w h i l e a t t h e same t i m e t h e column i s s e p a r a t i n g r e a c t a n t s and t h e v a r i o u s p r o d u c t s . L e t us t a k e , f o r example, a r e v e r s i b l e r e a c t i o n such as t h a t i n v o l v i n g t h e i n t e r c o n v e r s i o n o f t h e v a r i o u s hexane isomers o v e r a Pt/A1,0,

c a t a l y s t . Then by u s i n g h e a t e r d i s p l a c e m e n t chromatography

, n-hexane

may n o t o n l y te c o n v e r t e d i n t o i t s isomers and t h e v a r i o u s isomers t h e n separated, b u t one can a r r a n g e t h a t t h e lass-branched isomers ( s t a r t i n g w i t h n-hexane i t s e l f ) a r e s u c c e s s i v e l y and s e l e c t i v e l y r e i s o m e r i s e d . I n t h i s way t h e r e a c t i o n i s d r i v e n t o produce a l m o s t c o m p l e t e l y t h e most-branched isomer, 2,2-dimet h y 1 butane ( r e f . 4 ) . I t i s t h u s p o s s i b l e , i n p r i n c i p l e a t l e a s t , t o c o n t r o l chemical r e a c t i o n s so as t o produce almost 100 % o f a p r o d u c t w h i c h m i g h t o n l y be formed a t r a t h e r low y i e l d s by c o n v e n t i o n a l means. ( B ) T h i s l e a d s on t o a b r o a d e r aspect o f t h e r o l e o f chromatography i n syn-

t h e s i s . H i t h e r t o chromatography has been used a l m o s t e n t i r e l y as t h e handmaiden of t h e s y n t h e t i c chemist. He d e c i d e s what r e a c t i o n s and what t a r g e t m o l v u l e s t o s t u d y and m e r e l y c a l l s i n chromatographic a n a l y s i s t o t e l l him how w e l l he has achieved h i s g o a l s . I t seems t o me, however, t h a t chromatography s h o u l d a t t i m e s t a k e a more commanding p o s i t i o n a t t h e i n i t i a l d e s i g n o f t h e s y n t h e t i c experiments. Thus r e a c t i o n s c o u l d be u s e f u l l y s t u d i e d which were s e l e c t e d t o g i v e manv new p r o d u c t s i n s t e a d o f one t a r g e t molecule. As a s i m p l e examDle one may q u o t e some work i n which my group was i n v o l v e d many y e a r s ago i n t h e prepar a t i o n of s i l i c o n - g e r m a n i u m h y d r i d e s , b o r a z i n e s and Group I V a l k y l s . Here a h o s t o f h i t h e r t o unknown m o l e c u l e s were produced, separated and r a p i d l y i d e n t i f i e d by a v a r i e t y o f gas-chromatographic methods. Thus gas-chromatographic columns, v a r i o u s d e t e c t o r s , and a n c i l l i a r y apparatus were hooked up t o p r o v i d e t h e e q u i v a l e n t o f t h e t r a d i t i o n a l high-vacuum p r e p a r a t i v e l i n e , b u t w i t h f l o w i n g

343

gas streams r e p l a c i n g vacuum.(ref.

38).

I t c o u l d w e l l be o f c o n s i d e r a b l e v a l u e t o s t u d y a l l t h e p r o d u c t s o f a synt h e t i c process. T h i s c o u l d n o t o n l y p r o v i d e a much f u l l e r i n s i g h t i n t o t h e nat u r e of t h e s y n t h e t i c r e a c t i o n s b u t c o u l d l e a d t o new and more d i r e c t s y n t h e t i c r o u t e s . Thus q u i t e m i n o r p r o d u c t s o f t h e r e a c t i o n under one s e t o f c i r c u m s t a n ces may become s i g n i f i c a n t s y n t h e t i c p r o d u c t s o f t h e r e a c t i o n when t h e c i r c u m stances a r e a l t e r e d i n a manner suggested by t h e d e t a i l s o f chromatographic analyses. What I am

t h u s a d v o c a t i n g i s a s y s t e m a t i c s t u d y o f what a r e a t p r e -

s e n t o n l y s i d e r e a c t i o n s . To p u t i t a n o t h e r way, we should s u r e l y s t u d y t h e mu1 t i f a r i o u s p r o d u c t s o f a r t i f i c i a l s y n t h e t i c r e a c t i o n s j u s t as we have s t u d i e d t h e m u l t i f a r i o u s p r o d u c t s o f r e a c t i o n s o c c u r r i n g i n n a t u r e . How many f a s c i n a t i n g species a r e b e i n g thrown down t h e l a b o r a t o r y s i n k , as h i g h p o l y r i e r s once were b e f o r e a whole i n d u s t r y became founded upon them?

6.

CHROMATOGRAPHY I N EDUCATION My f i n a l peep i n t o t h e f u t u r e i s concerned w i t h t h e use o f chromatography

i n education. I f one l o o k s , f o r example, t h r o u g h t h e " J o u r n a l o f Chemical Educ a t i o n " one f i n d s each y e a r some dozen o r so r e f e r e n c e s t o chromatographic exp e r i m e n t s . However t h e y a r e v i r t u a l l y a l l concerned w i t h d e m o n s t r a t i n g how c h r o matography works o r w i t h i t s use f o r s p e c i f i c a n a l y s e s . Now I see chromatography h a v i n g a much more fundamental pedagogic r o l e , p a r t i c u l a r l y as chromatographs become w i d e l y a v a i l a b l e and as t h e computer and t h e VDU became f a m i l i a r i n o u r schools. What b e t t e r t o o l i s t h e r e t h a n chromatography f o r d e m o n s t r a t i n g

so many o f t h e b a s i c p r i n c i p l e s o f m o l e c u l a r c h e m i s t r y ? I t i s much more e f f e c t i ve t h a n t h e t e s t t u b e and s u r e l y more i n f o r m a t i v e , i f l e s s s p e c t a c u l a r , t h a n t h e t r a d i t i o n a l bangs and s m e l l s w i t h which t h e average s t u d e n t i s i n i t i a t e d i n t o t h e mysteries o f chemistry. L e t me b r i e f l y o u t l i n e a p o s s i b l e s e r i e s o f i n t r o d u c t o r y experiments. A m i x t u r e o f t h e l o w e r p a r a f f i n hydrocarbons i s f i r s t separated r a p i d l y , e.g.,

by

c a p i l l a r y gas chromatography. The s t r a i g h t - c h a i n molecules a r e a t once i d e n t i f i e d by t h e r e g u l a r p a t t e r n o f t h e i r r e t e n t i o n times, and t h e v a r i o u s isomers then p i c k e d o u t and t h e i r number and b e h a v i o u r r a t i o n a l i s e d . The d i s t i n c t i o n s between t h e d i f f e r e n t m o l e c u l e s can t h e n be f u r t h e r i n v e s t i g a t e d by t h e use of m o l e c u l a r s i e v e s , s e l e c t i v e v o l a t i l i t y and s e l e c t i v e d i f f u s i o n . Thus i n a v e r y s h o r t space o f t i m e t h e s t u d e n t becomes f a m i l i a r w i t h t h e n a t u r e and p r o p e r t i e s

344

o f a whole range o f s i g n i f i c a n t and n a t u r a l l y - a b u n d a n t species. From t h e n on i t i s a s t r a i g h t f o r w a r d m a t t e r t o develop i n t o m o l e c u l e s w i t h d i f f e r e n t a c t i v e groups and t o a v a r i e t y o f chemical r e a c t i o n s , a l l o f which may be s t u d i e d r a p i d l y and c o n v i n c i n g l y w i t h a r e l a t i v e l y s i m p l e gas chromatograph. What one needs f o r such a programme i s j u s t a simple, w i d e l y - u s e d and r e l a t i v e l y inexpens i v e chromatographic d e s i g n t o g e t h e r w i t h t h e d e d i c a t i o n o f an i n s p i r e d t e a c h e r t o d e v i s e a s e t o f w e l l - t e s t e d and graded experiments t o i n t r o d u c e s t u d e n t s e f f i c i e n t l y and c o n v i n c i n g l y t o t h e r i c h e s o f m o l e c u l a r c h e m i s t r y . 7.

WHY CHROMATOGRAPHY AND WHY NOT? F i n a l l y one may pose two r a t h e r b a s i c q u e s t i o n s . ( A ) Why s h o u l d one t r y t o

persuade people t o use chromatographic methods i n o t h e r t h a n t h e i r normal anal y t i c a l r o l e ? ( B ) Why a r e chromatographic methods as y e t so l i t t l e accepted outside analysis? (A) To t h e f i r s t q u e s t i o n , t h e r e a r e o f c o u r s e many answers well-known t o t h e p a r t i c i p a n t s a t t h i s meeting. I would w i s h t o h i g h l i g h t p a r t i c u l a r l y t h e f a c t t h a t " i t i s t h e r e " , t h a t i s t h a t chromatographic apparatus i s now v e r y w i d e l y d i s t r i b u t e d in a l l s o r t s o f s c i e n t i f i c l a b o r a t o r i e s , and indeed i n comp a r i s o n w i t h so many o t h e r t e c h n i q u e s i s v e r y easy t o s e t up and t o employ. Chromatographic experiments a r e u s u a l l y r e m a r k a b l y r a p i d . They r e q u i r e o n l y small amounts o f m a t e r i a l and i n many cases t h e s e need n o t even be pure, s i n c e t h e chromatographic method c o n t i n u o u s l y separates and d i s t i n g u i s h e s t h e v a r i o u s components o f a m i x t u r e . Yet t h e methods, e s p e c i a l l y w i t h a l i t t l e i n g e n u i t y can y i e l d a w e a l t h o f r a t h e r p r e c i s e i n f o r m a t i o n . Moreover t h e r e a r e now two q u i t e e x c e l l e r t books ( r e f . 2, 39) w h i c h s p e l l o u t e x t r e m e l y c l e a r l y a l l t h a t one needs t o know t o a p p l y gas-chromatographic methods t o physicochemical measurements. The e x t e n s i o n o f t h e s e i d e a s t o t h e use o f l i q u i d chromatography i s e s s e n t i a l l y s t r a i g h t f o r w a r d .

(B) Yet, and here I come t o t h e second q u e s t i o n , i t must be a d m i t t e d t h a t chromatographic methods a r e s u r p r i s i n g l y l i t t l e used o u t s i d e a n a l y s i s , and t h e n o n l y f o r t h e most p a r t by t h o s e whose background and c e n t r a l i n t e r e s t l i e i n a n a l y t i c a l chromatography ( s e e e.g.,

r e f . 40). Why i s t h i s ? I n p a r t I t h i n k i t

stems from t h e c u r i o u s i n t e l l e c t u a l c l o u d t h a t somehow hangs o v e r a n a l y t i c a l procedures. There seems t o be something o f magic and w i t c h c r a f t a b o u t them, a v i e w perhaps a s s i s t e d b y t h e e x o t i c chemicals t h e y o f t e n employ, and i n t h e ca-

345

se

o f chromatography t h e m u l t i p l i c i t y o f p h y s i c a l processes which a r e i n v o l v e d

and which need t o be u n r a v e l l e d . Now i t may appear t o some t h a t t h i s must a l ways be so, b u t I b e l i e v e t h a t science i s s t i l l v e r y o f t e n a m a t t e r o f f a s h i o n . Fashions change and i n t h e end wisdom may y e t p r e v a i l . A f t e r a l l gas chromatography was f i r s t e x e m p l i f i e d i n 1512 ( r e f . 41), was c l e a r l y expounded i n 1941 ( r e f . 42), b u t o n l y came i n t o i t s own a f t e r 1952 ( r e f . 43). For t h e c r u c i a l c o n t r i b u t i o n s on t h e l a s t two o f these occasions we a r e o f course indebted t o t h e man i n whose honour t h i s meeting i s held.

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P h i l l i p s , J. Chem. SOC. Dalton, (1973)

19 J.R. Chretien, K. Szymaniak, J.E. Dubois, R.F. H i r s c h and R.J. Gaydosh, J. Chromatogr., 294 (1984) 1-14. 20

A.G. M i t c h e l l , P.M. Lyne, K.F. S c o t t and C.S.G. raday Trans. 1 , 77 (1981 ) 241 7-2427.

21

J.J. van Deemter, F.J. Zuiderweg and A. Klinkenberg, Chem. Eng. Sci., ( 1 956) 271 -289.

22

J.C. Giddings, Anal. Chem.,

23

D.E. Damiani, E.M. 355-366.

24

G.J.S.

25

R.F. H i r s c h and C.S.G.

26

R.T. Kokes, H. Tobin and P.H. 5860-5862.

27

K.F. S c o t t and C.S.G.

28 A.F.

5

35 (1963) 1999-2002.

V a l l e s and C.E.

V i n t and C.S.G.

P h i l l i p s , J. Chem. SOC. Fa-

Gigola, J. Chromatogr.,

P h i l l i p s , J. Chromatogr.,

196 (1980)

292 (1984) 263-271.

P h i l l i p s , Anal. Chem., 49 (1977) 1549-1551. Emmett, J. Amer. Chem. SOC.,

77 (1955)

P h i l l i p s , J. Chromatogr., 112 (1975) 61-70. Prokhorova, J. Chromatogr., 283 (1984) 365-370.

Shushonova and L.Yu.

29

M. van Swaay, i n J.C. Giddings and R.A. K e l l e r (Eds.) Advances i n Chromatography Vol. 8, Marcel Dekker, New York, 1969, pp. 363-399.

30

S.H. Langer and J.E. Patton, i n J.H. P u r n e l l (Ed.), New Developments i n Gas Chromatography, John Wiley, New York, 1973, pp. 293-373.

31

R.W.

32

R. Kramer, J . Chromatogr.,l07

33

N. Burkle, H. Karfunkel and V. Schurig, J . Chromatogr.,

34

E. Cremer and R. Kramer, J. Chromatogr.,

F r e i , J. Chromatogr.,

165 (1979) 75-86. (1975) 241-252. 288 (1984) 1-14.

107 (1975) 253-263.

P h i l l i p s , J . Chem. SOC. Faraday I . , 76 (1980) 683-700.

35

K.F. S c o t t and C.S.G.

36

N.D.

37

K.F. Scott, J . Chem. SOC. Faraday I . , 76 (1980) 2065-2079.

38

C.S.G. P h i l l i p s , P. Powell, J.A. Semlyen and P.L. Timns, Z e i t . Anal. Chem., 197.2 (1963) 202-211.

39

R.J. Laub and R.L. Pecsok, Physicochemical A p p l i c a t i o n s o f Gas Chromatography, John Wiley, New York, 1978.

40

Faraday Symposium o f t h e Chemical Society, No. 15, Chromatography, E q u i l ibra and K i n e t i c s , 1980.

41

H. Brunschwig, L i b e r der a r t e d i s t i l l a n d i (1512) see E. Bayer, Gaschromatographie, Springer, B e r l i n , 1959, p4.

42

A.J.P.

43

A.T.

Perkins and C.S.G.

P h i l l i p s , J. C a t a l y s i s , 66 (1980) 248-250.

M a r t i n and R.L.M. James and A.J.P.

Synge, Biochem. J.,

35 (1941) 1358-1368.

Martin, Biochem. J., 50 (1952) 679-690.

341

THE SOLVENT EFFECT I N GAS LIQIJID CHROMATOGRAPHY

V PRETORIUS, K LAWSON, E ROHIIER, P APPS UNIVERSITY OF PRETORIA-INST. FOR CHROMATOGRAPHY 1.

-

0002 PRETORIA

SOUTH AFRICA

INTRODUCTION

The s o l v e n t e f f e c t i s a phenomenon which may be employed as one o f t h e many ways o f sample i n t r o d u c t i o n i n g a s - l i q u i d chromotography ( r e f . 1 ) . More p a r t i c u l a r l y i t i s one o f t h e t h r e e modes o f s o l u t e f o c u s i n g . I t i s an e s p e c i a l l y e f f e c t i v e means o f q u a n t i t a t i v e l y and r e p r o d u c i b l y t r a n s f e r r i n g s o l u t e s f r o m d i l u t e s o l u t i o n s t o t h e column ( r e f . 2) w i t h o u t o v e r l o a d i n g under m i l d c o n d i t i o n s . Consequently i t i s o f p a r t i c u l a r v a l u e i n t r a c e a n a l y s i s where t h e r m a l l y l a b i l e s o l u t e s a r e i n v o l v e d . Although t h e t e r m " s o l v e n t e f f e c t " was o n l y c o i n e d l a t e r ( r e f . 3 ) , t h e phenomenon was p r o b a b l y f i r s t observed i n 1969 by Grob ( r e f . 4 ) . U s i n g a d i l u t e s o l u t i o n o f r e l a t i v e l y n o n - v o l a t i l e s t e r o i d s i n b i s ( t r i m e t h y l s i l y l ) acetamide and an excess o f hexane he observed t h a t l a r g e amounts o f sample c o u l d be i n t r o d u c e d i n t o a c a p i l l a r y column w i t h o u t o v e r l o a d i n a " p r o v i d e d t h e columr! temperature i s a t l e a s t 100°C below t h e b o i l i n g p o i n t o f t h e most v o l a t i l e s o l u t e " . Under t h e s e circumstances s t a t i o n a r y phase f o c u s i n g (paragraph 2.1.1.(ii)),

and n o t t h e s o l v e n t e f f e c t . i s dominant.

The f i r s t unequivocal d e s c r i p t i o n and e x p l a n a t i o n o f t h e s o l v e n t e f f e c t , as we understand i t today, was p u b l i s h e d by Deans i n 1971 ( r e f . 5 ) . U n f o r t u n a t e l y he used t h e t i t l e "The sample as i t s own s t a t i o n a r y phase i n gas chromatography'; and r e g r e t t a b l y , b u t u n d e r s t a n d a b l y , t h e i m p o r t o f h i s work remained l a r g e l y unrecognized u n t i l 1982 ( r e f . 5 ) . S i n c e i t s d i s c o v e r y a g r e a t deal has been p u b l i s h e d on t h e s o l v e n t e f f e c t ( r e f s . 7-10). Although t h e p r a c t i c a l u s e f u l n e s s o f t h e e f f e c t became apparent y e a r s ago i t i s o n l y r e c e n t l y t h a t t h e u n d e r l y i n g processes have been u n r a v e l l e d and e x p l a i n e d . I n essence s o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t i n v o l v e s t h e f o l l o w i n g :

o

a d i l u t e vapour o r l i q u i d sample

o

t h e f o r m a t i o n o f a f i l m o f l i q u i d sample i n t h e i n l e t

348 o

t h e e v a p o r a t i o n o f t h e sample f i l m so t h a t s o l u t e s a r e focused by t h e solvent e f f e c t

o

t h e t r a n s f e r of focused s o l u t e s t o t h e column These i s s u e s may be a r b i t r a r i l y d i v i d e d i n t o fundamental ( p a r a g r a p h 2 ) and

p r a c t i c a l aspects (paragraph 3 ) . The former a r e concerned w i t h t h e s o l v e n t e f f e c t i t s e l f and t h e l a t t e r w i t h i t s p r a c t i c a l o p t i o n s . H i s t o r i c a l l y t h e s o l v e n t e f f e c t has been used f o r l i w i d samples i n open t u b u l a r i n l e t s . T h i s paper w i l l show t h a t b o t h l i q u i d and vapour samples can be handled, t h a t i n l e t geometries o t h e r t h a n open tubes can be used and t h e s o l v e n t e f f e c t can o p e r a t e e i t h e r s t a t i c a l l y o r d y n a m i c a l l y . The f u l l range o f o p t i o n s i s shown i n f i g u r e 1.

F i g . 1. P r a c t i c a l o p t i o n s a v a i l a b l e when.employing t h e s o l v e n t e f f e c t . L i n e s connect c o m p a t i b l e o p e r a t i o n a l parameters.

349

2.

FUNDAMENTAL ASPECTS It i s convenient t o consider t h e a p p l i c a t i o n o f the solvent e f f e c t t o l i q u i d

samples and t o vapour samples s e p a r a t e l y . 2.1

L i q u i d samples ( S t a t i c System) We d i s t i n g u i s h between an

e f f e c t s (paragraph 2 . 1 . 3 ( i ) ) ,

ideal s o l v e n t

e f f e c t (paragraph 2.1.2),

non-ideal

which a r e c l o s e l y connected w i t h t h e s o l v e n t

e f f e c t and a s s o c i a t e d e f f e c t s (paragraph 2.1 . l ) which a r e l e s s c l o s e l y connected. 2.1.1 A s s o c i a t e d e f f e c t s ( i ) Column o v e r l o a d i n g o

Volume o v e r l o a d i n g ( r e f . 2 ) o c c u r s when t h e w i d t h o f t h e vapour band emerging f r o m t h e i n l e t , and a f t e r s t a t i o n a r y phase f o c u s i n g ( p a r a g r a p h 2.1.1

( i i ) ) , i s comparable t o t h e

ncrease i n band w i d t h b r o u g h t about by

n o n - i d e a l chromatographic processes. Volume o v e r l o a d i n g reduces r e s o l u t i o n and r e s u l t s i n chromatographic peaks which a r e w i d e r t h a n t h o s e o b t a i n e d i n non-overloaded s i t u a t i o n s . Focusing t e c h n i q u e s ( e g s o l v e n t e f f e c t , t h e r m a l , s t a t i o n a r y phase) a r e methods o f combattifig volume o v e r l o a d i n g . I t i s i m p o r t a n t t o r e a l i z e , p a r t i c u l a r l y i n t h e p r e s e n t c o n t e x t , t h a t f o c u s i n g t o an e x t e n t much l e s s t h a n t h e column brc?adening i s unnecessary. o

c o n c e n t r a t i o n o v e r l o a d i n g ( r e f . 2 ) occurs when t h e p a r t i t i o n c o e f f i c i e n t s o f t h e s o l u t e s i n t h e f i r s t p o r t i o n o f t h e column change w i t h c o n c e n t r a t i o n . Overloaded peaks can show t a i l i n g o r f r o n t i n g ; t h e l a t t e r i s t h e most common. S o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t may be p a r t i c u l a r l y e f f i c i e n t and car- r e a d i l y l e a d t o c o n c e n t r a t i o n o v e r l o a d i n g . ( i i ) 5 t a t i o n a r y phase f o c u s i n g ( r e f . 1 1 ) i s always, t o some e x t e n t , a s s o c i a t e d

w i t h t h e s o l v e n t e f f e c t . I t i s caused by d i s s o l u t i o n o f s o l u t e emerging f r o m t h e i n l e t , i n t h e s t a t i o n a r y phase i n t h e f i r s t p o r t i o n o f t h e column. The e x t e n t

of

f o c u s i n g i s l a r g e l y d e t e r m i n e d by t h e p a r t i t i o n r a t i o o f t h e s o l u t e between t h e gas and s t a t i o n a r y phases, and t h u s by t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n . S t a t i o n a r y phase f o c u s i n g i s most p r o f i t a b l y a p p l i e d by d e c r e a s i n g t h e column temperature, o r a t l e a s t t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n d u r i n g t h e f o c u s i n g process and subsequently programming t h e t e m p e r a t u r e .

I n t h e p r e s e n t c o n t e x t two p o i n t s must be emphasized: o

S t a t i o n a r y phase f o c u s i n g can p a r t i a l l y o r c o m p l e t e l y e l i m i n a t e band d i s t o r t i o n a r i s i n g f r o m n o n - i d e a l s o l v e n t e f f e c t s (paragraph 2.1.3

( i ) ) . By

3 50

j u d i c i o u s use o f s t a t i o n a r y phase f o c u s i n g many o f t h e i l l s t o which t h e s o l v e n t e f f e c t i s prone may e f f e c t i v e l y be cured. o

S t a t i o n a r y phase f o c u s i n g masks, t o some degree, event< a t t h e e x i t o f t h e i n l e t . T h i s means t h a t t h e s t u d y o f s o l u t e and s o l v e n t band shapes a t t h e end o f t h e column does n o t n e c e s s a r i l y p r o v i d e a c o r r e c t p i c t u r e o f t h e band shapes i s s u i n g from t h e i n l e t . Most s t u d i e s o f t h e s o l v e n t e f f e c t have r e l i e d on i n f o r m a t i o n o b t a i n e d a t t h e comlumn e x i t .

( i i i ) S t a t i o n a r y phase s w e l l i n g R e l a t i v e l y l a r g e amounts o f s o l v e n t a r e a s s o c i a t e d w i t h t h e s o l v e n t e f f e c t . I f t h i s s o l v e n t i s n o t s p l i t . ( p a r a g r a p h 3.5) i t passes o n t o t h e column and may cause t h e s t a t i o n a r y phase t o s w e l l . The e x t e n t o f t h e s w e l l i n g , t h e dynamic behaviour o f t h e s w o l l e n f i l m and t h e consequences o f s w e l l i n g a r e discussed elsewhere ( r e f . 1 2 ) . T v p i c a l l y t h e s o l v e n t peak may be d i s t o r t e d by f r o n t i n g and a s o l u t e band immediately b e h i n d t h e s o l v e n t may be sharpened. T h i s process was f i r s t d e s c r i b e d by H a r r i s ( r e f . 13) and was i n v e s t gated by Grob ( r e f . 14) who termed i t 'phase s o a k i n g ' . 2.1.2

The i d e a l s o l v e n t e f f e c t o c c u r s when: n o n - i d e a l chromatographic processes ( d i f f u s i o n e t c i n l e t r e g i o n (paragraph 2.1.3

a r e absent f r o m t h e

(ii))

s o l u t e i n t e r a c t i o n s w i t h t h e surface o f t h e i n l e t a r e n e g l i g i b l e (paragraph 2.1.3

(iv))

s o l u t e c o n c e n t r a t i o n i n t h e gas phase, o u t s i d e t h e f o c u s i n g r e g i o n , i s n e g l i g i b l e (paragraph 2.1.3

(v))

thermal and c o n c e n t r a t i o n e q u i l i b r i a a r e l a t e r a l l y ( i e a t r i g h t angles t o t h e gas f l o w ) mantained (paragraph 2.1.3

(vi))

the solvent f i l m ,

-

once formed, i s s t a t i c

i e does n o t c r e e p

-

and s t a b l e

- i e i t does n o t break up (paragraph 2.1.3 ( v i i ) ) Consider a f i l m o f a d i l u t e s o l u t i o n o f a s i n g l e s o l u t e i n a solvent,spread on t h e s u r f a c e o f t h e i n l e t , which may be an open t u b e o r a packed bed. Consider i n p a r t i c u l a r t h e r e a r edge o f t h e f i l m as i s shown i n F i g u r e 2

351

Fig. 2 . The s t a t i c solvent e f f e c t as applied t o l i q u i d samples. 1 ) Carrier gas flow; 2 ) I n l e t surface; 3 ) Sample f i l m . U$equals t h e 'chromatographic' v e l o c i t y of s o l u t e x and Uf equals the velocity of t h e sample film r e a r edge. Carrier gas passing over t h e f i l m will cause t h e r e a r edge t o evaporate and move forward a x i a l l y . As t h e evaporation proceeds s o l u t e accumulates in t h e rear edge, causing a concomitant increase in the s o l u t e concentration in the gas above the r e a r edge. I f t h e l i n e a r axial v e l o c i t y of t h e r e a r edge of t h e f i l m i s f a s t e r than chromotographic movement of t h e s o l u t e the s o l u t e will accumulate ( i d e a l l y ) in an i n f i n i t e l y narrow band. In o t h e r words t h e s o l u t e i s focused and t h e focusing process i s t h e solvent e f f e c t . Grob ( r e f . 15) r e f e r s t o t h i s process as "solvent trapping".

I t has been shown ( r e f . 6 ) t h a t s o l u t e focusing occurs when, i n the i n i t i a l stages

-

K;c KO - (3 and i n the f i n a l stages

p > 0

(i)

(2)

where k, KO = p a r t i t i o n c o e f f i c i e n t s of t h e s o l u t e (z) and solvent

((1)

between the gas and l i q u i d phases

p

=

phase r a t i o

Solute focusing continues u n t i l t h e r e a r edge of the f i l m reaches t h e f r o n t edge a t which point t h e focused s o l u t e and residual solvent evaporate and pass into t h e column. Equations 1 and 2 suggest t h a t :

352

> KO w i l l

o

all

o

s o l u t e s w i t h Icic< ko can be focused t o an e x t e n t determined by

o

t h e shape o f t h e f i l m i s r e l a t i v e l y u n i m p o r t a n t

o

s o l u t e f o c u s i n g i s independent o f t h e c a r r i e r gas f l o w r a t e

o

f o c u s i n g w i l l occur a t a l l temperatures between t h e m e l t i n g p o i n t and t h e

s o l u t e s f o r which Icic

be focused

fi

b o i l i n g p o i n t o f t h e sample An i d e a l (one w i t h an i n f i n i t e l y f a s t response) d e t e c t o r a t t h e e x i t of t h e i n l e t would produce c o n c e n t r a t i o n - t i m e curves s i m i l a r t o t h o s e shown i n F i g . 3.

I F i g . 3. The i d e a l s o l v e n t e f f e c t . Peak shapes emerging f r o m an i n l e t w i t h an i d e a l s o l v e n t e f f e c t . a ) S o l v e n t s i g n a l ; b) S o l u t e s i g n a l . I n p r a c t i c e however, t h e s o l v e n t e f f e c t i s n o t i d e a l and t h e s o l u t e bands emerging f r o m t h e i n l e t can be d i s t o r t e d by a number o f phenomena. 2.1.3 Non-ideal e f f e c t s ( i ) Non-focusing Non-focusing occurs when e q u a t i o n 1 i s n o t v a l i d . F i g u r e 4 shows an example.

b

1 260

0

600

400

time

800

(s)

F i g . 4. Non-ideal e t t e c t s : non f o c u s i n g o f s o l u t e . S i g n a l s f r o m a mass spectrometer, i n s e l e c t i v e i o n m o n i t o r i n g , c o u p l e d d i r e c t l y t o a packed bed s o l v e n t e f f e c t i n l e t . a ) S o l u t e : n-decane; b) S o l v e n t :

353 methanol. The sample i s l o p 1 o f 1:lO3 n-decane i n methanol and i n j e c t e d a t 40°C under a f l o w r a t e o f 2 cm3 min-l o f helium. ( i i ) Back d i f f u s i o n Back d i f f u s i o n o f s o l u t e i n t h e gas phase i n t h e f o c u s i n g r e g i o n above t h e r e a r edge o f t h e sample f i l m broadens t h e f o c u s i n g s o l u t e band by an amount, g i v e n i n t i m e u n i t s b y ( r e f . 16) t =

Dg

.

n2 d i 4 E2 16 Vg L

(4)

where Dg

=

d i f f u s i o n c o e f f i c i e n t o f t h e s o l u t e i n t h e gas phase

di

=

i n t e r n a l diameter o f t h e i n l e t

€

=

porosity o f the i n l e t

Vg

=

volume f l o w r a t e i n t h e i n l e t

Back d i f f u s i o n can be s i g n i f i c a n t i n packed bed systems where t h e c a r r i e r gas f l o w r a t e i s s e t t o o p t i m i z e t h e column's chromatographic performance ( F i g . 5 ) .

a

b

F i g . 5. Non-ideal e f f e c t s : back d i f f u s i o n . 5a and 5b show t h e e f f e c t o f d i f f e r e n t c a r r i e r gases on peak w i d t h s emerging from packed bed s o l v e n t e f f e c t i n l e t s . I n b o t h cases t h e sam l e i s 1 5 4 of 1:103 n-octane i n n-hexane i n j e c t e d a t 30°C under 2 cm3 min-y o f c a r r i e r gas flow. The i n l e t b e i n g d i r e c t l y connected t o a mass spectrometer s e l e c t i v e l y m o n i t o r i n g f o r n-octane. I n 5a t h e c a r r i e r gas i s h e l i u m and i n 5b n i t r o g e n . D i f f u s i o n i s known t o be f a s t e r i n h e l i u m t h a n i n n i t r o g e n . Grob ( r e f . 3 3 )

has d i s c u s s e d a s i m i l a r e f f e c t i n an open t u b u l a r i n l e t .

The d e l e t e r i o u s e f f e c t s o f d i f f u s i o n can be b e s t overcome by u s i n g h i g h e r gas v e l o c i t i e s i n an o f f - l i n e system (paragraph 3 . 2 ) . ( i i i ) Chromatographic-type band-broadening processes i n p o r t i o n s o f t h e i n l e t n o t u t i l i z e d f o r t h e s o l v e n t e f f e c t , o r i n c o n n e c t i n g l i n e s between t h e i n l e t and t h e column can i n c r e a s e t h e w i d t h of t h e focused s o l u t e band ( r e f . 1 7 ) .

3 54

These processes should be m i n i m i z e d by procedures s e t o u t i n t h e p r e v i o u s paragraph. An e x p e r i m e n t a l s t u d y o f these processes i s s t i l l l a c k i n g . ( i v ) Solute l a g g a (ref.18) S o l u t e l a g g i n g occurs when

o

A d s o r p t i o n o f s o l u t e on t h e s u r f a c e o f t h e i n l e t i s s i g n i f i c a n t .

o

Slow e v a p o r a t i o n o f t h e focused s o l u t e s occurs i n t h e f i n a l stages o f t h e solvent effect. Both l e a d t o broadened peaks ( F i g u r e 6 ) .

a 1

I

5

10

15 MWS

5

m

15 YWS

F i g . 6. The e f f e c t o f r a p i d i n l e t h e a t i n g on s o l u t e peak shapes emerging f r o m packed bed solvenr; e f f e c t i n l e t s . 6a shows a chromatogram o f 1)n-octane; 2 ) n-nonane; 3 ) 2,4 d i m e t h y l hepan-3-one; 4) n-decane; 5 ) p - c r e s o l ; 6 ) n-undecane; 7 ) 2,4 d i m e t h y l a n i l i n e ; 8 ) n-dodecane; 9) n-decanol; 10) n - t r i d e c a n e ; 11) n - t e t r a d e c a n e ; 12) n-pentadecane. 20 1 o f a 1:lO7 s o l u t i o n i n n-hexane was i n j e c t e d o n t o a packed bed i n l e t connected t o a 19m, 0.3mm i . d . column c o a t e d w i t h c r o s s - l i n k e d SE-30. The sample was i n j e c t e d a t 50°C, on c o m p l e t i o n o f t h e s o l v e n t e f f e c t t h e i n l e t and column were temperat u r e programmed a t 10°C min-1. The c a r r i e r gas was hydrogen a t 50 cm sec-1. 6b was an i d e n t i c a l experiment except t h a t t h e i n l e t was b a l l i s t i c a l l y heated on c o m p l e t i o n o f t h e s o l v e n t e f f e c t . A s p e c i a l k i n d o f s o l u t e l a g g i n g a l s o o c c u r s when t h e i n l e t i s c o a t e d w i t h s t a t i o n a r y phase ( r e f . 1 9 ) . I n t h i s case s o l u t e s w i t h h i g h p a r t i t i o n r a t i o s between t h e s t a t i o n a r y phase and t h e gas phase chromatograph t o o s l o w l y t o remain i n t h e advancing back edge o f t h e s o l v e n t f i l m . F o c u s i n g , t h e r e f o r e ,

does

n o t occur. Grob ( r e f . 20) demonstrated t h i s phenomenon and c o i n e d t h e t e r m "band broadening i n space" t o d e s c r i b e i t . He termed t h e removal o f s t a t i o n a r y phase Prom t h e i n l e t r e g i o n t o be " c r e a t i n g a r e t e n t i o n gap". Both terms, i n

355

o u r view, a r e unnecessary. ( v ) S o l u t e escape S o l u t e escape ( r e f . 21) r e f e r s t o s o l u t e which i s t r a n s p o r t e d o u t of t h e i n l e t during t h e focusing period. The f r a c t i o n a l escape i s g i v e n by Mxe -

MXS

b+P

(5)

where Mxe 'M ,

=

mass o f s o l u t e t o escape

=

mass o f o r i g i n a l sample

S o l u t e escape i s c o n t r o l l e d s o l e l y by t h e vapour p r e s s u r e o f t h e s o l u t e o v e r t h e sample. The g r e a t e r t h e vapour p r e s s u r e t h e g r e a t e r t h e degree o f escape.

A

l a r g e p o l a r i t y d i f f e r e n c e between s o l v e n t and s o l u t e w i l l i n c r e a s e

t h e degree o f escape and s h o u l d be avoided i f p o s s i b l e . Grob J r ( r e f s . 20, 22,

23) has c o i n e d t h e terms "band broadening i n t i m e " , " p a r t i a l s o l v e n t t r a p p i n g " and "two s t e p chromatography" (among o t h e r s ) . Except f o r s o l u t e s which do n o t obey e q u a t i o n 1 s o l u t e escape may be prevented by p l a c i n g a f i l m o f pure s o l v e n t ahead o f t h e sample f i l m ( r e f . 21) I n p r a c t i c e t h i s i s t r i c k y t o manage and achieves l i t t l e . The degree o f escape f a l l s r a p i d l y w i t h p a r t i t i o n r a t i o ( e q u a t i o n 5 ) and i s n e g l i g i b l e f o r say n-decane d i s s o l v e d i n n-hexane. Escaped s o l u t e can o f t e n be r e f o c u s e d by phase s w e l l i n g ( r e f . 10). ( v i ) Thermal e f f e c t s o

E v a p o r a t i v e c o o l i n g o c c u r s on t h e r e a r edge o f t h e sample f i l m when t h e h e a t needed t o e v a p o r a t e t h e sample i s n o t s u p p l i e d q u i c k l y enough ( r e f . 2 4 ) . Temperature drops o f up t o 20°C have been measured i n packed bed systems made o f g l a s s . C o o l i n g i s p r o b a b l y i n s i g n i f i c a n t i n open t u b u l a r systems.

As f a r as we can a s c e r t a i n e v a p o r a t i v e c o o l i n g does n o t , i n p r a c t i c e , detrimentally a f f e c t the solvent e f f e c t .

o

L a t e r a l thermal g r a d i e n t s may a l s o o c c u r i n packed bed systems where t h e packing i s p o o r l y conducting. I n such s i t u a t i o n s t h e r e a r edge o f a sample f i l m w i l l move a t d i f f e r e n t

a x i a l v e l o c i t i e s across t h e t u b e and may broaden s o l u t e bands as a r e s u l t . D i r e c t e x p e r i m e n t a l e v i d e n c e o f such g r a d i e n t s i s s t i l l l a c k i n g .

356

( v i i ) Film i n s t a b i l i t y

o

F i l m creep occurs when, once t h e sample has been formed, t h e f i l m as a whole i s moved a x i a l l y f o r w a r d by shear f o r c e s e x e r t e d by t h e c a r r i e r gas. Creep i s g r e a t e s t i n smooth w a l l e d open t u b u l a r systems and l e a s t i n porous packing packed beds ( r e f . 2 5 ) . F i l m c r e e p does n o t a f f e c t t h e s o l v e n t f o c u s i n g b u t i n c r e a s e s t h e l e n g h t o f i n l e t r e q u i r e d f o r a p a r t i c u l a r s i z e o f sample.

o

F i l m break up may be a t t r i b u t e d t o f i l m i n s t a b i l i t y and shear f o r c e s . The former i s e v i d e n t when t h e s u r f a c e t e n s i o n o f t h e sample i s g r e a t e r t h a n t h e excess s u r f a c e f r e e - e n e r g y o f t h e i n n e r s u r f a c e s o f t h e i n l e t and i s most p r e v a l e n t w i t h p o l a r s o l v e n t s ( r e f . 2 6 ) . Shear f o r c e s f r o m t h e c a r r i e r gas can cause t h e w e t t e d f i l m on a smooth w a l l e d open t u b u l a r i n l e t t o break up f o r m i n g l e n s e s . Rough-wall open t u b e and packed bed i n l e t s a r e l e s s

2.2

prone t o s h e a r - f o r c e break up.

Vapour samples ( S t a t i c Systems) ( r e f . 27) F o r s o l v e n t e f f e c t f o c u s i n g t h e sample s h o u l d c o n s i s t o f vapours i n a non-

i n t e r a c t i n g gas. The sample may e x i s t as such, eg o r g a n i c p o l l u t a n t s i n a i r , o r may be formed by p a s s i n g c a r r i e r gas o v e r t h e sample (head-space s a m p l i n g ) .

A f i l m o f s o l v e n t i s l a i d down i n t h e i n l e t and sample vapour i s passed o v e r t h i s f i l m . Focusing o c c u r s by t h e mechanism o u t l i n e d i n paragraph 2.1.2 and t h e c o n d i t i o n f o r f o c u s i n g i s a l s o g i v e n by e q u a t i o n s 1 and 2. The sample f e e d i s c o n t i n u e d u n t i l t h e e v a p o r a t i n g r e a r edge approaches t h e f r o n t edge. Sample f e e d i s t e r m i n a t e d and p u r e c a r r i e r gas i s i n t r o d u c e d . Subsequent events p a r a l l e l t h o s e p r e v i o u s l y o u t l i n e d f o r l i q u i d samples. S o l u t e escape does n o t o c c u r i n t h i s sampling mode, except f o r non-focusinp s o l u t e s ; t h i s can be a m a j o r advantage. Vapour samples c o n t a i n i n g l a r g e amoun s o f water can he e a s i l y handled u s i n g t h i s t e c h n i q u e . Water i s a n o n - f o c u s i n g s o l u t e on a n o n - p o l a r s o l v e n t such as hexane and i s t h e r e f o r e n o t accumulated 2.3

Dynamic Systems ( L i q u i d samples) Dynamic systems a r e a r e l a t i v e l y new i n n o v a t i o n i n v o l v i n g a moving

(dynamic) sample " f i l m " and c o u n t e r c u r r e n t c a r r i e r gas f l o w ( r e f . 2 6 ) . They have n o t y e t been s t u d i e d i n as g r e a t d e t a i l as have s t a t i c systems and t h e t h e o r e t i c a l t r e a t m e n t and d i s c u s s i o n presented h e r e i s c o n s e q u e n t l y s k e t c h y . Consider a tube, c o n t a i n i n g an a x i a l porous l a y e r and a gas channel w i t h

357

one end dipped i n t o a l i q u i d sample and t h e o t h e r end connected t o a c a r r i e r gas s u p p l y ( F i g u r e 7 ) . The l i q u i d sample r i s e s by c a p i l l a r y a c t i o n i n t h e porous l a y e r w h i l e t h e c a r r i e r gas evaporates sample a t t h e upper edge, c a u s i n g i t t o move downwards. E q u i 1 i b r i u m . o c c u r s when t h e volume r a t e o f c a p i l l a r y

r i s e equals t h e volume r a t e o f e v a p o r a t i o n . S o l u t e i s focused i n t h e e v a p o r a t i o n zone by processes s i m i l a r t o t h o s e i n t h e s t a t i c systems p r e v i o u s l y d e s c r i b e d (paragraph 2 ) .

a

b 9

-2

F i g . 7a) Schematic diagram o f operation o f solvent e f f e c t i n t h e dynami c mode. 1 ) Pure gas ( f o r l i q u i d samples) o r head space sample; 2) Glass tube; 3) Pure s o l v e n t ( f o r head space samples) o r d i l u t e l i q u i d sample; 4) Region o f porous bed w e t t e d by l i q u i d ; 5 ) Solute/head space component m o l e c u l e c a r r i e d up by c a p i l l a r y r i s e o f l i q u i d ; 6) C a p i l l a r y r i s e o f l i q u i d ; 7 ) Chromatographic t r a n s p o r t o f s o l u t e s ; 8 ) S o l u t e s a c c u m u l a t i n g i n e v a p o r a t i o n zone when 5 and 6 a r e f a s t e r t h a n 7; 9) Dry r e g i o n o f porous bed. 7b) P l a n view. 3. 3.1

PRACTICAL ASPECTS S t a t i c vs dynamic systems

o

s t a t i c systems may be o n - l i n e o r o f f - l i n e ;

o

s t a t i c systems a r e p r e s e n t l y more w e l l - d e v e l o p e d t h a n a r e dynamic systems

o

dynamic systems can h a n d l e l a r g e r samples t h a n can s t a t i c systems

o

dynamic systems a r e more c o m p l i c a t e d t h a n s t a t i c systems

3.2 o

dynamic systems o n l y o f f - l i n e

O n - l i n e vs o f f - l i n e systems ' o f f - l i n e systems can employ h i g h c a r r i e r gas f l o w s t h e r e b y s h o r t e n i n g t h e

358

t i m e needed t o complete t h e s o l u t e f o c u s i n g

o

off-line

sampling can be done i n p a r a l l e l w i t h t h e chromatography; t h i s

s h o r t e n s a n a l y s i s t i m e i n r o u t i n e work o

o f f - l i n e systems a v o i d i n t r o d u c i n g n o n - v o l a t i l e r e s i d u e s i n t o t h e column

o

most o f t h e s o l v e n t i s removed d u r i n g o f f - l i n e o p e r a t i o n and t h i s reduces t h e amount o f s o l v e n t i n t r o d u c e d on t h e column

o 3.3

o

o n - l i n e systems a r e s i m p l e r t h a n o f f - l i n e systems Open vs packed tube i n l e t s open tubes, p a r t i c u l a r l y t h o s e w i t h smooth w a l l a r e prone t o f i l m creep and i n s t a b i l i t y . T h i s problem i s f a r l e s s i n packed tubes

o

packed tubes can handle l a r g e r samples p e r u n i t l e n g h t t h a n can open tubes

o

packed t u b e i n l e t s must be independenc'ently heated t o a v o i d l a g g i n g ; when open tubes a r e used t h e chromatograph oven i s s u f f i c i e n t

3.4

o

D i r e c t vs s p l i t l e s s i n j e c t i o n s p l i t l e s s i n j e c t i o n e n t a i l s v a p o r i s a t i o n and can l e a d t o decomposition o f thermally l a b i l e solutes

o

d i r e c t i n j e c t i o n does n o t r e q u i r e v a p o r i s a t i o n . I n p r o p e r l y d e a c t i v a t e d i n l e t s s o l u t e s can be t r a n s f e r r e d t o t h e column a t l o w temperatures

3.5

Solvent s p l i t t i n g r e f e r s t o t h e removal o f s o l v e n t d u r i n g f o c u s i n g b e f o r e t h e focused s o l u t e s a r e t r a n s f e r r e d t o t h e column s o l v e n t s p l i t t i n g occurs a u t o m a t i c a l l y i n o f f - l i n e systems and can be invoked i n o n - l i n e systems by u s i n g a Deans s w i t c h ( r e f . 2 9 ) . i f t h e s o l v e n t i s n o t s p l i t t h e l a r g e amounts o f s o l v e n t p a s s i n g i n t o t h e column can cause s t a t i o n a r y phase s w e l l i n g ( r e f . 14) and t a i l i n g o f solvent peaks i n a p r o p e r l y designed system n o n - s p l i t t i n g o f s o l v e n t does n o t cause problems ( F i g . 8 )

359 F i g . 8. A p r o t o t y p e i n l e t f o r t h e s t a t i c solvent e f f e c t 1 ) C a r r i e r gas i n l e t 2 ) Septum n u t 3 ) Septum purge 4 ) Gas chromatograph oven l i d 5 ) Packing 6 ) Fused s i l i c a l i n e 7) Connection 8 ) Heater c o i l s

3.6

tiardware A l a r g E number o f i n l e t s designed f o r s o l u t e f o c u s i n g u s i n g t h e s o l v e n t

e f f e c t have been d e s c r i b e d ( r e f s . 30, 31, 3 2 ) ; t h e f i n a l word has y e t t o be written. F i g u r e s 8 and 9 a r e schematic r e p r e s e n t a t i o n s o f s e v e r a l p r o t o t y p e i n l e t s b e i n g developed i n o u r l a b o r a t o r i e s .

4. PRACTICAL EXAMPLES F i g u r e 10 shows a chromatogram o f a headspace sample generated by a dynamic sol vent e f f e c t i n l e t . F i g u r e 6b shows a chromatogram o f a l i q u i d sample generated by a s t a t i c solvent e f f e c t operating on-line.

360

F i g . 9. Diagrammatic v e r t i c a l s e c t i o n o f system used t o t r a n s f e r sample f r o m an o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler t o a c a p i l l a r y GLC column

1) 2) 3) 4) 5) 6) 7) 8) 9)

9

10)

-7

9

C a r r i e r gas i n l e t Removable cap Ground g l a s s j o i n t O f f - l i n e , dynamic, s o l v e n t e f f e c t sampler Heater c o i l s Purge gas e x i t Porous packed bed To column E l e c t r i c a l power s u p p l y . S o l u t e s and s o l v e n t a r e evaporated f r o m t h e o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler by e l e c t r i c a l h e a t = i n g and condensed i n t o t h e porous bed where on-1 i n e , s t a t i c , nonsolvent s p l i t t i n g solvent e f f e c t focusing i s carried out. Seat

361

F i g . 10. A chromatogram o f v o l a t i l e s f r o m 10 m l o f Simonsig Colombar wine sampled by s t r i p p i n g w i t h 15 rnl m i n - I o f p u r e N2 f o r 30 min and t r a p p i n g by o f f - l i n e , dynamic s o l v e n t e f f e c t f o c u s i n g on p u r e n-hexane a t 30°C. Chromatographic c o n d i t i o n s as i n F i g u r e 6.

REFERENCES

1 V. P r e t o r i u s and W. B e r t s c h , J . HRC & CC, 6 (1983) 64. 2 V. P r e t o r i u s , K. Lawson and W. B e r t s c h , J. HRC & CC, 6 (1983 185. 3 K. Grob and K. Grob Jr, J. Chrom, 94 (1974) 53. 4 K. Grob and G. Grob, J. Chrom. S c i . , 7 (1969) 584. 5 D.R. Deans, Anal. Chem., 43 (1971) 2026. 6 V. P r e t o r i u s , C.S.G. P h i l l i p s and W. B e r t s c h , 3 . HRC & CC, 6 ( 1 983) 232. 7 F.J. Yang, A.C. Brown and S.P. Cram, 3 . Chrom., 158 (1978) 91 8 W.G. Jennings, R.R. Freeman and T.A. Rooney, J. HRC & CC, 1 1978) 275. 9 K. Knauss, J . Fulleman and M.P. T u r n e r . J . HRC & CC. 4 (1981 641. 10 R.G. J e n k i n s , i n "Proceedings o f t h e F o u r t h I n t e r n a t i o n a l Symposium on C a p i l l a r y Chromatography, Hindelang, 1981", R.E. K a i s e r ed., H e u t h i g , H e i d e l b e r g , 1981, 563 pp. 11 V. P r e t o r i u s , E.R. Rohwer and K.H. Lawson, S . A f r . J. Chem, 37(2) (1984) 65. 12 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 13 W.E. H a r r i s , J. Chrom. S c i . , 11 (1973) 184. 14 K. Grob J r and B. S c h i l l i n g , Chromatographia, 17 (1983) 361. 15 K. Grob J r , J . Chrom., 264 (1983) 7. 16 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 17 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 18 V. P r e t o r i u s , C.S.G. P h i l l i p s and W . B e r t s c h , J. HRC & CC, 6 (1983) 321. 19 V. P r e t o r i u s , K.H. Lawson, E.R. Rohwer and W. B e r t s c h , J . HRC & CC, 7 (1984) 92. 20 K. Grob Jr, J. Chrom., 213 (1981) 3. 21 V. P r e t o r i u s , K. Lawson and W . B e r t s c h , J . HRC & CC, 6 (1983) 419. 22 K. Grob J r , Chromatographia, 17 (1983) 357. 23 K. Grob J r , J. Chrom., 253 (1982) 17. 24 V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 210. 25 V. P r e t o r i u s , K.H. Lawson, P. Apps and W. B e r t s c h , J . Chrom., 279 (1983) 233. 26 P. Sandra, I . Temmerman and M. Verstappe, J. HRC & CC, 6 (1983) 501. 27 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n .

362

V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 212. 29 D.R. Deans, Chromatographia, 1/2 (1968) 18. 30 E. Geeraert, D. de Schepper and P. Sandra, J. HRC & CC, 6 (1983) 386. 31 K. Grob and K. Grob Jr, J . Chrom., 151 (1978) 311. 31 G. Schomburg, H. Behlau, R. Dielmann, F. Weeke and H. Husmann, J . Chrom., 142 (1977) 87. 33 K. Grob J r , J . HRC & CC, 8 (1984) 461. 28

363

WINDOW ANALYSIS: AN APPROACH TO TOTAL OPTIMISATION I N CHROMATOGRAPHY

J H PURNELL, Department o f Chemistry, U n i v e r s i t y C o l l e g e o f Swansea, S i n g l e t o n Park, Swansea, Wales, U.K.

SUMMARY An account i s g i v e n o f t h e development o v e r a decade o f t h e window a n a l y s i s approach t o chromatographic o p t i m i s a t i o n .

Examples a r e g i v e n o f

o p t i m i s a t i o n s conducted w i t h r e s p e c t t o one or o t h e r o f t h e many v a r i a b l e s t h a t i n f l u e n c e t h e performance o f GC and HPLC systems.

I t i s emphasised t h a t

a g i v e n a n a l y t i c a l problem may be s a t i s f a c t o r i l y r e s o l v e d i n many ways, e i g h t e e n d i f f e r e n t p r a c t i c a l s o l u t i o n s a r e i l l u s t r a t e d f o r t h e GC s e p a r a t i o n o f a C1-C3

chlorohydrocarbon mixture.

B u t i t i s shown t h a t , i f o v e r a l l

a n a l y s i s t i m e i s i n c l u d e d as a c r i t e r i o n o f o p t i m i s a t i o n , one column system emerges as u n i q u e l y b e s t , t h a t i s , as t h e optimum o f t h e a v a i l a b l e optima. To t h i s e x t e n t , t h e a n a l y s i s i s t o t a l l y o p t i m i s e d . T h i s paper i s d e d i c a t e d t o A r c h e r M a r t i n as a t o k e n o f t h e a u t h o r ' s r e g a r d and a d m i r a t i o n f o r h i s massive c o n t r i b u t i o n t o s c i e n c e and, t h r o u g h t h i s , t o t h e q u a l i t y o f l i f e f o r mankind. INTRODUCTION S c i e n t i f i c t e c h n i q u e s r e a c h m a t u r i t y when we g a i n a d e t a i l e d u n d e r s t a n d i n g o f t h e u n d e r l y i n g processes and can p r o v i d e t h e t e c h n i c a l w h e r e w i t h a l t o exploit this.

From t h i s p o i n t , t h e scope f o r f u r t h e r advance i s r e l a t i v e l y

l i m i t e d and so, a t t e n t i o n i n e v i t a b l y t u r n s t o more f r u i t f u l use of what i s available.

I t i s a t t h i s p o i n t t h a t t h e concept o f o p t i m i s a t i o n a t t a i n s r e a l

significance.

A l t h o u g h t h e r e may be some d i f f e r e n c e o f o p i n i o n i n d e t a i l , i t

i s g e n e r a l l y accepted t h a t gas chromatography reached t h i s degree of m a t u r i t y somewhere i n t h e m i d ~ O ' S ,some t w e n t y y e a r s a f t e r t h e i n i t i a l , d r a m a t i c impact o f t h e work o f James and M a r t i n [l]. two counts;

T h i s s t a t e m e n t i s s u p p o r t a b l e on

f i r s t , t h e r e has been l i t t l e advance i n o u r fundamental under-

s t a n d i n g o r i n b a s i c t e c h n o l o g y i n t h e l a s t decade, and, secondly, t h e g r e a t mass o f l i t e r a t u r e devoted t o o p t i m i s a t i o n i n one o r o t h e r o f i t s s e v e r a l forms can be d a t e d t o t h e same p e r i o d . The s e v e r a l a l t e r n a t i v e forms o f chromatography t h a t have come i n t o

364

general use so successfully in recent years, largely as a spin-off of knowledge gained in the developing years of gas chromatography, can be correspondingly argued to be, as yet, immature. In liquid chromatography for instance, general understanding of the theory of the liquid state and of solution, as well as of interaction at the liquid-solid interface, is so diffuse that the fundamentals of HPLC processes are known only in outline and, as a consequence, are the subject of current debate. Whilst some measure of optimisation is, of course, feasible, it must not be overlooked that to optimise a basically inefficient process is no more than a temporary palliative. Essentially in the light of the foregoing views, we turned our attention in 1974 to the problem of attaining greater substrate selectivity in GLC than was then attainable. The range o f available liquid substrates was very great, yet many relatively simple separations demanded very long columns, long analysis times and, commonly, recourse to the advantages offered in this context by open tube systems. Despite early interest in the potential of mixed substrates, the 1 iterature was surprisingly sparse and, furthermore, rather discouraging. The single most pressing problem emerged as the need for a large amount of empirical information which then presented great difficulty of interpretation since no simple way then existed to ascertain from the data whether an optimum mixture composition existed let alone to identify it uniquely. It was to this particular problem that we first appl ied ourselves. WINDOW ANALYSIS The starting point was the recognition that, if a packed column contained a substrate comprising two non-interacting liquids A and 6, the individual contributions of A and B to total retention must be additive in terms of liquid weights. That is, for a given solute,

vAB=w v

O

A gA

+ wB vogB

(1)

where W represents a liquid weight whilst Vo is a specific retention volume. g The specific retention volume with the binary system, Vo , must then be gAB

ViAB

= WA

ViA + w

B

vogB

where w represents the weight fraction of A or B in the column. Self-evidently, if A and B act as assumed, there is no requirement to construct a series of columns of wA = 0 to 1 and evaluate Vo gAB experimentally. It is sufficient to determine Vo and Vo and calculate the gA gB corresponding values of Vo for all compositions of substrate. Indeed, gAB si nce

365 WA

+

vogAB

WB

= 1

=

ViB +

wA(v;A-v;B)

(3)

V i A and Vo would r e p r e s e n t t h e extremes o f a s t r a i g h t l i n e on a p l o t o f Vo gB gAB a g a i n s t wA. Given o n l y t h a t we know Vo and Vo f o r each o f t h e m i x t u r e components gA gB t o be separated we can t h u s , v i a equn. (3), c a l c u l a t e a l l r e l a t i v e r e t e n t i o n s , a, d i r e c t l y and t h e n c o n s t r u c t a p l o t o f a a g a i n s t wA as an a i d t o l o c a t i o n of an optimum v a l u e o f wA.

But, f u r t h e r , s i n c e we w i l l i d e n t i f y

t h e most u n f a v o u r a b l e v a l u e o f @ f o r t h e whole sample m i x t u r e as t h a t c o r r e s p o n d i n g t o wAopt

we a r e a b l e , v i a t h e w e l l known r e s o l u t i o n e q u a t i o n [ 2 ]

f o r t h e s e p a r a t i o n o f any p a i r , i n t h i s case t h e most d i f f i c u l t t o separate, 2 1+ k' Nreq = 36

[ T I

(4)

( k ' b e i n g t h e c a p a c i t y f a c t o r o f t h e second e l u t e d o f t h e p a i r ) t o c a l c u l a t e t h e number o f t h e o r e t i c a l p l a t e s t h a t w i l l be r e q u i r e d f o r b a s e - l i n e r e s o l u t i o n o f a l l sample components s i n c e , o b v i o u s l y i f t h e most d i f f i c u l t p a i r i s separated so w i l l a l l o t h e r s .

We w i l l , o f course, have r e q u i r e d t o

c o n s t r u c t p u r e phase columns t o e v a l u a t e Vo and Vo o r i g i n a l l y and, f r o m t h e gA gB d a t a p r o v i d i n g t h e s e q u a n t i t i e s , can e v a l u a t e b o t h k ' and t h e p r o b a b l e theoretical p l a t e height,

H, a v a i l a b l e .

Thus, we can c a l c u l a t e t h e n e c e s s a r y

column l e n g t h t o p r o v i d e Nreq. F i g u r e 1 i l l u s t r a t e s t h i s procedure.

I n f i g l a we show t h r e e

h y p o t h e t i c a l p l o t s ( f o r sample components 1, 2 and 3 ) a c c o r d i n g t o equn ( 3 ) f r o m which we see t h a t o n l y two peaks can be r e s o l v e d i n a chromatogram d e r i v e d w i t h e i t h e r p u r e A o r p u r e B o r , indeed, w i t h a m i x t u r e o f t h e p a r t i c u l a r c o m p o s i t i o n c o r r e s p o n d i n g t o t h e wA a t which t h e l i n e s f o r components 1 and 2 c r o s s .

The r e l a t i v e r e t e n t i o n s c a l c u l a t e d f r o m t h e s e d a t a

a r e p l o t t e d a g a i n s t wA i n f i g . l b ;

s i n c e we a r e u n i n t e r e s t e d i n t h e o r d e r o f

e l u t i o n i t i s c o n v e n i e n t always t o r e p r e s e n t a

>

1.

form, o f a s e r i e s o f approximate t r i a n g l e s (windows); two windows w i t h i n w h i c h s e p a r a t i o n i s p o s s i b l e .

Such a p l o t t a k e s t h e i n t h i s case t h e r e a r e

S i n c e t h e "window diagram"

i s a c t u a l l y made up o f t h e envelope o f v a l u e s o f t h e l e a s t f a v o u r a b l e any wA (amin) m i n i m i s e s Nreq.

at

we must l o o k f o r t h e h i g h e s t v a l u e o f t h i s q u a n t i t y s i n c e t h i s I n t h e example shown t h e r e a r e two windows w i t h one

m a r g i n a l l y b e t t e r i n terms o f amin.

R e t u r n i n g t o f i g . l a we can now r e a d u p

a t t h e c o r r e s p o n d i n g v a l u e o f wA, c a l c u l a t e Vo f o r each sample component 9AB and t h u s p r o j e c t t h e r e s u l t i n g chromatogram. Knowing t h e r e l e v a n t v a l u e s o f k ' f o r each component, and t h e average H f o r t h e t e s t experiments, as w e l l as

366

we can f u l l y determine t h e exact experimental conditions needed f o r t h e Nreq separation.

90

80

70

60

50

0.0

0.25

0.50

0.75

1.o

@* Figure l a .

Hypothetical p l o t o f s o l u t e p a r t i t i o n c o e f f i c i e n t (KR) against

volume f r a c t i o n o f solvent A i n A + B mixture.

Solutes 3 and 2 cannot be

separated w i t h pure B ($A = 0); solutes 3 and 1 cannot be separated w i t h pure

A ( $ A = 1); solutes 1 and 2 cannot be separated a t

Figure l b .

= OB = 0.5.

Window diagram derived from KR r a t i o s ( a ) o f f i g . l a .

The window

on t h e l e f t i s marginally t h e b e t t e r i n terms o f amin a t the window peak.

If t h e o r e t i c a l p l a t e heights are t h e same i n both windows, the l e f t hand window

demands a shorter column and o f f e r s f a s t e r analysis o v e r a l l (note KR f o r component 3 i n both windows).

361

A t t h i s p o i n t i t i s worth observing t h a t t h e o r i g i n a l assumption t h a t A and B do n o t i n t e r a c t can, i n p r a c t i c e , be v i r t u a l l y guaranteed by c o n s t r u c t i n g t h e packing n o t as A + B + support b u t by mechanically m i x i n g A

+ support w i t h B + support. publications,

[3,4]

obtained e i t h e r way.

I n f a c t , as we have shown i n a number o f

i n t h e m a j o r i t y of cases, e x a c t l y t h e same r e s u l t s a r e Even where r e t e n t i o n d i f f e r e n c e s a r e observed these a r e

g e n e r a l l y small (ca. 10%) and r e l a t i v e r e t e n t i o n s a r e b a r e l y a f f e c t e d .

The

i m p l i c a t i o n s o f t h i s f o r s o l u t i o n t h e o r y remain t o be f u l l y e x p l o r e d and reconciled, b u t t h i s aspect i s o u t s i d e t h e bounds o f t h e d i s c u s s i o n i n hand. Success was achieved so e a r l y i n t h e programme, w i t h 30 and even 40 component hydrocarbon m i x t u r e s being base-1 i n e separated [4,5]

t h a t immediate

extension o f t h e procedural concept t o o t h e r aspects o f o p t i m a l s e l e c t i o n was indicated. The immediately obvious r o u t e t o t a k e f o l l o w e d from t h e o b s e r v a t i o n t h a t , w i t h some e x t e n s i o n of t h e experimental side, i t should be p o s s i b l e t o o p t i m i s e a s e p a r a t i o n i n ignorance of t h e i d e n t i t i e s o f t h e sample components.

B r i e f l y , i f a s e r i e s o f columns o f v a r y i n g wA a r e c o n s t r u c t e d

and t h e unknown m i x t u r e r u n w i t h each, t h e d e r i v e d values o f Vo f o r a given gAB component must a l l l i e on a s t r a i g h t l i n e p l o t o f Vo a g a i n s t wA. We need SAB only, t h e r e f o r e , p l a c e experimental values o f Vo on a diagram a t t h e gAB a p p r o p r i a t e values o f wA and draw a l l t h e s t r a i g h t l i n e s p o s s i b l e through t h e p o i n t s . O f course, some o f these l i n e s may r e p r e s e n t coincidence b u t we can a s c e r t a i n t h e t r u t h o r otherwise o f t h i s as f o l l o w s .

From t h i s f i r s t

s p e c u l a t i v e p l o t we can c o n s t r u c t a window diagram and, hence, compute t h e column and c o n d i t i o n s needed f o r f u l l b a s e - l i n e r e s o l u t i o n o f t h e supposed x-component m i x t u r e .

I f now we do t h i s e x p e r i m e n t a l l y , every component

I f o n l y y peaks appear, t h e l i n e s

present must show up as a r e s o l v e d peak.

on t h e ViAB/wA p l o t corresponding t o t h e group y

-f

x a r e spurious.

can be e l i m i n a t e d and t h e c o r r e c t new window diagram constructed. p o i n t t h e procedure i s as before.

So, t h e y From t h i s

I t must be almost unique i n a n a l y s i s t o

achieve an o p t i m i s e d s e p a r a t i o n i n ignorance o f t h e n a t u r e o f t h e sample.

Of

course, i n p r a c t i c e , one i s never t o t a l l y i g n o r a n t and i t i s t h i s t h a t guides one t o t h e o r i g i n a l choice o f A and 6. unachieved s e p a r a t i o n performed by us

Figure 2 i l l u s t r a t e s a previously

161

on behalf of t h e A g r i c u l t u r a l

Research Council L a b o r a t o r i e s e x a c t l y as described above.

The v e r y modest

e x t e n t o f o u r knowledge o f t h e component i d e n t i t i e s i s made c l e a r i n t h e 1egend

.

368 The window a n a l y s i s approach can be based upon o t h e r q u a n t i t i e s t h a n s o l v e n t w e i g h t f r a c t i o n and s p e c i f i c r e t e n t i o n volume. F o r example, equn. (1) i s d e r i v e d on t h e b a s i s t h a t r e t e n t i o n a d d i t i v i t y means t h a t 'RAB = V~~ + 'RB and, s i n c e , b y d e f i n i t i o n , r e t e n t i o n volume i s r e l a t e d t o t h e l i q u i d gas p a r t i t i o n c o e f f i c i e n t ( K ) and s o l v e n t volume ( V ) v i a VR = K.V i t follows that

KAB(VA+VB) = KAVA that is,

+

KBVB

(5) KAB = @AKA + 6BKB where 6 r e p r e s e n t s t h e volume f r a c t i o n o f

a s o l v e n t component. 8

Figure 2.

4

TIME (MINS)

This representation

i s j u s t as u s e f u l as i s equn. (1) s i n c e

0

t h e p r a c t i c a l d e t e r m i n a t i o n of K ' s i s no

Optimised separation

o f a m i x t u r e o f phenols, c r e s o l s

d i f f e r e n t t o t h a t o f V o ' s and volumes o f 9 s o l v e n t are, i f a n y t h i n g , e a s i e r t o work

and t h e i r c h l o r i n a t e d d e r i v a t i v e s . w i t h t h a n a r e w e i g h t s . I n d i v i d u a l i d e n t i t i e s unknown.

However, i n a

p r a c t i c a l s i t u a t i o n t h e r e may be

C o n d i t i o n s as d e t a i l e d i n r e f [6]. c o n s i d e r a b l e advantages i n t h e a l t e r n a t i v e use o f c a p a c i t y f a c t o r s ( k ' ) o r even t h e i r u n c o r r e c t e d analogue ( l t k ' ) s i n c e b o t h can be r e a d d i r e c t l y f r o m any chromato gram c o n t a i n i n g an a i r peak.

We have shown [7] how t h e whole p r o c e d u r e can

be c a r r i e d o u t i n such terms w h i l e

Laub and h i s c o l l e a g u e s [8] have even

shown how i t can be a p p l i e d i n terms o f Kovats i n d i c e s , f o r i n s t a n c e . F i n a l l y , as can r e a d i l y be seen, t h e procedure i s i d e a l l y s u i t e d t o c o m p u t e r i s a t i o n and programmes have been p u b l i s h e d [9].

T h i s aspect a t t a i n s

most r e l e v a n c e when t e r n a r y o r h i g h e r c o m p l e x i t y s o l v e n t m i x t u r e s a r e contemplated f o r use s i n c e , then, p i c t o r i a l r e p r e s e n t a t i o n o f t h e window a n a l y s i s diagrams i s d i f f i c u l t , i f n o t i m p o s s i b l e . I n t e r e s t i n g l y , i n a s t u d y of t h e u t i l i t y o f t h r e e component s o l v e n t systems [3] we found l i t t l e advantage o v e r b i n a r y systems i n t h a t , w i t h t h e range o f b i n a r y m i x t u r e s a v a i l a b l e , i t was always p o s s i b l e t o a c h i e v e a comparable s e p a r a t i o n . Ternary systems would, on t h i s evidence, have t o be seen as an u l t i m a t e f i n e t u r n i n g approach f o r p a r t i c u l a r l y d i f f i c u l t s e p a r a t i o n s .

369

WIDER EXTENSION OF THE TECHNIQUE The window analysis approach has, in subsequent years, been extended by us and others in a wide variety of ways. For instance, Pretorius [lo] has applied it to optimising choice of mixed carrier gas composition in GC, in particular in the context of large scale separations involving air or steam. We have shown how optimum analysis temperature can be selected, not only in GC but in HPLC as well. Indeed, in a non-chromatographic application we have shown [ l l ] how lanthanide-shift n.m.r. may be optimised. The method is, clearly, suited for other aspects of HPLC optimisation. For example, since almost all HPLC separations are conducted with binary solvents, the eluent composition is a critical factor in determining the degree of analytical success achieved. Even though experimental retention values are often far from linear in solvent composition so that the procedure is less facile than for GC, it is necessary only to produce a minimum amount of direct experimental data for various solvent compositions to construct a window diagram which will identify the optimum eluent composition. Many HPLC separations depend also on attainment of specific pH values of the flowing eluent. Given that some preliminary evaluation of solvent composition is available, studies of retention variation with pH then allow construction o f a window diagram of VR against pH, and thus location of the best pH value. Fig. 3 illustrates our recent [12] separation o f some aromatic acids optimised for eluent composition, pH and, indeed, temperature as well; not only is it an excellent separation but a rapid one of a mixture previously difficult to deal with. Were workers interested in that direction, the window approach would obviously allow facile location of optimised weight ratios of mixed HPLC adsorbents. The foregoing illustrates one important point, of even greater relevance in liquid than in gas chromatography, total optimisation demands consideration of more than one variable upon which analytical attainment depends. Thus, in HPLC, it can arise that a total optimisation depends upon decisions based on the interplay of, for instance, temperature, substrate identity and composition, solvent identity and composition and pH or additive concentration. This is, indeed, a formidable extra complication but, given a willingness to ascertain a modest volume of experimental data, the subsequent computation is straightforward in that the necessary computer programme can readily be devised.

370

i

..-C

J -1

1

I

I

I

16

12

0

1

I

4

0

T i m e ,mins.

Figure 3

Separation o f .aromatic acids by HPLC optimised f o r eluent

composition, pH, temperature and analysis time.

Conditions as d e t a i l e d i n

r e f . [121. This brings us t o an important issue i n t h e general context o f optimisation. What i s i t desired t o optimise? So f a r , we have l a r g e l y discussed a p p l i c a t i o n s designed o n l y t o l e a d t o the attainment o f acceptable separation, and by i m p l i c a t i o n , w i t h t h e s h o r t e s t possible column. t h a t alone would be enough.

For many,

For others t h e r e might be other e x t r a demands,

the most obvious o f which i s analysis time.

Once we introduce t h i s idea we

immediately recognise t h a t i n what has gone before i t has been e x p l i c i t t h a t there are innumerable s o l u t i o n s v i a the window, o r any o t h e r technique, t o any p a r t i c u l a r separation problem.

I n p r i n c i p l e , a given separation can be

achieved w i t h a large, p o s s i b l y i n f i n i t e , range o f a l t e r n a t i v e substrate or eluent mixtures, and so f o r t h .

When, however, we l o o k also f o r f a s t e s t

analysis, t h e options reduce d r a m a t i c a l l y since o n l y a few systems from the many a v a i l a b l e w i l l provide the conditions f o r t r u l y f a s t analysis.

Fig. 4a and 4b, t o which we address ourselves l a t e r i l l u s t r a t e s both the points. I n other words, we now need t o extend our horizons t o include both an approach

371 f o r t h e comparison o f t h e r e l a t i v e m e r i t s o f a l t e r n a t i v e s u b s t r a t e systems and t o a n a l y s i s time theory. TOTAL OPTIMISATION I N GLC I n attempting an approach t o t h i s matter we must f i r s t consider t h e I n t h e e a r l y 1960's, several

background t o a n a l y s i s time t h e o r y i n GC.

groups attacked t h i s problem more o r l e s s simultaneously [13-161 and, w h i l e t h e i r approaches d i f f e r e d i n d e t a i l , t h e general conclusions drawn were v e r y similar.

Since t h a t due t o P u r n e l l and Quinn [13] i s t h e s i m p l e s t i n

a p p l i c a t i o n we have chosen t o use i t as a b a s i s f o r our demonstration. We s t a r t by accepting t h a t , f o r p r a c t i c a l purposes, t h e simple van Deemter equation i s an e n t i r e l y adequate d e s c r i p t i o n o f t h e r e l a t i o n s h i p o f H w i t h t h e average c a r r i e r gas v e l o c i t y , H = A + B/l +

whence, Hmin

Ci

= A

+ 2(BC) t a t

zmin

i.

= (B/C)'

Thus and a t t h e minimum o f t h e van

Deemter curve, t h e r e f o r e ,

and t h e very smallest value o f t h i s q u a n t i t y i s 2C when A=O. high

I n contrast, a t

E,

H z Cu whence, (H/i)min

C.

On t h e van Deemter p l o t , (H/i)min

represents t h e slope o f a l i n e from t h e

o r i g i n t o t h e p o i n t where t h i s l i n e becomes, e f f e c t i v e l y , t h e assymptote t o t h e curve. Despite t h e d e t e r i o r a t i n g H, and consequent need f o r a l o n g e r column, i t i s nevertheless f a s t e r t o work a t h i g h

i

than

Urnin.

The e l u t i o n time, t, o f some peak i s given i n terms o f column l e n g t h (L) and peak c a p a c i t y f a c t o r , by t = ( L / c ) ( l + k ' ) = (H/c)N(l + k ' ) .

(6) We have already shown (equn. 4) how t h e number o f t h e o r e t i c a l p l a t e s needed f o r a base-line separation o f some p a i r ( N second o f t h a t p a i r .

) i s r e l a t e d t o a and k ' f o r t h e req I n t r o d u c i n g t h i s r e l a t i o n s h i p f o r N i n equn. 6 then

yields t = 36(H/u)min

[(a/a-l)'

(1+k')3/(k')2]

(7)

I f we assume t h a t H / i i s independent o f k ' , d i f f e r e n t i a t i o n o f t h e above y i e l d s a minimum a n a l y s i s time a t k'=2.

This assumption i s , o f course, o n l y

approximate, b u t i s e n t i r e l y adequate i n p r a c t i c e s i n c e t h e f u n c t i o n 3 2 v a r i e s o n l y between about 7 and 8 over t h e range k ' = 1 t o 4. (l+k') /(k') I f k ' = 2, equn. ( 7 ) reduces t o tmin= 243(H/fi)min(a/a-1) 2.

372 But o u r concern i s w i t h o v e r a l l a n a l y s i s t i m e , n o t t h a t o f e l u t i o n o f t h e second o f t h e most d i f f i c u l t p a i r t o separate. L e t us assume t h a t t h e r a t i o o f k ' f o r t h e l a s t peak t o t h a t f o r t h e second o f t h e most d i f f i c u l t p a i r i s

n. Then t o v e r a l 1 = t (l t n k ' ) / ( l t k ' ) and w i t h k ' = 2,

Thus, o v e r a l l a n a l y s i s t i m e i s n o t j u s t a f u n c t i o n of

amin

and Hmin,

as i n

t h e e a r l i e r examples, b u t o f amin, (H/L~),,,~,, and n. S e l f - e v i d e n t l y , t h e most f a v o u r a b l e s i t u a t i o n , a l l o t h e r t h i n g s b e i n g equal, i s when t h e l a s t p a i r o f peaks t o emerge i s t h e most d i f f i c u l t t o separate.

T h i s i s , o f course, t r u e

i r r e s p e c t i v e o f what t h e o r e t i c a l approach i s adopted. I n t h e above e q u a t i o n we can assume r e a s o n a b l y t h a t b o t h a and n a r e independent o f s o l v e n t / s u p p o r t r a t i o a t a l l r e a s o n a b l e v a l u e s o f p r a c t i c a l variables.

Thus, d a t a o b t a i n e d w i t h t e s t columns can be t a k e n t o a p p l y t o

t h e s o l v e n t / s u p p o r t r a t i o e v e n t u a l l y i d e n t i f i e d as t h a t needed t o p l a c e t h e most d i f f i c u l t p a i r a t k ' = 2. (H/i)min,

however, v a r i e s q u i t e m a r k e d l y w i t h

s o l v e n t l o a d i n g and s o , n o t o n l y must e f f i c i e n c y s t u d i e s be conducted w i t h t h e t e s t columns b u t , p o s s i b l y , w i t h t h o s e o f t h e c a l c u l a t e d l o a d i n g t o o . However, i n g e n e r a l , we f i n d t h a t when o p t i m a l l o a d i n g s a r e l e s s t h a n t h e t e s t l o a d i n g s , (H/G) m i n i s a l s o l e s s , and so a n a l y s i s i s f a s t e r t h a n m i g h t be expected b y c a l c u l a t i o n . I t remains now t o d e c i d e on some method o f d e a l i n g w i t h c o m p a r a t i v e

solvent s e l e c t i v i t i e s .

A number o f approaches a r e a v a i l a b l e b u t , e a r l y experience i n d i c a t e d t h a t f o r o u r p r e s e n t purposes a Rohrschneider t y p e o f approach o f f e r e d t h e g r e a t e s t advantage.

As an example o f t h e a p p l i c a t i o n o f t h i s extended approach we have s t u d i e d t h e s e p a r a t i o n o f a seven component C1-C3 c h l o r o h y d r o c a r b o n m i x t u r e by GLC [17,18].

The s t e p s i n t h e procedure a r e as f o l l o w s .

(1) From l i t e r a t u r e d a t a we a s c e r t a i n t h o s e GLC s o l v e n t s t h a t appear t o span a wide " p o l a r i t y " range and then, u s i n g c o r r e s p o n d i n g t e s t columns

t o p r o v i d e data, c o n s t r u c t a Rohrschneider p l o t .

(2)

The d a t a of t h e Rohrschneider p l o t a r e t h e n c o n v e r t e d t o a window diagram by p l o t t i n g v a l u e s o f l n ( a ) as a f u n c t i o n o f t h e v a r i o u s d e r i v e d " p o l a r i t i e s " (on a s c a l e ' 0-100).

T h i s procedure i d e n t i f i e s t h e " p o l a r i t i e s " c o r r e s p o n d i n g t o encouraging windows.

373 For each window a mixed s o l v e n t composition f o r a l l p o s s i b l e p a i r s o f solvents t h a t i n p r i n c i p l e w i l l reproduce t h e necessary p o l a r i t y i s calculated.

There may be many such p a i r s .

For instance, i f we a r e

c o n s i d e r i n g o n l y f i v e p o s s i b l e substrates, t h r e e o f which l i e on one s i d e o f t h e window and two on t h e other, t h e r e a r e s i x mixed l i q u i d combinations t h a t , given enough column e f f i c i e n c y , w i l l p r o v i d e separation.

Ifmore substrates a r e considered, and numerous windows

appear, a v a s t number of p o s s i b l e s o l v e n t systems capable o f p r o v i d i n g separation presents i t s e l f . We now c o n s t r u c t H/u p l o t s from t h e t e s t data and evaluate (H/i)min

f o r each t e s t solvent, and so estimate (H/i)min

each mixed column composition i n d i c a t e d .

for

We have, i n f a c t , found [19]

t h a t simple (weight f r a c t i o n ) a r i t h m e t i c averaging i s e n t i r e l y adequate. Using a, k ' and H data, v i a equn. ( 4 ) we a r e now a b l e t o c a l c u l a t e a t k ' = 2 f o r t h e second of t h e most d i f f i c u l t p a i r t o separate, Nreq and hence t h e r e q u i s i t e column l e n g t h , f o r a l l i n d i c a t e d s o l v e n t mixtures.

This a l s o a l l o w s

u

t o be f i x e d since we know (H/u),~~.

I n p r a c t i c e , o f course, when we go from t h e t e s t c o n d i t i o n t o some o t h e r s o l v e n t l o a d i n g as i s necessary f o r time o p t i m i s a t i o n , we may (see equn.

4 ) reduce k ' f o r o t h e r p a i r s so much t h a t they, i n f a c t , take over t h e r o l e o f t h e most d i f f i c u l t p a i r .

Thus Nreq f o r a l l p a i r s must

be c a l c u l a t e d , and where necessary, f u r t h e r c a l c u l a t i o n c a r r i e d out. F i n a l l y , having t i m e optimised a l l p o t e n t i a l systems by c a l c u l a t i o n s i n t r o d u c i n g t h e t e s t data values of n, many can be discarded immediately since they can o f f e r minimum o v e r a l l a n a l y s i s times so f a r removed from t h e b e s t t h a t no amount o f approximation i n t h e theory could p o s s i b l y be r e l e v a n t . I t then remains o n l y t o c o n s t r u c t and operate t h e system o r systems

i n d i c a t e d , i n t h e manner c a l c u l a t e d , t o achieve what i s , i n p r i n c i p l e , t h e v e r y b e s t separation t h a t can be achieved i n any circumstances.

I n o t h e r words, s u b j e c t t o the o p t i m i s a t i o n c r i t e r i a adopted, t o i d e n t i f y t h e optimum o f a l l optima. Lengthy though t h e procedure seems i t i s n o t p a r t i c u l a r l y so i n practice.

Ifone be1 ieves i n t h e v a l i d i t y o f Rohrschneider-type p o l a r i t y

scales, f o r instance, no more than f i v e o r s i x l i q u i d substrates need ever be taken i n t o account provided o n l y t h a t they o f f e r acceptable values o f (H/G)min

and o f n.

Further, t h e experimental work i n v o l v e s o n l y then the

determinations o f r e l a t i v e r e t e n t i o n s and o f k ' w i t h t e s t columns a t t h e few f l o w r a t e s needed t o assess (H/6)min.

Subsequently, t h e whole o f t h e Indeed, t h e most

q u a n t i t a t i v e assessment i s c a r r i e d through by the computer.

tedious p a r t a r i s e s when i t i s d e s i r e d t o o p t i m i s e a l s o f o r a n a l y s i s

374

temperature,

when t e s t data a t t h r e e o r f o u r temperatures must be added.

In

the example t o be quoted here which r e l a t e s t o complete separation o f seven C1-C3

c h l orohydrocarbons, t h i s too has been done. The t e s t solvents chosen f o r the exercise were squalane (SQ),

d i b u t y l tetrachlorophthalate (DBTC), dinonylphthalate (DNP), t r i c r e s y l phosphate (TCP) and polyethylene g l y c o l (PEG 1500) which, i n Rohrschneider terms, do i n f a c t give a wide spread o f n o t i o n a l p o l a r i t i e s . For t h e sample mixture specified, pure squalane o f f e r e d prospects i n terms o f amin and

(H/i)min

a t l e a s t as good as d i d any other possible system a t any temperature

i n the range 40-120°C.

However, i n terms o f n, i t was so much l e s s w e l l

favoured t h a t several solvent mixtures emerged as competitive, w i t h a TCP/PEG 1500 mixture proving s u b s t a n t i a l l y b e t t e r o v e r a l l , so much so t h a t i t provides an analysis time approaching one-half t h e b e s t a t t a i n a b l e w i t h squal ane a1one. Fig. 4a i l l u s t r a t e s a s e l e c t i o n o f t h e minimum time chromatograms obtained using n i t r o g e n as c a r r i e r gas, t h e experimental d e t a i l s being l i s t e d i n the legend. We see here twelve acceptable, a l t e r n a t i v e , ways t o achieve f u l l separation and many more were i d e n t i f i e d b u t are n o t shown.

Using

analysis time as a c r i t e r i o n , t h e r e i s , however, a unique optimum system, t h a t i l l u s t r a t e d i n t h e bottom r i g h t hand corner o f f i g . 4a. I t i s worthwhile t o note t h a t , f o r f a s t e s t analysis, hydrogen as c a r r i e r gas i s best, followed by helium which, i n turn, i s f a r b e t t e r than i s nitrogen, The reasons are well known [ZO].

Fig. 4b i l l u s t r a t e s t h i s c l e a r l y , analysis time being

e f f e c t i v e l y halved when we go from N2 t o H2 as c a r r i e r . The examples c i t e d have a l l r e l a t e d t o p,acked columns.

The theory,

however, i s general and we have shown examples o f i t s a p p l i c a t i o n t o c a p i l l a r y systems [17,21].

The d i f f i c u l t y here l i e s i n t h e problems

associated w i t h producing mixed solvent open-tube columns and, i n p r i n c i p l e , the use o f s e r i a l columns looks more straightforward. course,

There i s no reason, o f

why s e r i a l packed columns should n o t be used, and we have r e c e n t l y

discussed [22] t h i s too. Column s e r i a l i s a t i o n , however, b r i n g s us t o a new problem.

On account o f t h e continuing expansion o f c a r r i e r along a column,

the average v e l o c i t y

i i n i n d i v i d u a l s e r i a l columns i s a f u n c t i o n o f t h e i r

r e l a t i v e l e n g t h and sequence.

Thus, not o n l y do a, k ' , H and n vary

depending on whether columns I and I 1 are operated i n the mode 1/11 or II/I, but they depend a l s o upon r e l a t i v e solvent loading, p a r t i c l e s i z e and tube diameters.

I t was a f a i l u r e t o recognise these important f a c t s t h a t l e d t o

i n i t i a l f a i l u r e o f an attempt [23,24]

t o optimise lengths i n b i n a r y s e r i a l

c a p i l l a r y columns v i a the window analysis method.

375

e

Figure 4a. ( a ) SQ, 1.27% w/w, 50"C, 183 cm, 150 sec; ( b ) SQ, 2.38% w/w, 75°C. 183 cm, 125 sec; ( c ) SQ, 2.91% w/w, 82"C, 183 cm, 198 sec; ( d ) DNP/PEG 1500, wPEG = 0.47, 0.44% w/w, 50°C. 204 cm, 82 sec; ( e ) SQ/PEG 1500, wPEG = 0.61. 0.53% w/w, 50"C, 205 cm, 75 sec; ( f ) SQ/PEG, wPEG = 0.65, 1.27 % w/w, 75"C, 366 cm, 125 sec; ( 9 ) SQ/PEG 1500, wPEG = 0.69, 2.63% w/w, lOO"C, 366 cm, 199 sec; ( h ) SQ/PEG 1500, wPEG = 0.048, 2.38% w/w, 75OC, 183 cm, 156 sec; ( i ) DBTC/PEG 1500, wPEG = 0.47, 0.50 % w/w, 5OoC, 230 cm, 123 sec; (j) 1500, wPEG = 0.43, 1.21 % w/w, 75"C, 438 cm, 449 sec; ( k ) TCP/PEG 1500, wPEG = 0.21, 0.54% w/w, 50"C, 210 cm, 73 sec; ( 1 ) TCP/PEG 1500, wPEG = 0.22, 1.49% w/w, 75"C, 260 cm, 171 sec. [Sequence: System, weight fraction, solvent/support ratio, analysis temperature, column length, overall analysis time.] Carrier gas in all examples: N;..

376

. -

Figure 4b.

Top row: column (e) o f f i g . 4a eluted, l e f t t o r i g h t , by N2,

H2 i n times 75, 59, 45 sec.

sec.

He,

Middle row: column ( d ) , e l u t i o n times 82, 61, 49

Botom row: column ( k ) e l u t i o n times 73, 56, 40 sec.

Bottom r i g h t

chromatogram represents unique optimal s o l u t i o n . The above problem has been addressed i n t h e l i t e r a t u r e o n l y t h r i c e i n t h e past.

F i r s t , by Hildebrand and R e i l l e y [25] secondly, by Buys and Smuts

[26] and, f i n a l l y , by Krupcik, Guiochon and Schmitter [27].

I n each instance,

o n l y special cases were d e a l t w i t h and s o , f o r t h e general p r a c t i c a l s i t u a t i o n , t h e i r derived equations are o n l y o f modest value and, i n some aspects, misleading.

I n consequence, we have r e c e n t l y generated t h e

generalised theory o f s e r i a l column operation [22] o f any type o f column and shown how i t s consequences modify t h e window method f o r

optimisation o f

s e r i a l systems. The subsequent agreement between t h e o r y and p r a c t i c e has been found t o be e x c e l l e n t .

W h i l s t i t i s e n t i r e l y p o s s i b l e t o achieve o v e r a l l

r e s u l t s f o r s e r i a l packed columns comparable w i t h those f o r mechanically mixed columns i t i s the case t h a t e x t r a t e s t data, r e l a t i n g t o column p e r m e a b i l i t i e s , a r e needed.

Given such, however, t h e c a l c u l a t i o n a l s i d e

o f f e r s no problem s i n c e i t i s a s t r a i g h t f o r w a r d m a t t e r t o e x t e n d t h e computer programme.

Where m u l t i - s u b s t r a t e s a r e r e q u i r e d f o r use w i t h c a p i l l a r i e s , on

t h e o t h e r hand, s e r i a l columns a r e more o r l e s s e s s e n t i a l .

The t h e o r y and

procedure, however, a r e now a v a i l a b l e , as e x p l a i n e d above. N o t a l l analyses a r e s u s c e p t i b l e t o i s o t h e r m a l o p e r a t i o n , o f course, and temperature programming i s so w i d e l y employed t h a t t h e necessary f a c i l i t i e s a r e s t a n d a r d on modern equipment.

The p r e c i s e way t o a c h i e v e an o p t i m i s a t i o n

o f t h i s t e c h n i q u e i s n o t obvious s i n c e t h e r e a r e numerous o p t i o n s i n v o l v i n g

c o n t i n u o u s programming, programming f o r m a t and r a t e and i n t e r m i t t e n t p l a t e a u operation.

The t h e o r y o f t e m p e r a t u r e programming has a t t r a c t e d l i t t l e

a t t e n t i o n s i n c e t h e m i d d l e 60's. e s s e n t i a l l y because i t appeared t o have been developed as f a r as was p r a c t i c a b l e .

However, f o r o p t i m i s a t i o n purposes, t h i s l e v e l o f development was e s t a b l i s h e d by us as inadequate, a p r o b l e m t h a t

has s i n c e been e l e g a n t l y r e s o l v e d [28] i n o u r l a b o r a t o r y by D r . P. S. W i l l i a m s who has n o t o n l y extended t h e t h e o r y a p p r o p r i a t e l y b u t has c a s t i t i n t o p r a c t i c a l terms a l l o w i n g r e a d y use o f window a n a l y s i s based on a minimum o f experimental information.

T h i s work, i n c o u r s e o f p u b l i c a t i o n ,

i l l u s t r a t e s d r a m a t i c a l l y t h a t s i g n i f i c a n t improvements a r e a t t a i n a b l e i n programmed o p e r a t i o n i n terms, n o t o n l y o f s e p a r a t i o n , b u t o f a n a l y s i s t i m e . POSTSCRIPT I n t h i s paper, one o f a number o f approaches t o chromatographic o p t i m i s a t i o n c u r r e n t l y b e i n g e x p l o i t e d has been d i s c u s s e d i n terms o f i t s i n i t i a t i o n , and development o v e r a decade, i n o u r own l a b o r a t o r i e s .

T h i s was

t h e a u t h o r ' s s p e c i f i c r e m i t and i t i s on t h i s account t h a t c o n t r i b u t i o n s l e a d i n g t o t h e expansion o f t h e h o r i z o n s o f t h e window method, n o t a b l y by Laub and by Deming, have been l a r g e l y i g n o r e d .

We a r e f u l l y aware t o o t h a t ,

c o n c u r r e n t w i t h t h e work d e s c r i b e d here, s e v e r a l o t h e r approaches t o chromatographic o p t i m i s a t i o n have been e x p l o r e d i n some d e p t h a t a number o f c e n t r e s world-wide.

Any a t t e m p t t o summarise and compare t h e a l t e r n a t i v e s

must b e done i n a n o t h e r p l a c e and a t a n o t h e r time.

Indeed, t h a t

which i n t h e end proves t h e most p r o f i t a b l e r o u t e cannot now be p r e d i c t e d

w i t h any confidence.

I t may w e l l be t h a t some o r a l l may f i n d an i m p o r t a n t

niche i n t h e f u t u r e . The a t t r a c t i o n s o f what we have c a l l e d window a n a l y s i s , a name perhaps l e s s o b v i o u s l y s u i t a b l e t o d a y t h a n when f i r s t c o i n e d ( b u t t h e n one can say t h e same o f chromatography, and t h a t s u r v i v e s ) , i s i t s e v i d e n t s i m p l i c i t y and extraordinary generality.

U s i n g e s t a b l i s h e d t h e o r y as i t s base, and t h u s

a l l o w i n g a q u a n t i t a t i v e connexion o f t h e v a r i a b l e s c o n t r i b u t i n g t o s e p a r a t i v e achievement, i t p r o v i d e s a r o u t e t o s i g n i f i c a n t improvement i n performance i n v o l v i n g a minimum o f e x p l o r a t i v e s t u d y and, i n consequence, i n t r o d u c e s a

378

degree of redundancy that allows us t o contemplate even more difficult separations than w e currently attempt. In concluding, I take the opportunity t o thank my erstwtiile collaborators, Richard Laub and Steven Williams, for their considerable contributions t o the development described. REFERENCES 1. A.T. James and A.J.P. Martin, Biochem.J., 50 (1952) 679. 2. J.H. Purnell , J.Chem.Soc. , (1960) 1268. 3. R.J. Laub and J.H. Purnell, J.Chromatog., 112 (1975) 71. 4. R.J. Laub and J.H. Purnell, Anal.Chem., 48 (1976) 799. 5. J.H. Purnell , Proc.Ana1yt.Div.Chern.Soc. , 16 (1979) 136. 6. R.J. Laub and J.H. Purnell, J.Chromatog., 161 (1978) 59. 7. R.J. Laub, J.H. Purnell, D.M. Summers and P.S. Williams, J.Chromatog. , 155, (1978) 1. 8. R.J. Laub, Anal.Chem., 52(1980) 1219. See also R.L. Pecsok and J.Apffe1 , Anal .Chem. , 51 (1979) 594. 9. R.J. Laub, J.H. Purnell and P.S. Williams, J.Chromatog., 134 (1977) 249; Anal .Chim.Acta, 95 (1977) 135. 10. V.Pretorius, J.High Res. Chrom., 1 (1978) 199. 11. R.J. Laub, A.Pelter and J.H. Purnell, Anal.Chem., 51 (1979) 1878. 12. B.Pate1, J.H. Purnell and C.A. Wellington, J.High Res.Chrom., 7 (1984) 375. 13. J.H. Purnell and C.P. Quinn, Gas Chromatography 1960, Butterworths, London, ed. R.P.W. Scott, p.184. 14. J.H. Knox, J. Chem. SOC., (1961) 433. 15. D.D. DeFord, R.J. Lloyd and B.O. Ayers, Anal.Chem., 35 (1963) 426. 16. J.C. Giddings and P.D. Schettler, Anal.Chem., 36 (1964) 1483. 17. J.H. Purnell, P.S. Williams and G.A. Zabierek, Proc. 4th 1nt.Symp.Capillary Chrom., 1981, Dr. Alfred Heuthig Verl Reg, Hei del berg , p. 573. 18. M.Y.B. Othman, J.H. Purnell, P. Wainwright and P.S. Williams, J. Chromatog. , 289 (1984) 1. 19. M.Y.B. Othman and P. Wainwright, Ph.D. Theses, University of Wales, 1985. 20. J.H. Purnell, Gas Chromatography, 1962, John Wiley, New York, p.150. 21. J.H. Purnell and P.S. Williams, J. High Res. Chrom., 6 (1983) 569. 22. J. H. Purnell and P. S. Williams, J. Chromatog. 292 (1984) 187; see also generalised theory to appear J. Chromatog. , 1985. 23. D.F.Ingraham, C.F.Shoemaker and W.Jennings, J.Chromatog., 239 (1982) 39.

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24. G.Takeoka, H.M. Richard, M.Mehran and W.Jennings, J . High Res. Chrom., 6 (1983) 145. 25. G.P. Hildebrand and C.N. Reilley, Anal.Chem., 36 (1964) 47. 26. T.S. Buys and T.W. Smuts, J. High Res. Chrom., 3 (1980) 461. 27. J. Krupcik, G. Guiochon and J.M. Schmitter, J. Chromatog., 213 (1981) 189. 28. P.S. Williams, in course o f publication.

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381

FROM WIDEBORE V I A NARROWBORE AND ULTRA NARROWBORE TO WIDEBORE COLUMNS I N CAPILLARY GAS CHROIIATOGRAPHY. A POTPOUBRI ? P.

Sandra.

L a b o r a t o r y o f O r g a n i c C h e m i s t r y , U n i v e r s i t y of G h e n t , K r i j g s l a a n 281 ( S . 4 ) , B - 9 0 0 0 Gent ( B e l g i u m ) . 1. INTRODUCTION

I n t h e t w e n t y f i v e y e a r s which have p a s s e d s i n c e Golay p r e s e n t e d h i s t h e o r e t i c a l and p r a c t i c a l work on c a p i l l a r y columns ( r e f . 1 , 2 ) s i g n i f i c a n t p r o g r e s s h a s been made i n p r a c t i c a l c a p i l l a r y g a s chromatography.

Developments s u c h a s t h e s t a t i c c o a -

t i n g technique, high temperature s i l y l a t i o n , t h e introduction of new gum p h a s e s and t h e i r i m m o b i l i z a t i o n , on column i n j e c t i o n , m u l t i d i m e n s i o n a l c a p i l l a r y g c , c a p i l l a r y gc c a p i l l a r y gc

-

-

mass s p e c t r o m e t r y ,

FTIR and many o t h e r s , have r e s u l t e d i n a h i g h t e c h -

n o l o g i c a l v a l u e of t h e t e c h n i q u e and t h e r e f o r e i n a w i d e s p r e a d u s e i n a l l f i e l d s of c h e m i c a l a n a l y s i s .

I n t h e s e twenty f i v e

y e a r s however, o n l y few m o d i f i c a t i o n s have b e e n made t o t h e t h e o r y of c a p i l l a r y g a s chromatography.

Notwithstanding t h i s , a l a r g e

number of c h r o m a t o g r a p h e r s a t p r e s e n t i s c o n f u s e d by t h e p e r f o r mances of t h e d i f f e r e n t column t y p e s which a r e c o m m e r c i a l l y a v a i lable. I t i n d e e d i s n o t e a s y t o s e l e c t t h e p r o p e r column d i a m e t e r ,

t h e column l e n g t h , t h e f i l m t h i c k n e s s , t h e s t a t i o n a r y p h a s e e t c . f o r a g i v e n s e p a r a t i o n problem.

Widebore t h i c k f i l m columns

r e c e i v e d much a t t e n t i o n o v e r t h e l a s t y e a r s and t h e y p r o b a b l y

w i l l become more i m p o r t a n t i n t h e n e a r f u t u r e .

On t h e o t h e r h a n d ,

u l t r a n a r r o w b o r e columns have b e e n i n t r o d u c e d and t h e y t o o a r e gaining i n popularity. I n t h i s c o n t r i b u t i o n b o t h a s p e c t s , column i n t e r n a l d i a m e t e r and f i l m t h i c k n e s s w i l l b e emphasized and o n r e q u e s t of t h e e d i t o r o f t h i s book,

t h e p h i l o s o p h y o f o u r l a b o r a t o r y on t h e s e

a s p e c t s w i l l b:? a d v a n c e d .

E x c e l l e n t c o n t r i b u t i o n s have r e c e n t l y

been p u b l i s h e d on t h e p e r f o r m a n c e of open t u b u l a r columns a s a f u n c t i o n o f t h e t u b e d i a m e t e r ( r e f . 3,4) thickness ( r e f . 3 ) .

and t h e l i q u i d p h a s e

F o r a more d e t a i l e d d i s c u s s i o n w e r e f e r t o

t h e s e p u b l i c a t i o n s and t h e r e f e r e n c e s c i t e d t h e r e i n .

382

2. FUNDAMENTAL EQUATIONS. F o r a b e t t e r u n d e r s t a n d i n g of t h e p h i l o s o p h y b e h i n d t h e select i o n of t h e i n t e r n a l d i a m e t e r and t h e f i l m t h i c k n e s s , t h e knowledge o f t h e f u n d a m e n t a l e q u a t i o n s of c a p i l l a r y g a s c h r o m a t o g r a p h y i s necessary. 1) R e s o l u t i o n of two p e a k s

2AtR

dFi (-a-1 )(--) k 2 R = 4 dk2+1

=

Wbl

+

Wb2

2 ) n : number of t h e o r e t i c a l p l a t e s L

2

n : LL

= + C,;

+

h : B

CL;

U

2

. -2DG -

+

U

-

u : -

(1+6k+llk2)r2 2 4 ( l + k )2 DG

- 2 k u + s -( 1-+ k ) 2

df DL

L tM

3) a : s e l e c t i v i t y f a c t o r

t' a:-

t'

r e l a t i v e r e t e n t i o n of t w o p e a k s

R2 R1

4) k : capacity factor

k : -

tR-tM tM

5 ) TZ v a l u e : t R ( z + l ) - t R ( z ) - 1 W h ( z + l )+ w h ( z ) z : h y d r o c a r b o n z C-atoms z + l : hydrocarbon z + l C - a t o m s

6) Coating e f f i c i e n c y theor CE %

: hmin

hmin exp

x 100

=

*

x

theor

100

-

U

(5)

3 83

7 ) Minimum a n a l y s i s t i m e f o r a two component m i x t u r e

t R i s assumed t o b e e q u a l t o t h e r e t e n t i o n t i m e o f t h e second peak.

8 ) Sample c a p a c i t y

SC ? n r 2 ( l + k ) JLh

3 . L I S T OF SYMBOLS. tR

re t e n t i o n t i m e

tM

r e t e n t i o n t i m e of u n r e t a i n e d s o l u t e ( d e a d t i m e )

t’R

adjusted retention t i m e t q R : tR-tM

Wh Wb

peak w i d t h a t h a l f h e i g h t peak w i d t h a t b a s e

L

column l e n g t h

n

number of t h e o r e t i c a l p l a t e s

n’m

number of t h e o r e t i c a l p l a t e s p e r m e t e r

h

h e i g h t e q u i v a l e n t t o one t h e o r e t i c a l p l a t e

U

average l i n e a r gas velocity

K

distribution constant

-

B

vG

: phase r a t i o : -

vL

=

2df

vG

: volume o f

vL

: volume o f t h e l i q u i d p h a s e i n t h e column

dC

r df

DG DL B

cG cL

t h e g a s p h a s e i n t h e column

: column d i a m e t e r

: column r a d i u s : film thickness : d i f f u s i o n c o e f f i c i e n t i n t h e g a s phase : d i f f u s i o n c o e f f i c i e n t i n t h e l i q u i d phase : longitudinal diffusion t e r m : r e s i s t a n c e t o mass t r a n s f e r i n t h e g a s p h a s e : resistance

t o mass t r a n s f e r i n t h e l i q u i d p h a s e

384 4 . DISCUSSION The c o n t r i b u t i o n o f t h e t h r e e t e r m s o f t h e column e q u a t i o n h =

B

+

C,;

+ ; C,

i s g r a p h i c a l l y p r e s e n t e d i n Fig:

1.

U

H

4

t,/

DGf

-

uopt

U

F i g . 1. C o n t r i b u t i o n t o B , CG and CL on h . P r a c t i c a l l y , three d i s t i n c t s i t u a t i o n s can occur. 1. The r e s i s t a n c e t o mass t r a n s f e r i n t h e g a s p h a s e

(C ) G

i s controlling h. 2. The r e s i s t a n c e t o mass t r a n s f e r i n t h e l i q u i d p h a s e (C,)

i s controlling h.

3. Both CG and CL a r e c o n t r i b u t i n g t o b a n d b r o a d e n i n g . Emphasis w i l l be g i v e n t o s i t u a t i o n 1 and 2. 4.1.

R e s i s t a n c e t o mass t r a n s f e r i n t h e g a s p h a s e i s c o n t r o l l i n g h

The l i q u i d phase t e r m CL c a n be n e g l e c t e d f o r columns w i t h a l a r g e @ - v a l u e . A s t h e B-value i s p r o p o r t i o n a l t o 2 b o t h t h e 2% r a d i u s and t h e f i l m t h i c k n e s s have t o be c o n s i d e r e d . I g n o r i n g t h e CL

hmin = r

t e r m , hmin i s e q u a l t o 2 ( B C G ) l i 2 o r

v"

F o r h i g h k - v a l u e s hmin a p p r o a c h e s t h e column d i a m e t e r dc. hmin i s i n d e p e n d e n t of t h e d i f f u s i o n c o e f f i c i e n t i n t h e g a s p h a s e and t h u s hmin

(N2) i s e q u a l t o hmin

(H2). On t h e o t h e r hand t h e

(B/CG) i s proportional t o the d i f f u s i t i v i t y opt A s DG i s i n v e r s e l y p r o p o r t i o n a l t o t h e s q u a r e i n t h e gas phase.

corresponding

r o o t of t h e m o l e c u l a r w e i g h t o f t h e c a r r i e r g a s U o p t ( H 2 )

2

3.75

385

-

u

(N2). I n p r a c t i c a l c a p i l l a r y GC o n t h i n f i l m c o l u m n s , h y d r o OP t gen i s t h u s t h e b e s t c h o i c e a s t h e a n a l y s i s t i m e i s r e d u c e d by a

f a c t o r o f 3 . 7 5 w h i l e t h e s e n s i t i v i t y i s i n c r e a s e d b y t h e same order. The i n f l u e n c e o f t h e column d i a m e t e r o n t h e e f f i c i e n c y o f a c a p i l l a r y column h a s b e e n i n v e s t i g a t e d b y e x p e r i m e n t a l l y r e c o r d i n g h;

and T Z ;

c u r v e s f o r columns v a r y i n g i n i n t e r n a l d i a m e t e r .

All

columns w e r e coated w i t h t h e same c o a t i n g s o l u t i o n ( 0 . 3 3 % w / v OV-1 i n p e n t a n e ) a n d t h u s h a v e t h e same B - v a l u e The d a t a are r e p r e s e n t e d i n F i g . 3 .

(297).

The c h a r a c t e r i s t i c s o f t h e

columns are l i s t e d i n Table 1 .

Fig.

2. E x p e r i m e n t a l h i

-

TZ

u

c u r v e s f o r columns v a r y i n g i n I . D .

h w a s c a l c u l a t e d f o r C12 a t 1 0 0 O C . TZ w a s c a l c u l a t e d f o r C12-C13

a t 100°C.

carrier g a s : hydrogen. TABLE 1 Data f o r c o l u m n s i n Fic;. 2 . ID

(mm) ( m ) ~~~~~~

TZ/m HEPTmin

L TZ 1' 2-'13

HEP Tmi

Exp. ( m m ) T h e o r . (mm)

U

CE

df

(cm/sec) ( % ) ( p m )

~

0.18 0.27 0.51 0.70 0.88

37 60 58 50 49

54 53 35 30 26

1.45 0.88 0.60 0.60 0.53

0.19 0.27 0.49 0.71 0.88

0.178 0.24 0.46 0.66 0.78

45 32 25 16 14

94 89 93 86 88

0.15 0.23 0.43 0.59 0.74

386 From t h e s e d a t a t h e f o l l o w i n g c o n c l u s i o n s c a n be drawn :

-

f o r columns w i t h l a r g e 6 - v a l u e s , column d i a m e t e r . t h e hmin-values,

hmin

is proportional to the

F o r t h e c o m m e r c i a l l y a v a i l a b l e FSOT c o l u m n s , maximum p l a t e s p e r meter a n d minimum l e n g t h

n e e d e d f o r 50.000 p l a t e s c a n be c a l c u l a t e d . dc(mm)

hmin

n/m

0.05 0.10

0.05 0.10

20.000 10.000

0.25 0.32

0.25 0.32 0.50 0.53

4.000

0.50

0.53

L f o r 50.000 p l a t e s

2.5 m

5 12.5 16 25 27

3.125 2.000

1.887

m m m m m

The d e c r e a s i n g e f f i c i e n c y w i t h i n c r e a s i n g i n t e r n a l d i a m e t e r c a n e a s i l y be compensated by i n c r e a s i n g t h e column l e n g t h by t h e

same f a c t o r .

T h i s i s i l l u s t r a t e d i n t h e a n a l y s i s of t h e oxygen

f r a c t i o n o f hop e s s e n t i a l o i l ( F i g . 3 ) on a n a r r o w b o r e column ( 2 5 m x 0 . 2 5 nun) a n d a widebore column ( 5 0 m x 0 . 5 m m ) .

Both

columns were coated w i t h S u p e r o x 4 , r e s p e c t i v e l y w i t h 0 . 3 and 0 . 3 5 pm.

The r e s o l u t i o n i n b o t h chromatograms i s t h e same. The

a n a l y s i s t i m e however i s much l o n g e r on t h e w i d e b o r e column a l t h o u g h i n F i g . 3 it is p a r t i a l l y compensated by t h e temperat u r e program mode.

I f speed i s a l l i m p o r t a n t , t h e narrowbore

column must b e f a v o u r e d .

The sample w a s d i r e c t l y i n t r o d u c e d f o r

t h e w i d e b o r e column w h e r e a s s p l i t t i n g w a s employed f o r t h e n a r rowbore column.

The w i d e b o r e column i n d e e d h a s a much l a r g e r

sample c a p a c i t y (see e q u a t i o n 1 2 , r t , kf, L f , h t compared t o t h e n a r r o w b o r e column) and d i r e c t s a m p l i n g c a n e a s i l y be p e r f o r m e d . Widebore columns c a n manage l a r g e s o l v e n t s a m p l e s w i t h o u t u n d u l y l a r g e s o l v e n t peaks.

On w i d e b o r e columns o f t e n i m p r o v e d q u a n t i -

t a t i v e r e s u l t s are o b t a i n e d .

The r e q u i r e m e n t s on i n s t r u m e n t

c o n s t r u c t i o n are less s t r i n g e n t ( d e a d v o l u m e s , make-up g a s ) and c o n v e n t i o n a l GC i n s t r u m e n t s c a n be u s e d . Moreover more r e l i a b l e d a t a are o b t a i n e d w i t h w i d e b o r e columns i n t h e c o m b i n a t i o n CGC-hyphenated

-

-

u

techniques.

decreases with increasing i n t e r n a l diameter.

A t hmin, t h e opt l a r g e r t h e column diameter, t h e l o n g e r t h e a n a l y s i s t i m e . This

a l s o f o l l o w s from e q u a t i o n 11, h i n c r e a s e s w h i l e u decreases. A l o n g e r r e s i d e n c e t i m e i n t h e column c o r r e s p o n d s t o more band-

broadening.

The s e n s i t i v i t y t h u s a l s o d e c r e a s e s i n f u n c t i o n o f

387

A

1 I

70"

c ib.

I

I

110.

I

110'

3,

I 1

m'

I

SO'

Fig. 3. Chromatogram of the oxygenated fraction of hop essential oil on a narrowbore (A) and widebore (B) capillary. Chromatographic conditions : (A) column 2 5 m x 0.25 mm ID Superox-4; temperamin-1; injection via an allture-programmed 70 to 190°C at 2'C glass inlet splitter (1:20). (B) column 5 0 m x 0.5 mm ID Superox-4 temperature-programmed 70 to 190°C at 2 O C min-l; direct injection.

t h e column d i a m e t e r .

-

The s l o p e o f t h e h;

c u r v e i s much s t e e p e r f o r w i d e b o r e columns

( d e t e r m i n e d by t h e CG c o n t r i b u t i o n a s t h e CL t e r m a c c o u n t s f o r 5 % o n l y i n a l l columns).

To o b t a i n a good e f f i c i e n c y on wide-

b o r e columns, one must work v e r y c l o s e t o t h e o p t i m a l g a s v e l o T h i s i s n o t t h e c a s e f o r n a r r o w b o r e columns.

city. 4.2.

R e s i s t a n c e t o mass t r a n s f e r i n t h e l i q u i d p h a s e i s cont r o l l i n g h.

F o r low B - v a l u e s o r h i g h f i l m t h i c k n e s s e s , t h e CL t e r m no l o n g e r c a n be i g n o r e d .

Depending on t h e d i f f u s i t i v i t y of t h e s o l u t e s i n

t h e s t a t i o n a r y p h a s e (DL) and t h e f i l m t h i c k n e s s ( d f ) I CL becomes more i m p o r t a n t and f i n a l l y i s c o m p l e t e l y g o v e r n i n g h

The min' chromatographic c h a r a c t e r i s t i c s of 5 pm p o l y d i m e t h y l s i l o x a n e (RSL 1 6 0 ) f i l m s i n 0.32 mm columns h a s b e e n d e s c r i b e d ( r e f . 5 ) .

E x p e r i m e n t a l h i c u r v e s a r e r e p r e s e n t e d i n F i g . 4A f o r n i t r o g e n and hydrogen f o r o c t a n e ( k = 1 0 . 5 ) and i n F i g . 4B f o r nonane (k = 20).

The TZ-;

c u r v e s a r e shown i n F i g . 4 C .

-

T a b l e 2 l i s t s t h e measured hminl u

and c o r r e s p o n d i n g TZ v a l u e s . OP t From t h e s e d a t a t h e f o l l o w i n g c o n c l u s i o n s c a n be drawn :

-

hmin

i s i n b o t h c a s e s ( n i t r o g e n and h y d r o g e n ) much h i g h e r t h a n

f o r t h i n f i l m columns where hmin i s 0 . 2 9 f o r k = 10 and 0 . 3 0 f o r k = 20.

The e f f i c i e n c y i s o n l y s l i g h t l y a f f e c t e d by t h e

c a p a c i t y r a t i o f o r t h i n f i l m columns.

The c h r o m a t o g r a p h i c p e r -

formance o f t h e 5 p m column compared t o c l a s s i c a l c a p i l l a r y columns i s o n l y 5 0 % f o r k = 10 u s i n g n i t r o g e n ] 32 % u s i n g h y d r o g e n ] 65 %

f o r k = 2 0 u s i n g n i t r o g e n ] and 43 %

k = 2 0 u s i n g hydrogen.

capacity r a t i o .

f o r k = 10 for

Moreover hmin d e c r e a s e s w i t h i n c r e a s i n g

The c h r o m a t o g r a p h i c p e r f o r m a n c e d a t a do n o t

r e f l e c t t h e r e a l v a l u e o f t h e column f o r p r a c t i c a l c a p i l l a r y GC.

The TZ-value p e r m e t e r f o r a normal f i l m c a p i l l a r y column

is 2 -2.1

TZjm;

f o r t h e t h i c k f i l m c a p i l l a r y column a t t h e o p t i -

mal g a s v e l o c i t i e s t h i s v a l u e i s 1 . 4 - 1 . 5 (70 %

-

(56 % ) .

TZ/m

f o r nitroqen

c o r r e s p o n d i n g t o r e f . 1) and 1 . 1 - 1 . 2 TZ/m f o r hydrogen

; (15 - 2 0 opt r e d u c e s t h e TZ-value

Compared t o n i t r o g e n , u s e o f hydrogen a t

c m / s f o r the capacity r a t i o s considered !)

t o 8 0 % , b u t t h e a n a l y s i s t i m e i s s t i l l roughly 2.5 t i m e s s h o r -

ter.

However, F i g . 4C a l s o r e v e a l s t h a t t h e e f f i c i e n c y f o r n i -

f o r hydrogen i s a p p r o x i m a t e l y e q u a l t o t h e t r o g e n a t t h e i~ opt e f f i c i e n c y f o r hydrogen ( c r o s s i n g p o i n t o f t h e two c u r v e s ) .

389 I f analysis t i m e

Therefore nitrogen surely i s the b e s t choice.

i s i m p o r t a n t , t h e n hydrogen must be f a v o r e d above 2 0 c m / s

-

opt

i s roughly half t h e

rier gases.

-

opt

!

o f t h i n f i l m columns f o r b o t h c a r -

The s l o p e s of t h e c u r v e s a r e o f c o u r s e much s t e e p e r t h a n f o r t h i n f i l m columns b u t , c o n t r a r y t o t h i n f i l m c o l u m n s , t h e c u r v e s The s l o p e s o f t h e c u r v e s a r e

f l a t t e n with increasing k values !

c o m p l e t e l y g o v e r n e d by t h e CL c o n t r i b u t i o n .

12'

H'

U'

Imnl.

Iml.

C

22

1

f'

L-

1.4-

7'

I l -

.-

10-

1.2-

2-

10-

10-

0.8-

8-

0.6-

0 6-

0 L-

0.A-

0.2-

c.2-

o

o

m

x)

0

Y I - -

ir Icm/uo I

0 Icm/uc I

0 lUn/Ul

Fig. 4

.Hz

A. h i p l o t f o r k = 10

B. h i p l o t f o r

C. TZ;

p l o t C8-Cg

k = 20

TABLE 2 .

Experimental h

min

and

I

opt

f o r a column 15 m x 0 . 3 2 mm

N2 k

10

20

10

20 0.69

(mm)

0.58

0.46

0.96

u

(cm/s)

6

7

14

-

OP t

I

H2

hmin

18.5 17.5

22

TZ

FSOT.

The e f f i c i e n c y d r o p i n f u n c t i o n o f i n c r e a s i n g f i l m t h i c k n e s s i s f u r t h e r i l l u s t r a t e d by t h e c o m p a r i s o n ( F i g . 5 ) of t h e TZ-value (C12-C13)

d e t e r m i n e d a t a f i x e d f l o w r a t e o f 2 m l min-'

hydrogen

f o r a s e r i e s o f 2 0 m x 0 . 5 mm columns c o a t e d w i t h d i f f e r e n t f i l m thickness (0.1, 0.2,

0.4,

0.6,

1.2,

2 and 3 p m ) .

To o b t a i n t h e

390 same c a p a c i t y f a c t o r s on a l l columns t h e a n a l y s i s t e m p e r a t u r e was a d j u s t e d a c c o r d i n g t o t h e p r i n c i p l e o f an i n c r e a s e o f t h e f i l m t h i c k n e s s i s doubled.

5

15OC i f

As c a n s e e n from F i g . 5 , t h e s e p a -

r a t i o n power i s h a l v e d g o i n g from a 0.1 p m f i l m t o a 3 pm f i l m .

3

1

0.1 0.2

1

0.8 1

1

I

I

1.2

2

3

b

dflpl

F i g . 5 . TZ v e r s u s d f f o r 2 0 m x 0.5 mm OV-1 columns. The main a d v a n t a g e o f t h i c k f i l m columns l i e s i n t h e i r h i g h sample capacity. To have an i d e a o f t h e sample c a p a c i t y o f t h e columns, w e cons i d e r e d maximal sample c a p a c i t y a s b e i n g t h i s amount o f p r o d u c t f o r which ,the peak w i d t h a t h a l f peak h e i g h t i n t h e a s c e n d i n g p a r t o f t h e c u r v e ( a ) i s t w i c e t h e peak w i d t h i n t h e d e s c e n d i n g p a r t o f t h e c u r v e (b)

.

This r e s u l t e d i n Fig. 6 .

The sample c a p a c i t y e x p o n e n t i a l l y i n c r e a s e s w i t h i n c r e a s i n g film thickness.

The f a c t t h a t t h e c a p a c i t y f o r d o d e c a n e ( k

2

6)

i s h i g h e r t h a n f o r t r i d e c a n e (k 2 11) c a n n o t be e x p l a i n e d and i s i n c o n t r a d i c t i o n w i t h t h e o r y ( e q u a t i o n 1 2 ) . The a p p l i e d c r i terium of course only i s a r b i t r a r i l y s e l e c t e d . F i g . 6 , c l e a r l y i l l u s t r a t e s t h a t t h e maximal sample c a p a c i t y f o r a 0.1 p m column

i s a f a c t o r 100 lower t h a n f o r t h e 3 p m column. 0 . 4 p m w i d e b o r e columns however s t i l l have a sample c a p a c i t y above 100 ng which i s o f t e n enough f o r t h e c o m b i n a t i o n CGC-hyphenated t e c h n i q u e s .

391 df

Or1 u

a/b C12=2

a/b C13=2

n9

ng

70

35

Or2 P

120

80

0,4 u

190

125

0 , 8 IJ

3 80

24 0

1,2 P

500

300

2 , o IJ

1900

1200

3,O

7600

5500

p

c F i g . 6 . Sample c a p a c i t y i n f u n c t i o n of t h e f i l m t h i c k n e s s f o r 2 0 m x 0 . 5 mm OV-1 c o l u m n s . An i m p o r t a n t p a r a m e t e r i n t h e G o l a y e q u a t i o n w h i c h h a s n o t

been c o n s i d e r e d so f a r i s t h e d i f f u s i t i v i t y i n t h e l i q u i d p h a s e . F o r t h i c k f i l m c o l u m n s t h i s c a n be a v e r y i m p o r t a n t p a r a m e t e r a s i l l u s t r a t e d i n Fig. 7.

I

m Fig.

I

20

I

30 cm/nc

I

u) ij

7 . I n f l u e n c e o f DL o n h .

392 The computer r e c o n s t r u c t e d h;

p l o t s o f t h e f o l l o w i n g columns were

recorded. 1. p o l y d i m e t h y l s i l o x a n e 11

2.

(RSL 1 6 0 )

0.32 mm I . D .

0,

0 . 5 3 mm I . D .

1,

3. polyphenylmethylsiloxane (RSL 300) 0 . 3 2 mm I . D . I,

4.

0 . 5 3 mm I . D .

11

-

3 um 3 pm 3 pm 3 pm

The d a t a u s e d f o r t h e c a l c u l a t i o n s were : DG : 0.10 cmL/sec ( n i t r o g e n ) , k = 10,- DL (diMe) 3 . 3 2

c m /sec.

c m 2 /sec a n d DL ( $ M e )

3.8

low7

The DL v a l u e s were measured from e x p e r i m e n t a l c u r v e s ,

p e r f e c t l y f i t t i n g t h e o r e t i c a l curves ( r e f . 5 , 6 ) . In t h i s series, t h e B-term

i s e q u a l f o r t h e f o u r columns, t h e

CG t e r m i s e q u a l f o r column one and t h r e e a n d f o r column two and

f o u r w h i l e t h e CL term i s e q u a l f o r column o n e and two a n d f o r c o lumn t h r e e and f o u r .

The d i f d u s i o n i n t h e p o l y p h e n y l m e t h y l s i l o -

xane i s so s l o w t h a t t h e e f f i c i e n c y i s v e r y l o w , e s p e c i a l l y a t flow rates l a r g e r than t h e optim al ones. Moreover, due t o t h e l a r g e CL term t h e e f f i c i e n c y o f t h e 0 . 5 3 mm a n d t h e 0 . 3 2 mm column a r e n e a r l y e q u a l . T h i s i s a c l e a r i l l u s t r a t i o n o f a v e r y low e f f i c i e n c y p h a s e f o r t h i c k f i l m columns w h a t e v e r t h e i n t e r n a l d i a m e t e r o f t h e column.

F o r polyphenylme-

t h y l s i l o x a n e s s u c h a s RSL 300, 1 p m i s t h e h i g h e s t u s e f u l f i l m thickness.

Columns w i t h f i l m t h i c k n e s s o f 0.1 t o 0 . 3 um e x h i b i t

t h e same e f f i c i e n c y a s p o l y d i m e t h y l s i l o x a n e columns

(ref. 7,8).

5 . APPLICATIONS The s e l e c t i o n c r i t e r i a c a n b e s t b e i l l u s t r a t e d w i t h some a p plications.

The u l t i m a t e g o a l b e i n g t o s e p a r a t e t h e compounds

o f i n t e r e s t , t h e column c h o i c e c a n b e b a s e d o n t h e e f f i c i e n c y , o n t h e s e l e c t i v i t y o f t h e s t a t i o n a r y phase o r on t h e o p t i m i z a t i o n o f t h e c a p a c i t y f a c t o r s o f t h e compounds.

The o p t i m i z a t i o n o f

a l l t h r e e parameters of c o u r s e i s t h e b e s t approach.

5.1.

A n a l y s i s of n a t u r a l g a s .

Due t o t h e v o l a t i l i t y o f t h e compounds p r e s e n t i n n a t u r a l g a s , a t h i c k f i l m c a p i l l a r y column i s r e q u i r e d . "

a

given

Based upon t h e r u l e

m a t e r i a l b e s t d i s s o l v e s i n a l i k e one

'I,

an a p o l a r

phase such as polydim ethyls iloxane i s t o be p r e f e r r e d .

Fig.

6

shows t h e a n a l y s i s o f n a t u r a l g a s on a 30 m x 0 . 3 2 mm FSOT column c o a t e d w i t h a 5 rlm l a y e r of RSL 1 6 0 .

RSL 1 6 0 i s a p o l y d i m e t h y l -

siloxane w i t h a s e l e c t e d v i s c o s i t y , allowing t h e p r e p a r a t i o n of t h i c k f i l m columns.

The a n a l y s i s w a s p e r f o r m e d i s o t h e r m a l l y a t

3 93

55OC w i t h a n i n l e t p r e s s u r e o f 0 . 1 7 5 kg/cm2 n i t r o g e n .

The p r e -

v i o u s d i s c u s s i o n h a s shown t h a t f o r s u c h c o l u m n s , n i t r o g e n i s t h e b e s t carrier gas. s e p a r a t i o n can

The t o t a l a n a l y s i s t i m e i s 10 m i n u t e s .

a l s o b e o b t a i n e d on widebore columns.

Good

Fig. 9

shows t h e a n a l y s i s o f n a t u r a l g a s on a 100 m x 0 . 7 5 nun I.D. g l a s s column w i t h a f i l m t h i c k n e s s o f 1 . 2 v m OV-1.

The a n a l y s i s o f t h e

medium v o l a t i l e s i s e m p h a s i z e d i n t h i s a p p l i c a t i o n .

C c3

I F i g . 8. A n a l y s i s o f n a t u r a l g a s . column 30 m x 0 . 3 2 mm FSOT 5 pm RSL 1 6 0 . i s o t h e r m a l a t 55OC carrier gas nitrogen - i n l e t p r e s s u r e 0.175 kg/cm2 s p l i t i n j e c t i o n 1 ~ 1 1( 1 : 4 0 0 ) 5.2.

J

Fig. 9. Analysis o f n a t u r a l gas. column 100 m x 0 . 7 5 mm g l a s s 1 , 2 u m OV-1 t e m p e r a t u r e programmed 3OoC t o 5OoC a t l 0 C min-l carrier g a s hydrogen - i n l e t p r e s s u r e 0 . 1 kg/cm2 d i r e c t i n j e c t i o n 1 ~1

Analysis of f r e e f a t t y acids.

The a n a l y s i s o f f r e e f a t t y a c i d s i n w a t e r , i n b e v e r a g e s , f o r t h e i d e n t i f i c a t i o n of bacteria etc. i s an important a n a l y s i s .

Free

f a t t y a c i d s c a n be a n a l y s e d on n i t r o t e r e p h t a l i c a c i d d e r i v a t i z e d p o l y e t h y l e n e g l y c o l s s u c h a s S u p e r o x FA o r A T 1 0 0 0 ( r e f . 9 ) .

Fig.

10 shows t h e s e p a r a t i o n o f t h e f r e e f a t t y a c i d s f r o m a c e t i c a c i d

t o d e c a n o i c a c i d on a 2 0 m x 0 . 3 2 mm FSOT column c o a t e d w i t h a f i l m t h i c k n e s s o f 0 . 2 5 urn S u p e r o x FA.

The c r i t i c a l p a i r i n t h i s

s e p a r a t i o n i s p r o p i o n i c a c i d (C3) and i s o b u t y r i c a c i d ( i C 4 ) and

394

t h e p a i r i s i n f a c t o v e r - r e s o l v e d o n t h e n a r r o w b o r e column.

By

s h o r t e n i n g t h e column, t h e a n a l y s i s t i m e c a n be d r a s t i c a l l y reduced. Fig.

A n o t h e r a p p r o a c h i s t h e u s e of a s h o r t w i d e b o r e column.

11 shows t h e a n a l y s i s of t h e same compounds o n a 10 m x

0 . 5 3 nun FSOT column c o a t e d w i t h 0 . 5 p m S u p e r o x FA.

The c r i . t i c a l

p a i r i s b a s e l i n e s e p a r a t e d . The a d v a n t a g e of t h i s s e l e c t i o n i s mainly t h a t automation i s f a c i l i a t e d .

The a n a l y s i s of F i g .

was r u n w i t h a C a r l o E r b a on-column a u t o s a m p l e r AS-550,

11

giving us

s t a n d a r d d e v i a t i o n s on a b s o l u t e p e a k a r e a of below 1 % and on p e r Such good d a t a c o u l d c e n t a g e s o f below 0 . 4 8 f o r t w e n t y s a m p l e s . n o t b e o b t a i n e d on a narrowbore column.

"C5

iC6

m

m m

'6

t h

m

I F i g . 10. F r e e f a t t y a c i d s on a n a r r o w b o r e column column : 2 0 m x 0 . 3 2 mm FSOT Superox FA CB - t e m p e r a t u r e programmed 100°C t o 15OoC a t 2OC min-1 - f l o w r a t e : 2 ml min-l H 2 - s p l i t i n j e c t i o n : 2 10 ng on t h e column

-

F i g . 11. F r e e f a t t y a c i d s on a w i d e b o r e column - column : 10 m x 0 . 5 3 mm FSOT Superox. FA CB - t e m p e r a t u r e programmed i n j e c t i o n 6OoC b a l i s t i c a l l y t o 100°C 2OC min-1 t o 1 4 O o C - f l o w r a t e : 4 m l min-1 H~ - a u t o m a t i c on column i n j e c t i o n 1 p l sample r a n g e 5 t o 50 ppm of f a t t y a c i d s

395

I f f o r m i c a c i d h a s t o b e a n a l y s e d as w e l l , h o t w i r e d e t e c t i o n

i s a p p l i e d . The s e n s i t i v i t y of t h e h o t w i r e d e t e c t o r i s r o u g h l y 100 t i m e s l e s s t h a n FID d e t e c t i o n and w e t h e r e f o r e need h i g h s a m p l e c a p a c i t y columns.

Formic a c i d i s e l u t i n g between a c e t i c a c i d

and p r o p i o n i c a c i d and t h e s e p a r a t i o n i s n o t so c r i t i c a l a s f o r Due t o t h e l o w s e n s i t i v i t y of t h e

p r o p i o n i c and i s o b u t y r i c a c i d .

h o t w i r e d e t e c t o r , w e have t o n o t e t h a t v e r y s m a l l t r a c e s of f o r -

m i c a c i d c a n o n l y b e a n a l y s e d by t h e c o m b i n a t i o n CGC-MS. We have s t u d i e d t h e i n f l u e n c e o f t h e f i l m t h i c k n e s s o n t h e c r i t i c a l p a i r by m e a s u r i n g t h e r e s o l u t i o n i n f u n c t i o n o f t h e f i l m thickness (Fig. 1 2 ) .

The c a r r i e r g a s f l o w r a t e was 2.5 rnl/min-'

hydrogen f o r a l l columns.

To o b t a i n t h e same c a p a c i t y f a c t o r s

and a n a l y s i s t i m e , t h e t e m p e r a t u r e was i n c r e a s e d w i t h 15°C f o r each doubling of t h e f i l m t h i c k n e s s .

A s c a n b e deduced from F i g .

1 2 t h e r e s o l u t i o n f o r a 4 pm c o a t i n g s t i l l i s s u f f i c i e n t .

The

sample c a p a c i t y i s i n c r e a s e d by a t l e a s t a f a c t o r 100 o f f e r i n g no d e t e c t i o n p r o b l e m s u s i n g h o t w i r e d e t e c t i o n .

We however have t o emphasize t h a t i f h i g h sample c a p a c i t y i s n o t r e q u i r e d , w e p r e f e r t h i n n e r f i l m columns.

The e f f i c i e n c y i s

h i g h e r , t h e a n a l y s i s t i m e i s r e d u c e d , t h e t e m p e r a t u r e c a n be l o w e r e d which o f t e n i s needed f o r t h e a n a l y s i s of t h e r m o l a b i l e compounds and moreover t h e c a p a c i t y s t i l l i s h i g h enough f o r m o s t applications.

A c o n t r a argument c a n be t h a t t h e i n e r t n e s s o f a

column i n c r e a s e s w i t h i n c r e a s i n g f i l m t h i c k n e s s .

Deactivation of

FSOT t u b i n g however i s d e v e l o p e d t o s u c h a n e x t e n t t h a t w e h a r d l y o b s e r v e any d i f f e r e n c e between t h i c k and t h i n f i l m columns i n t h i s respect. 5.3.

A n a l y s i s o f GC r e f e r e n c e FAME m i x t u r e s .

F o r t h e a n a l y s i s of o i l s and f a t s , s t a n d a r d r e f e r e n c e f a t t y a c i d m e t h y l e s t e r (FAME'S) m i x t u r e s a r e a v a i l a b l e t h r o u g h t h e Amer i c a n O i l Chemistry S o c i e t y .

The c o m p l e t e s e p a r a t i o n r e q u i r e s

p h a s e s o f h i g h p o l a r i t y and s e l e c t i v i t y .

RSL 1000 i s a p o l y b i s -

c y a n o p r o p y l s i l o x a n e w i t h gum p r o p e r t i e s h a v i n g t h e same p o l a r i t y and s e l e c t i v i t y a s S i l a r 1 O C ( r e f . 1 0 ) .

The a n a l y s i s o f AOCS

m i x t u r e 18813 on a 10 m x 0 . 5 3 mm FSOT column c o a t e d w i t h 0 . 5 pm RSL 1000 i s r e p r e s e n t e d i n F i g .

13.

A l l the f a t t y acids present

i n t h e s a m p l e , a c c o r d i n g t h e AOCS s p e c i f i c a t i o n s , a r e w e l l s e p a r a t e d i n 2 0 minutes.

On a p a c k e d S i l a r 1 O C column t h e a n a l y s i s

t i m e exceeds 4 0 minutes.

A n a l y s i s on a n a r r o w b o r e column e . g .

396

11 2

I

3

logdflnml+

F i g . 12. I n f l u e n c e o f t h e f i l m t h i c k n e s s o n t h e r e s o l u t i o n o f propionic acid : isobutyric acid. 2 5 m x 0.25 mm FSOT column c o a t e d w i t h 0.3 iJm RSL 1000 however r e v e a l s t h a t some e x t r a compounds are p r e s e n t i n t h e s t a n d a r d mixtures.

F i g . 1 4 shows t h e sdme a n a l y s i s a s on t h e w i d e b o r e co-

lumn and w e c a n o b s e r v e t h e s e p a r a t i o n o f t h e C22:l FAME i n i t s The same h o l d s f o r re-

t r a n s ( p e a k 9 ' ) and c i s i s o m e r ( p e a k 9 ) . f e r e n c e m i x t u r e AOCS 18803 ( F i g . 1 5 ) .

The compound a c c o u n t i n g

f o r 35.0 0 namely m e t h y l o l e a t e ( p e a k 4 amounts of m e t h y l e l a i d a t e ( p e a k 4 '

-

-

Cis: 1 c i s ) c o n t a i n s s m a l l

C18:l t r a n s ) .

The s e p a r a t i o n

o f c i s - t r a n s i s o m e r s o f f a t t y a c i d s a s w e l l as t h e s e p a r a t i o n o f i s o m e r s d i f f e r i n g i n t h e p o s i t i o n of d o u b l e bounds ( r e f . 11) req u i r e s b o t h t h e s e l e c t i v i t y of a polybiscyanopropylsiloxane p h a s e and t h e e f f i c i e n c y o f a n a r r o w b o r e column.

5.4.

S e p a r a t i o n of b i o l o g i c a l m a r k e r s i n p e t r o l e u m p r o d u c t s .

The b a s e l i n e s e p a r a t i o n of t h e d i a s t e r e o i s o m e r s of n o r p r i s t a n e , p r i s t a n e and p h y t a n e i s d i f f i c u l t t o r e a l i z e .

High e f f i c i e n c y ,

h i g h s e l e c t i v i t y and o p t i m i z e d c a p a c i t y f a c t o r s have t o b e comb i n e d ( r e f . 12).

F i g . 1 6 compares t h e r e l e v a n t r e g i o o f t h e ana-

l y s i s of d i e s e l o i l o n a 25 m x 0.25 mm I.D. and on a 25 m x 0.10

mrn I . D .

FSOT column b o t h c o a t e d w i t h OV-1.

The a n a l y s i s i n b o t h

c a s e s was o p t i m i z e d t o g i v e o p t i m a l r e s o l u t i o n .

The 250.000 t h e -

397

I

I Bmin

F i g . 1 3 . A n a l y s i s AOCS s t a n d a r d 1 8 8 1 3 . Column 10 m x 0 . 5 3 mm FSOT RSL 1000. I s o t h e r m a l a t 167OC, f l o w r a t e 2 . 0 m l min-’

H2

rDCS 1880: 1

AOCS

3

18813

>

*

30min

r-

,IL

398 C o m p o s i t i o n of t h e AOCS s t a n d a r d s . Chain 18813 Ci4: 0 C16:O C18:O C18: 1 C18: 2 C18: 3 c20: 0

c22:o c22: 1 C24:O

item

p e a k no

Methyl Methyl Methyl Metby1 Methyl Methyl Methyl Methyl Methyl Methyl

myristate palmi t a t e stearate oleate linoleate linoienate arachidate behenate erucate lignocerate

Methyl Methyl Methyl Methyl Methyl Methyl

palmitate stearate

% by weight

1.o

1 2 3 4 5 6 7 8

4 .O 3 .O 45 .o 1 5 .O 3.0 3.0 3.0

9 10

20.0 3 .O

2 3 4 5 7 6

6 .O 3 .O 35 .O 50.0 3 .O 3.0

18803 C16:O

C18:O C18: 1 C18: 2 Ci8: 3 c20: 0

oleate linoleate linolenate arachidate

o r e t i c a l p l a t e s of t h e 0.10 mm column are n e e d e d f o r b a s e l i n e separation.

Compared t o t h e 0.10 mm column, t h e s e p a r a t i o n on t h e

0 . 2 5 mm column i s n o t b a d a t a l l .

T h i s i s a consequence o f t h e

f a c t t h a t r e s o l u t i o n ( e q u a t i o n 1) i s p r o p o r t i o n a l t o t h e s q u a r e r o o t o f t h e p l a t e number. On t h e o t h e r h a n d , t h e s e p a r a t i o n of t h e monomethyl d i b e n z o thiophenes,

important markers t o e l u c i d a t e o i l s p i l l a g e , can o n l y

be p e r f o r m e d on a n a r r o w b o r e column c o a t e d w i t h a polyphenylme-

thylsiloxane

( R S L 300) a s shown i n F i g .

17 ( r e f . 1 3 ) .

The s e p a -

r a t i o n i s f e a s i b l e on 0 . 5 mm columns o n l y i f t h e l e n g t h i s i n c r e a s e d t o 4 0 - 5 0 m.

However d u e t o e x c e s s i v e l y l o n g r e t e n t i o n

t i m e s , t h e s e n s i t i v i t y of t h e s y s t e m i s l o w e r e d t o s u c h an e x t e n t t h a t t h e d e t e c t i o n o f traces w i t h flame photometric d e t e c t i o n can n o t e a s i l y be r e a l i z e d .

399 16

I

c17

3

F i g . 1 6 . A n a l y s i s of b i o l o g i c a l m a r k e r s i n d i e s e l o i l . Column A : 25 m x 0.25 m FSOT OV-1. t e m p e r a t u r e programmed 6OoC t o 2OO0C a t 0.2OC m i n - l . i n l e t p r e s s u r e 0.65 a t m H2 Column B : 25 m x 0.10 nun FSOT OV-1. t e m p e r a t u r e p r o g r a m e d 60°C t o 2OO0C a t 0.2OC m i n - l . i n l e t p r e s s u r e 4 a t m H2 P e a k s : 1. n o r p r i s t a n e , 2 . p r i s t a n e , 3 . p h y t a n e .

1 7 . D i b e n z o t h i o p h e n e d e r i v a t i v e s . Column 15 m x 0 . 3 2 mm FSOT; 0 . 1 5 p m f i l m of RSL 300. I n j e c t i o n o n column. Temperature

Fig.

50' t 120' - 3"/min-280°. p h e n e isomers.

Peaks : 1.2.3.4.

methyldibenzothio-

400 6 . CONCLUSION.

The p h i l o s o p h y o f column s e l e c t i o n i n r e s p e c t t o i n t e r n a l d i a -

meter a n d f i l m t h i c k n e s s c a n be summarized a s f o l l o w s . The p o t e n t i a l o f w i d e b o r e columns i s u n d e r e s t i m a t e d .

They o f -

f e r a good a l t e r n a t i v e t o h i g h r e s o l u t i o n g a s c h r o m a t o g r a p h y i f t h e d i f f e r e n t p a r a m e t e r s are o p t i m i z e d . I.D.

Columns o f 25 m x 0 . 5 mm

o f f e r 50.000 p l a t e s a t o p t i m a l g a s v e l o c i t i e s and i f t h i s

e f f i c i e n c y i s combined w i t h a h i g h s e l e c t i v i t y most a p p l i c a t i o n s c a n be h a n d l e d .

S e v e r a l a p p l i c a t i o n s on widebore columns have

b e e n p u b l i s h e d by o u r l a b o r a t o r y i n t h e l a s t f i f t e e n y e a r s ( r e f . 14-22).

The s e l e c t i o n o f t h e s t a t i o n a r y p h a s e i n w i d e b o r e c a p i l -

l a r y g a s c h r o m a t o g r a p h y i s more i m p o r t a n t t h a n i n n a r r o w b o r e c a p i l l a r y g a s chromatography t o compensate f o r t h e i n h e r e n t lower e f f i c i e n c y .

Considering the film thickness i n our opi-

n i o n t h e columns c o m m e r c i a l l y a v a i l a b l e a r e o v e r - c o a t e d . b e t t e r r e s u l t s a r e o b t a i n e d on t h i n n e r f i l m s .

Much

The p a c k e d column

a l t e r n a t i v e , by o p e r a t i n g s h o r t w i d e b o r e t h i c k f i l m columns a t high flow rates, can without d i f f i c u l t i e s r a p i d l y be converted i n t o h i g h r e s o l u t i o n g a s chromatography ( r e f . 2 3 ) .

Therefore we

would n o t be s u r p r i s e d i f t h e n e x t s t e p i n t h e p o p u l a r i z a t i o n o f open t u b u l a r columns w i l l b e t h e i n t r o d u c t i o n o f 25 m x 0 . 5 3 mm columns w i t h f i l m t h i c k n e s s o f 0 . 3

-

0.5

pm.

I n t h e p e r i o d 1972-

1975 w e d i s c u s s e d t h e i r a d v a n t a g e s a n d d i s a d v a n t a g e s t h o r o u g h l y

( r e f . 1 4 ~ 1 5 ) . Maybe t h e p h i l o s o p h y a d v a n c e d t h e n , w i l l be accepted i n t h e near future. I t indeed looks l i k e a potpouri.

For t h e 12th I n t e r n a t i o n a l

Symposium on Chromatography h e l d i n Baden-Baden i n 1 9 7 8 , w e s u b m i t t e d a p a p e r e n t i t l e d : P o s s i b i l i t i e s a n d l i m i t a t i o n s o f wideb o r e c a p i l l a r y columns.

The p a p e r was n o t a c c e p t e d b e c a u s e a t

t h a t t i m e it was considered o l d fashioned.

Widebore columns

i n d e e d have b e e n u s e d e x t e n s i v e l y by t h e p i o n e e r s o f CGC.

In

1984 however, a t t h e 1 5 t h I n t e r n a t i o n a l Symposium on Chromatography h e l d i n N u r n b e r g , s e v e r a l c o n t r i b u t i o n s e m p h a s i z e d t h e p o s s i b i l i t i e s o f widebore columns, a l t h o u g h o n l y w i t h t h i c k f i l m coatings. I t however i s o b v i o u s , t h a t t h e f u t u r e b e l o n g s t o t h e u l t r a

narrowbore c a p i l l a r y columns.

Making u l t r a n a r r o w b o r e c o l u m n s

i s no p r o b l e m b u t i n s t r u m e n t s t o f u l l y e x p l o i t t h e p o t e n t i a l o f

t h e s e columns a r e n o t a v a i l a b l e y e t . The r e a l w o r k h o r s e i n CGC i s , a n d w i l l m o s t p r o b a b l y r e m a i n

401 f o r a long period,

t h e n a r r o w b o r e column o f 0 . 2 5 - 0 . 3

mm I . D .

w i t h a l e n g t h of a p p r o x i m a t e l y 2 0 m a n d f i l m t h i c k n e s s v a r y i n g from 0.05 t o 5

pm.

ACKNOWLEDGEMENTS. I t h a n k t h e " N a t i o n a a l Fonds v o o r W e t e n s c h a p p e l i j k O n d e r z o e k

N.F.W.O.",

-

t h e " I n s t i t u u t t o t Aanmoediging v a n h e t W e t e n s c h a p p e -

l i j k O n d e r z o e k i n N i j v e r h e i d e n Landbouw

-

I.W.O.N.L."

and t h e

" M i n i s t e r i e voor Wetenschapsbeleid" f o r f i n a n c i a l s u p p o r t t o t h e laboratory. REFERENCES. 1

2.

3 4. 5. 6 7 8 9 10 11

12 13. 14 15 16

17 18 19 20 21

22

23

G o l a y , i n V . J . C o a t e s , H.J. N o e b e l s a n d 1 . S F a q e r s o n . ( E d i t o r s ) , G a s C h r o m a t o g r a p h y , Academic P r e s s , N e w Y o r k , 1 9 5 8 , p.1. M.J.E. Golav. i n D. Destv . ' .( E d i t o r ) , G a s C h r o m a t o g r a p h y , B u t t e r w o r t h s , London, 1 9 5 8 , p . 3 6 . L.S. E t t r e , C h r o m a t o g r a p h i a , 8 ( 1 9 8 4 ) 477 K . S c h u t j e s , PhD d i s s e r t a t i o n E i n d h o v e n U n i v e r s i t y o f Technol o g y , 1984. P . S a n d r a , I . Temmerman a n d M V e r s t a p p e , HRC & C C , 6 ( 1 9 8 3 ) 501. F. D a v i d , M . P r o o t , P . S a n d r a a n d M . V e r z e l e , P r o c . 6 t h I n t . Symp. Cap. Chrom., P. S a n d r a E d i t o r ) , H u e t h i g V e r l a g , H e i d e l b e r g , 1985, i n p r e s s . E . V a n l u c h e n e , D . V a n d e k e r c k h o v e , P . S a n d r a a n d F. D a v i d , HRC & C C , 7 ( 1 9 8 4 ) 6 4 6 . E . Geeraert a n d P. S a n d r a , HRC & C C , 7 ( 1 9 8 4 ) 431. M. V e r z e l e , P . S a n d r a and J . V e r z e l e , I n t . Lab. March 1 9 8 3 , p. 49. D. Duquet, H . V e r l e t , D. Vanbeneden, K . N o t t e and J . V e r z e l e , P r o c . 6 t h I n t . Symp. Cap. Chrom., P . S a n d r a ( E d i t o r ) , H u e t h i g V e r l a g , H e i d e l b e r g , 1985, i n p r e s s . P . S a n d r a , M . V e r s t a p p e a n d M . V e r z e l e , HRC & C C , 1 ( 1 9 7 8 ) 28. M. P r o o t , F. David, P. S a n d r a and M . V e r z e l e , P r o c . 6 t h I n t . Symp. Cap. Chrom., P . S a n d r a ( E d i t o r ) , H u e t h i g V e r l a g , Heidelb e r g , 1985, i n p r e s s . F . B e r t h o u , Y. D r e a n o and P . S a n d r a , HRC & C C , 1 2 ( 1 9 8 4 ) 679686. M . V e r z e l e , M. V e r s t a p p e , P . S a n d r a , E . V a n l u c h e n e a n d A. Vuye, J . Chrom. S c i . , 10 ( 1 9 7 2 ) 6 6 6 . P . S a n d r a , M. V e r z e l e a n d E . V a n l u c h e n e , C h r o m a t o g r a p h i a 8 (1975) 499. P . S a n d r a a n d M. V e r z e l e , P r o c . 1 5 t h EBC C o n g r e s s , N i c e 1 9 7 5 , E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company, Amsterdam, 1 9 7 5 , p . 1 0 7 . M . V e r z e l e a n d P. S a n d r a , J . C h r o m a t o g r . , 1 5 8 ( 1 9 7 8 ) 111. T . S a e e d , G . R e d a n t a n d P . S a n d r a , HRC & C C , 2 ( 1 9 7 9 ) 7 5 . E . W a u t e r s , P . S a n d r a and M . V e r z e l e , J . C h r o m a t o g . , 1 7 0 ( 1 9 7 9 ) 1 2 5 and 1 3 3 . P . S a n d r a , T a l a t S a e e d , G. R e d a n t , M . G o d e f r o o t , M . V e r s t a p p e a n d M . V e r z e l e , HRC & C C , 3 ( 1 9 8 0 ) 1 0 7 . M . G o d e f r o o t , M . Van R o e l e n b o s c h , M . V e r s t a p p e , P . S a n d r a a n d M. V e r z e l e , HRC & C C , 3 ( 1 9 8 0 ) 3 3 7 . M. V e r z e l e , G . R e d a n t , S . Q u r e s h i a n d P . S a n d r a , J . C h r o m a t o g . , 199 ( 1 9 8 0 ) 105. H e w l e t t P a c k a r d P u b l i c a t i o n N o . 43-5953-1735, 1983. M.J.E.

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403

E.D. Katz, K. Ogan and R.P.W. Scott The Perkin-Elmer Corporation Norwalk, Ct. 06856 The heart of the chromatograph is the column where the separation takes place. Two processes must occur simultaneously and progressively during the development of the chromatogram in order to achieve a separation. These two processes proceed more or less independently of one another. Firstly, the individual solutes are moved spatially apart as a result of the different molecular interactions that take place between the molecules of the two phases and those of the solutes. Secondly, by careful column design, the migrating solute bands are kept sufficiently narrow such that each band is eluted discretely. Unless exclusion processes are being employed to aid in the separation, the movement of the peaks apart in the column is solely controlled by the phase system selected. The control of peak dispersion, however, will depend on the physical characteristics of the column, such as length, particle size, etc. It follows that to design the optimum column for a given separation it is necessary to have a valid and experimentally proved theory of chromatography and in particular an explicit equation that describes column dispersion. Chromatography theory has developed extensively since the invention of gas chromatography (GC) nearly thirty years ago. In 1959, Purnell (1) derived the equation to calculate the number of theoretical plates necessary to effect a given separation in GC. Shortly after, Purnell and Quinn (Z), Desty and Goldup ( 3 ) and Scott and Hazeldean (4) developed equations to calculate the total analysis time required for GC separations. Later, Snyder ( 5 ) , Martin et a1 ( 6 ) , Halasz et a1 ( 7 ) and Guiochon (8) developed similar equations for liquid chromatographic (LC) separations. Equations that could be used to calculate the length of the column necessary to achieve a given separation were developed for GC by Scott and Hazeldean (4), and for LC by Snyder (S), Martin et al. ( 6 ) and The effect of pressure on chromatographic performance which

Halasz et al. ( 7 ) .

404 can have a major pertinence to LC was also investigated by Snyder ( 5 ) and Martin et al. ( 6 ) , and also by Guiochon ( 8 ) , Kraak et al. ( 9 ) and Knox and Saleem ( 1 0 ) . More recently, equations have been developed to calculate the minimum column radius for maximum solvent economy and maximum mass sensitivity in LC ( 1 1 , 1 2 ) and the maximum permissible response time of the detecting system ( 1 3 , 1 4 , 1 5 , 16).

The calculation of all column parameters and operating conditions is

ultimately contingent, however, on knowing the explicit equation that accurately predicts the variance per unit length of a column [more commonly known as the height equivalent t o a theoretical plate, HETP or HI from a knowledge of the mobile phase velocity. Furthermore, the explicit terms of the equation must accurately establish the interrelationship of the physical and chemical properties of the solute, phase system and column contents. Due to the fact that in GC the mobile phase is a gas and consequently compressible, while in contrast the liquids used as mobile phases in LC are only very slightly compressible, the equations describing dispersion for the two systems, although apparently similar, are quite different in detail.

This difference, although

somewhat related to the fact that solute diffusivity in a gas varies with the absolute pressure, mainly arises from the nonlinear change in mobile phase velocity that occurs along a GC column in contrast to the LC column where the mobile phase velocity is sensibly constant along the whole of its length.

The

dispersion for each system will therefore be considered separately, commencing with that for LC.

Dispersion in LC Colums The LC literature is not wanting in equations that purport to describe solute dispersion that takes place in a column.

The first HETP equation was

derived by Van Deemter et al. ( 1 7 ) in 1 9 5 6 and experimental support for the equation was published by Keulemans and Kwantes for CC ( 1 8 ) at the first Gas Chromatography Symposium held in London in the same year. However, when the equation was applied to LC, it was found that the relat.ionship predicted by Van Deemter et al. did not appear to hold, probably due to the presence of artifacts such as those caused by extra column dispersion, large amplifier time constants, etc.

Nevertheless, this poor agreement between theory and experiment

provoked a number of workers in the field to develop alternative HETP equations in the hope that a more exact relationship between HETP and mobile phase linear velocity

u

could be obtained that would be compatible with experimental data. In

1 9 6 1 , Giddings ( 1 9 ) produced an HETP equation, of which the Van Deemter equation

was a special case.

Giddings was dissatisfied with the Van Deemter equation

insomuch as it predicted a finite contribution to dispersion independent of the solute diffusivity in the limit of zero mobile phase linear velocity, which appeared to him to be unreasonable.

Consequently, Giddings suggested that there

was a coupling term that accounted for increased diffusion that resulted from the tortuous path followed by the solvent between the particles.

However, when

the mobile phase velocity was sufficiently high, the equation simplified to the Van Deemter equation as, at practical mobile phase velocities, the other functions in the equation were similar to those of Van Deemter.

The coupling

function introduced by Giddings was not strictly a term that described a multipath effect, although at high values of

u,

the function extrapolated to a

constant, independent of the mobile phase velocity. The dispersion phenomenon that the coupling function described was more comparable to a resistance-to-mass transfer contribution arising solely within the interparticulate voids in the column packing.

In fact, a dispersion effect independent of solvent velocity is

not necessarily unacceptable.

There must be a range of paths of different

length that a molecule can take when passing through the interstices of a packed bed and this range of pathlengths must lead to dispersion that is independent of the solvent velocity.

Nevertheless, the constant in the Van Deemter equation

independent of mobile phase velocity could include both the multipath term, as such, together with the limiting value of the coupling term of Giddings at high linear velocities. The next HETP equation to be developed was that of Huber and Hulsman in 1967 ( 2 0 ) .

These authors introduced a term similar to the coupling term of

Giddings which also allowed the dispersion factor due to the multipath effect to become zero,

at zero mobile phase velocity.

different model from

These authors, however, used a

that of Van Deemter or Giddings in deriving the resistance

to mass transfer in the mobile phase located solely between the particles.

They

arrived at an additional term involving the square root of the mobile phase velocity, as opposed to the linear function postulated by Van Deemter and Giddings.

However, this mobile phase mass transfer factor of Huber has a

distinct similarity to that of the coupling term of Giddings in its physical interpretation and the form of its velocity dependence.

It is difficult to

evoke a physical process that would reduce the resistance to mass transfer between the particles other than the coupling effect postulated by Giddings.

It

appears, therefore, that both the resistance-to-mass transfer effect containing the fractional power of u and the coupling term in the Huber equation could be describing the same dispersion phenomenon. During 1 9 7 2 and 1 9 7 3 , Knox and co-workers ( 2 1 , 2 2 , 2 3 ) carried out a considerable amount of work on different packing materials with particular reference to the effect of particle size on the reduced plate height of a column.

These workers produced a fourth HETP equation which was significantly

different from those mentioned previously and was developed from a curve fitting procedure applied to their fairly extensive experimental data. Consequently,

406

although empirically interesting, the equation of Knox and his co-workers is not explicit with respect to the physical and chemical properties of solvent-solute and column packing and thus cannot be used for column design. Finally, Horvath and Lin (24, 25) used yet another model to derive an HETP equation similar to that of Huber and Hulsman, but the term describing the resistance to mass transfer in the mobile phase involved the square of the cube root of the mobile phase velocity and not the square root.

Once more, the

resistance-to-mass transfer term involving the fractional power of the linear velocity in the Horvath equation was introduced to Characterize a dispersion effect similar to the coupling term of Giddings.

In fact, it may be accounting

for the same phenomenon that is described by a coupling term he also included in his equation. The five equations are as follows: Van Deemter et al. (1956) (17) H-AtBtCu Giddings (1961) (19) A t -B t c u l t E/u u Huber and Hulsman (1967) (20)

Ha-

H=-

A

1 t E/u1I2

t

2 t u

cu t h 1 / 2

Kennedy and Knox (1972) (21) H =

t

t Cu

Horvath and Lin (1976) (24) H=A 2 cU ~ ~ 2 1 3 1 t E/u1l3 u where A,B,C,D and E are appropriate constants for a given solute chromatographed on a given column and phase system. In 1983 Katz, Ogan and Scott (26) carried out a series of carefully planned experiments to identify which equation accurately described the dispersion that took place in an LC column. At first sight, it might appear adequate to test the above equations to a number of data sets of H a n d u and to identify the equation that gives the best fit. Unfortunately, in practice this is of little use as, due to the nature of the functions, all five equations would provide an excellent fit to any given experimentally derived data set provided it was obtained with adequate precision. However, all the individual terms for each equation purport to describe a specific dispersive effect; that being

all the constants for the above equations derived from a curve fitting procedure so,

must be positive and real if the dispersion effect described is to be physically significant over the mobile phase velocity range examined. Any equation that

407

did not consistently provide positive and real values for all constants would obviously not be an appropriate explicit equation to describe the dispersion effects occurring over the said range of velocities.

However, any equation that

d i d p r o v i d e a good f i t to a series of experimentally determined data sets and

did meet the requirement that all constants were positive and real w o u l d n o t u n i q u e l y i d e n t i f y t h e a p p r o p r i a t e equation for use.

The coefficients A , B, C

etc., would then have to be replaced by the explicit functions, derived from the specific theory employed, incorporating the physical properties of solvent, solute and stationary phase.

The physical properties of the solute and phase

system could then be varied in a defined manner and the change in the nature of the constants A,B,C etc. tested against the relationship predicted by the explicit functions. Katz, et al. made 750 accurate measurements of solute band dispersion, each measurement being the average of three replicates.

They examined columns packed

with particles of different diameter, solutes of different k' values and a number of mobile phases in which the solutes had significantly different diffusivities.

They found that virtually any hyperbolic or pseudo-hyperbolic

function will algebraically fit experimentally determined H, u data sets, but will not necessarily provide the correct physical interpretation of the dispersion processes that take place.

The HETP equation that most accurately

describes the relationship between H a n d u over the velocity range of 0.02 to

1.00 cm/sec for columns packed with porous silica is that of Van Deemter which, itself, appears to be a special case of the Giddings equation.

The Van Deemter

equation, in the following form, can be used with confidence in column design

where

dp is the the particle diameter u e is the linear velocity (measured from the retention time of

a fully excluded solute) Dm is the diffusivity of the solute in the mobile phase ke is the capacity factor of the solute (measurement from the retention time of fully excluded solute) and where A and 7 may vary with the quality of the packing but for a reasonably well packed column can be taken as 0.5 and 0.8; a, b and c can be taken as 0 . 3 7 , 4.69 and 4.04, respectively.

It would appear from equation ( 1 ) that there is no contribution to dispersion from the resistance to mass transfer in the stationary phase.

In

point of fact the contribution from ,he stationary phase to the overall resistance to mass transfer in an LC column does indeed appear to be small; however, it is not assumed to be zero in equation ( 1 ) .

It can be assumed,

408 however, as the contribution is small, that the diffusivity of the solute in the stationary phase D, is linearly related to the diffusivity of the solute in the mobile phase Dm; consequently, due to the nature of the Van Deemter function for the resistance to mass transfer in the stationary phase, its contribution is contained in the constant b in the quadratic function of k,.

In fact, equation

(1) was confirmed only for silica gel as the stationary phase.

It is likely,

however, that the resistance to mass transfer in the stationary phase of a reversed-phase column is also very much less than the resistance-to-mass transfer in the mobile phase.

In fact, preliminary experiments indicate that

this is true. An example of an HETP curve obtained is given in Figure 1. The precise fit of the data with the theoretical curve is clearly illustrated.

u,(cm/sec) Figure 1. H versus u curve. Partisil-10; 5 . 4 % ethyl acetate in n -hexane; benzyl acetate. Fit to Van Deemter eqn.; r = 0 . 9 9 9 6 9 9 . Dispersion in GC Columns A s opposed to LC, there are two types of column in common use in GC, the

packed column and the capillary column. Although the use of capillary columns for LC are presently being investigated, they are not yet sufficiently advanced for general use.

However, in GC it is necessary to consider two dispersion

equations, one for packed columns and one for capillary columns. The equation pertinent to capillary columns will be considered first. In 1 9 5 8 , Golay ( 2 7 ) published a theoretical treatment of peak dispersion occurring in a straight open tube.

He originally took this point of view a s a

theoretical concept, the ultimate goal being an accurate development of the dispersion equation for a packed column.

Golay's theory predicted chroma-

tographic performance for an open tube that was significantly better than the

409 experimental results obtained for packed columns at that time.

Consequently,

experiments were performed with a n open tubular column and these largely confirmed the validity of the Golay equation.

In essence, the Golay equation is

given by

B t (Cg t Cs) u H = -

where

H

is the plate height or variance per unit length

Dg is the diffusivity of the solute in the gas phase. D, is the diffusivity of the solute in the stationary phase. r is the radius of the column df is the film thickness o f stationary phase u

is the average gas velocity.

and fg(k') =

1 t 6k' t

llk2

24(1 t k')I

Equation (2) i s a subset of the Van Deemter equation in that it includes both the longitudinal diffusion and the resistance-to-mass transfer terms, but it lacks the multipath term because there are no particles in this column and hence there is n o diversity of possible pathlengths that a solute molecule can follow in passing through the column.

Furthermore, empirical factors found in

the Van Deemter equation are not present in the Golay equation because the geometry of an open tubular column lends itself to exact analytical solutions. Gases are compressible and hence there is a nonlinear change in pressure along the length o f the capillary.

Tho solute diffusivity in the gas

phase also

varies with position along the column as a consequence of the compressibility of the carrier gas.

Thus, the Golay equation is much more complex than the simple

form of equation (2) would suggest. Ogan and Scott (28) developed a modified form of the Golay equation that took into account the compressibility o f the gas, the dependence of solute diffusivity in the gas phase on pressure and the nonlinear change in velocity o f the gas along the column.

where

The equation they derived is as follows,

uo is the exi't velocity

410 Do is the diffusivity of the solute in the mobile phase at atmospheric pressure, y' is the inlet/outlet pressure ratio. and the other symbols have the meanings previously ascribed to them.

In an analogous manner the equation of Van Deemter for the packed column can also be modified to take into account the compressibility of the gas and the dependence of solute diffusivity on pressure.

The modified form of the Van

Deemter equation takes the following form,

where all the symbols have the meanings previously ascribed to them. It is now possible to use the respective dispersion equation as a basis for column design.

The design of GC columns will first be considered together with

a comparison of the performance of packed and capillary columns.

The Design of Packed and Capillary Columns The columns most commonly used in gas chromatography today are the packed and the capillary columns. The packed column was introduced by the inventors of gas chromatography, Martin and James (29), in the early 50's. The capillary column, invented by Marcel Golay, was first described at a meeting of the American Chemical Society in 1 9 5 6 (30) and the full details presented at the International Meeting on Gas Chromatography, held in Amsterdam in 1958 ( 2 7 ) . When the capillary column concept was first advanced, it was thought that it would completely replace packed columns for general gas chromatographic analysis. However, this did not happen and, in fact, has still not happened 26 years later.

During the 60's some competition developed between workers with

each extolling the virtues of their respective columns which occasionally resulted in some trivial and sometimes irrational arguments.

In fact, the

capillary columns, although capable of providing very fast analyses and very high efficiencies, had (and still have) certain disadvantages.

The early

columns exhibited adsorption properties which often impaired the separation obtained, furthermore, capillary columns in general require special injection devices which, besides being clumsy to use, can also seriously reduce the dynamic range of the chromatographic system. The introduction of the glass capillary by Desty et.al. ( 3 1 ) helped solve some of the adsorption problems and with the introduction of the flexible fused-silica columys by Dandenau (32) in 1 9 7 9 and the flexible soft-glass capillaries by Ogan et. al. ( 3 3 ) the capillary

columns improved still further. The problem of injection still remains, and it should also be noted that packed columns are still extensively used due to their simplicity and ease of operation as well as their larger sample capacity. Today the respective proponents of the two columns have become more polarized, and

411 claims are again being made that the capillary columns will completely replace packed columns in much the same way as was suggested in 1 9 5 8 .

One voice,

however, Purnell and Quinn ( 2 ) , suggested that packed columns can do many analyses as efficiently as capillary columns, but this has remained an open question since 1 9 5 8 .

It is important for the analyst to be able to choose the

right column for a particular analytical problem.

It is unlikely that any

specific device is the analyst's "Philosopher's Stone" for all applications of a particular technique and it is likely that both the capillary column and the packed column have a place of value in the analytical service laboratory.

It i s

therefore useful to compare the performance of both capillary and packed columns that have been optimally designed to effect a specific separation. For this reason the design of both capillary columns and packed columns will be discussed in terms of meeting the same chromatographic challenge.

Defining the Cbrolatographic Problem In order to design a column to effect a particular separation it is necessary to quantitatively define the chromatographic problem. Any mixture of substances to be separated can be reduced to a simple separation that involves three solutes:

if the three solutes are separated and eluted then, in almost

all cases, the complete mixture will be resolved. (Unlikely exceptions would occur where early peaks might have a very high molecular weight and consequently have unusually wide peaks ( 3 4 ) ) .

Such a chromatogram can be termed a reduced

chromatogram an example of which is given in Figure 2 .

A Figure 2 .

Reference Chromatogram for the Critical Pair

The two peaks close together represent the two solutes in the mixture that, for the chosen phase system, are eluted closest together and are termed the critical pair.

The third peak is the last eluted and determines the total analysis time.

412 Consequently, the chromatographic system has to be designed to separate the critical pair and elute the last peak in the minimum time.

This procedure

applies to both GC and LC and will now be discussed in detail.

The Key Equations The key equations used in the calculation of the optimum chomatographic parameters to effect a given separation are as follows. N =

(4(1

-

t k')/k'(a

1))2

(5)

where N is the number of theoretical plates necessary to effect the separation of a pair of solutes having a separation ratio a and the first of the pair being

eluted at a capacity factor of k'. Equation ( 5 ) was developed for gas chromatography by Purnell ( 1 ) and allows the number of theoretical plates required to separate the critical pair to be calculated. The next equations of importance are those that give expressions for column length. (6)

L = NH

Thus, for a packed column,

or

L = N(A t

where

A = 2Xdp B = 2yD0

and

B t Cu,) UO

lk' )dpc ['1tbk'24(ltk') Do t1

and for a capillary column H = %- .+ ( 1t6k'tl lkt2)r2uo UO 24(ltk')L Do

4k' 3(ltk')z

t

%f-2 (-12

4k' 3(ltk')L

df-2

3

D,

(y'tl)

0

-

L = N ( B t Cu,) UO

where in this case B = 2D, and

= [(lt6k'tllkt2)r2

24(ltk')z

Do

Another set of equations relating the column length with exit velocity will be the Poiseuille Equation for the capillary column and the Darcy Equation for the packed column.

For a packed column: L =

J,

-Pd P-

nu0 where JI is the appropriate constant for a packed column and n is the mobile phase viscosity

( 9A)

413 For a,capillary column: (9B) where $ ' is the appropriate constant for a capillary column Now by equating equation (7B) and equation ( 9 A ) and solving for u, for a packed column NCn and for a capillary column

It is seen that by employing equations (10A) or (lOB), the value of the exit velocity uo can be calculated for either the capillary column or the packed column from the parameters of the respective HETP equation, the viscosity o f the mobile phase and the pressure drop across the column. Consequently, by inserting the appropriate values for the mobile phase velocity,

so

calculated, back into

equations (9A) or ( 9 B ) the respective column length can be calculated. There are two further equations that have to be defined and the first is the equation for the analysis time,

where k'l is the capacity factor of the last eluted peak, and

is the reciprocal of the Martin-James pressure correction factor.

The second equation is that for the capacity factor which for the capillary column is given by

( 12A)

k' =K(2df/r) where K is the partition coefficient of the solute between the two phases.

The expression for k' for a packed column however, is considerably different. The volume o f mobile phase in the column will be the difference between the total volume of the column and the volume of support and stationary phase Thus

Vg = nR2L - Lg/p

-

LgAdf

where R is the radius of the packed column g

is the mass of support per unit length of column

414

p is the density of the support A is the surface area per gram of the support.

Hence, VL = LgAdf Thus, a = (nR2L Lg/p - LgAdf)/LgAdf where a is the phase ratio of the column

-

Now nR2L

-

Lg/p>>LgAdf Thus a = (nR2 g/p)/gAdf.

-

Hence k' = KgAdf/(wR2

-

g/p)

( 12B)

It is interesting to note that the packing density does not change very significantly with the particle diameter (dp) and furthermore nr2= 1Og/p. Consequently, the value of k' for a given solute is sensibly independent of the particle diameter and varies only with the film thickness (df). The Effect of Column Radius and Particle Diameter or Analysis Time in a GC Separation

Assuming a separation ratio of 1.01, a partition coefficient for the first peak of the critical pair of 100 (and either equation (12A) or (12B) to calculate k'),

the number of theoretical plates required to effect the

separation can be calculated for both a packed column and a capillary column from equation (5). Then employing equation (10A) or (lOB), the constants in the Van Deemter equation shown in Table 1 , and an arbitrary film thickness of 0.05 micron the value of u can be calculated for both a capillary column and a packed column for different particle diameters and different column radii. TABLE 1 PHYSICAL PROPERTIES OF GAS CHROMTOGRIUJ€~ICSYSTENS

Diffusivity of Solute in Mobile Phase Diffusivity of Solute in Stationary Phase Viscosity of Mobile Phase Van Deemter Multipath Constant ( A ) Van Deemter Diffusivity Constant ( y ) Packed Column Diameter

0.5 cm2/sec 5 x 10-6 cm2/sec 0.00025 poises 0.5 0.6 3 mm

Finally, by employing equation (Il), the time taken to achieve the separation can be calculated and plotted against particle diameter or column radius to demonstrate how the analysis time varies with these parameters. It is found that there is both an optimum particle diameter and an optimum radius that provides the minimum analysis time. The curve relating analysis time against column radius for a capillary column is shown in Figure 3 . It is seen that the minimum is quite sharp, with radii less than the optimum resulting in a very sharp increase in analysis time whereas the increase with radii in excess of the

415 optimum being less severe. It follows that there is a specific column radius or particle diameter that will give the minimum analysis time for any given mixture.

9

8000 7000

P

Dlffumlvlty of Solute In Stationary Phare 5 x 10-*cm2/rec.

Film Thlcknere 0.5 mlcrons

P

I

6000

e

Partltlon Coeflkbnt 100 Separation Ratio 1.01 InbVOuUer Prerrure RaUo 4 VISCOSltY of Mobllo Phare 0.00025 Dlffurlvlty of Solute h Y o b b Phaao 0.6 cmz/rec.

P

\

5000

d 0

2

E

4000

3000 2000 1000 I

I

1

I

I

Radius (mm) Figure 3 .

Graph of analysis time against column radius.

The Effect of Film Thickness on Analysis T i m e in a GC Separation Exactly the same procedure can be used to examine the effect of stationary phase film thickness on analysis time.

The same parameters were used except

that the column radius and particle diameter were fixed and the film thickness varied, and the analysis time calculated for each film thickness. It was found that an optimum film thickness also existed which would produce the minimum analysis time.

The curve obtained for a capillary column having a radius of 0.3

mm (300 micron) is shown in Figure 4. It is clearly seen that an optimum film thickness does exist which was predicted by Scott and Hazeldean ae long ago as 1960 ( 4 ) . The nature of the curve, however, differs significantly from the curve relating analysis time with column radius. Although a film thickness significantly less than the optimum causes a rapid increase in analysis time, a film thickness greater than the optimum resulted in a relatively slow increase. Consequently, although the optimum film thickness should be sought, it would be far better to err on the side of greater than optimum film thickness than to make the film too thin.

416 Partition Coefficient 100 Separation Ratio 1.01 Iniet/Outlet Pressure Ratio 4 Viscosity of Mobile Phase 0.00025 P Diffusivity of Solute in Mobile Phase 0.5 cm2/sec.

5000

DHfusivity of Solute in Stationary Phase 5 x 10'6cm2/sec. Column Radius 0.3 mm

4000

3000 2000

1000 I

I

I

I

0.5

1.o

1.5

2 .o

1

2.5

Film thickness (microns) Figure 4 .

Graph of analysis time against film thickness. TABLE 2 c E R ~ o G R A P E YEXAMPLES

PARTITION COEFF'ICIKNT

50

500

1000

2,2-Dimethylbutane on squalane at 8OoC Cyclopentane on squalane at 80°C Toluene on squalane at 135°C (Solutes above their boiling points) p-Xylene on squalane at 8OoC Ethylbenzene on squalane at 80°C n-Hexane on decane at 3OoC (Solutes just below their boiling points) n-Hexane on squalane at 3OoC (Interpolated) Methylcyclohexane on squalane at 3OoC (Interpolated) (Solutes considerably below their boiling points)

Optimum Column Conditions for Gas Chromatographic Separations of Different DeErees of Mfficulty The need for an optimum film thickness and an optimum column radius (or implicitly particle diameter) is clearly seen from Figures 3 and 4. However, in GC an explicit set of equations have, as yet, not been developed to permit the direct calculation of these parameters.

Nevertheless, they can be arrived at by

an iterative procedure employing the ubiquitous computer.

The optimum

417 parameters were calculated in this way employing the chromatographic conditions previously defined for a range of separation ratios of the critical pair and for two values of the partition coefficient of the first peak of the critical pair. The two values of the partition coefficient chosen were 50 and 1000. Chromatographic examples of solutes that have different partition coefficients are shown in Table 2, the data being taken from the work of Kwantes and Rijnders (35). It is seen from Table 2 that a partition coefficient of 50 represents substances chromatographed above their boiling points, such as cyclopentane on squalane at 80°C or toluene on squalane at 135OC. A partition coefficient of 1000, on the other hand, represents substances chromatographed at.temperatures well below their boiling points such as n-hexane or methylcyclohexane on squalane at 30 'C.

It follows that a choice of 50 and 1000 for the limiting

partition coefficients brackets the extremes of sample types that are most likely to be analysed by gas chromatography. The optimum radius and film thickness calculated in this way for separation ratios of the critical pair of 1.01 to 1.12 for a capillary column are shown in Figure 5.

It is seen that there is not a great difference in the optimum radius

for solutes having the extreme values for the partition coefficient and that columns of wide radius are optimum for difficult separations and conversely, columns of very small radius are optimum for very simple separations.

More

important, it appears that for the optimum separation of solute mixtures with the separation ratio of the critical pair greater than 1.03 would require an optimum column radius of less than 75 micron and for a separation ratio of 1.12 a radius as small as 25 micron.

In practice a column having a radius of much

less than 75 micron is extremely difficult to coat and furthermore is very liable to blockage.

It follows that the optimum column radius for simple

separations is not at present very practical. It is also seen that there is a significant difference in the optimum film thickness for the two extremes of partition coefficient indicating that the film thickness should be adjusted for the analysis of different solute types irrespective of the difficulty of separation.

Samples having a separation ratio

of 1.12 for the critical pair require a very thin film of stationary phase for optimum performance, whereas the more difficult separations typified by separation ratios of 1.01 or 1.02 require much thicker films, particularly for solutes that are being chromatographed much above their boiling points.

In

general, it is difficult, if not impossible, to coat stable films on capillary tubes in a reproducible manner with films much below 0.05 micron. Consequently, the optimum film thickness cannot be used for solutes eluted significantly below their boiling points and where the separation ratio of the critical pair is

418 greater than 1.03. It appears that there are at present significant restraints on the complete optimization of a capillary column which can be defined as a minimum radius of 75 micron and a minimum film thickness of 0.05 micron; in fact these limits may be somewhat generous. .024

Film Thickness (micron) a20

1.8 -

.016

.012

Minimum Practlcal Column Radlu6 .008

Minimum Practical .004

0 1.

1.02

1.04

1.06

1.08

Separation Ratio a

1.10

1.12 1.00

1.02

1.04

1.06

1.08

1.10

1.12

Separation Ratio a

Figure 5. Graph of optimum capillary column radius and optimum film thickness against separation ratio. The complimentary curves for a packed column giving the optimum particle radius and optimum film thickness for the separation of solute pairs of different difficulty are shown in Figure 6. It is seen that in an analogous manner to the capillary column radius, large particles are optimum for difficult separations, (separation ratios from 1.01 to 1.03), whereas small particles are optimum for simple separations (separation ratios from 1.08 to 1.12). However, due to the fact that the column radiuslparticle radius ratio must not be less than twenty for efficient packing, the maximum particle radius that can be used in a 3 mm i.d. column will be 300 micron. Consequently, as seen from Figure 6 , optimum particle radii cannot be used for very difficult separations; i.e. for separation ratios of the critical pair of less than 1.02.

It is interesting to

also note that the optimum film thickness does not depend on the separation ratio of the critical pair but only on the partition coefficient of the solute. This arises from the fact that the phase ratio of a packed column is one to two orders of magnitude less than a capillary column and that the density of packing is sensibly independent of the particle diameter. It is also interesting to note

419 that the practical limit of film thickness (equivalent to a 1% loading of stationary phase on the support) allows an optimum film thickness to be used almost throughout the whole of the partition coefficient range.

Graph of Optimum Particle Radius against Separation Ratio

.06

I

r

:c

I

Maximum Practkai Partkk Radius for 3 mm. 1.D. Column

K=50

Graph of Optimum Film Thickness against Partition Coefficient

+

Practical Llmlt of Mlnlmum F l h Thkkness (1.0% w/w Loading of

- 1000

-0 1

-.

1.00

1.02

1.04

1.06

1.09

Separatlon Ratio

Figure 6 .

1.10

I 1.12 0

600

1000

Partition Coefficient K

Optimum Parameters for a Packed Column.

Employing the optimum radius and film thickness for the capillary column and the optimum particle diameter and film thickness for the packed column the analysis time and the length of column necessary can be calculated for both column types using the equations already discussed.

This can be carried out for

a range of separation ratios of the critical pair and the results from such calculations are shown as curves relating analysis time to separation ratio in Figure 7.

It is

seen

that in all cases, if fully optimized columns are employed and

practical limitations are ignored, then the capillary column will always provide a shorter analysis time than the packed column irrespective of the complexity of the separation.

It should also be noted that the difference in analysis times

between the two column types can be as much a s two orders of magnitude.

For

example, separating a mixture where the separation ratio of the critical pair was 1.08 and the partition.coefficient 1000 the capillary column would effect the separation in 0 . 3 second whereas the packed column would take 30 seconds. Another point of interest is the separation of a mixture of substances having

420

separation ratios as little as 1 . 0 3 can be achieved in slightly more than 10 seconds (requiring 90000 theoretical plates), a separation very similar to that achieved by Desty as long ago as 1960 ( 3 ) .

I 1.00

Separation Ratio a

Separation Ratio a

Figure 7 . Graph of log analysis time against separation ratio for packed and capillary columns. If the same calculations are repeated, but the practical constraints of 7 5 micron as the minimum capillary column radius, 0.05 micron as the minimum capillary column film thickness are imposed, together with a maximum particle radius for the packed column of 300 micron and a minimum stationary phase loading of 1 X , the relative performance of the two column types is seen to be much different.

The results are shown in Figure 8.

It is seen that the

relative performance of the two column types is much closer, particularly for solutes chromatographed at temperatures significantly above their boiling points and for other types of compounds where the separation ratio of the critical pair is greater than 1 . 0 7 .

In many cases the ratio of the analysis time for the

packed column to that of the capillary column is only a factor of two, which for separations that take 10 or less seconds to complete is not a significant price to pay. This is particularly

so

since the packed column provides a much wider

sample concentration range and allows the use of a simplier injection system which is significantly less expensive and far more reproducible.

421

In an attempt to reduce the injection problems associated with capillary columns and extend the sample concentration range to that similar to the packed column, the idea was put forward to employ wide capillary columns; i.e. 0.05 in or 1 . 2 7 mm i.d. and a thick film of stationary phase ( 1 micron thick).

Such

columns would permit on-column injection and allow larger samples to be placed on the column to increase the concentration range o f solutes that could be analysed.

I

y

Partition Coefficient= 50

Partition Coefficient = 1000

X

Capillary Column 1.27 mm I.D. Film Thkkness 1 p

-

Capillary Column 1.27 mm I.D. Film Thickness 1 y

-

Optimized Packed Column

-

Capillary Column (Limited Opthization)

-‘\ Capillary Column’ (Limited Optlmizatlon) \

Capillary Column (Fully Optimized)

Capillary Column (Fully Optimized)

/\\.-

-It -1

r n

1.00

1.02

1.04

1.06

1.08

1.10

Separation Ratlo a

I

I

I

i

1.02

1.04

I 1.06.

I

I

1.08

1.10

Separation Ratio

(I!

Figure 8. Graph o f log analysis time against separation ratio for packed and capillary columns under different conditions of optimization. Using the computer procedures already discussed the analysis times obtained from such columns operated under the same conditions were calculated, and the curves obtained are also included in Figure 8.

It is seen that the performance of the

wide column is 10 to 100 times worse than the packed column and consequently is not a viable alternative to the use of packed columns. There is only o n e optimum column that will effect a separation in the minimum time and for a

given set of chromatographic conditions,

this column

must have a specific radius or particle diameter, a specific film thickness and specific length.

The theory of chromatography is sufficiently advanced to

permit these parameters to be precisely calculated. Unless these calculations are carried out, the minimum analysis time cannot be achieved and if optimum columns are designed for GC it will be found that analysis times are at least an

422

order l e s s than those presently tolerated. The Design of Packed LC Colums

The approach to the design of LC columns will follow the logical procedures that were employed for GC column design. However, in this case only packed columns will be considered as LC capillary columns have not been developed to an adequate level of performance for general analytical use. In a similar mariner to the development of GC column design, the equations employed will first be discussed.

In order to maintain the integrity of the

protocol, some equations will be repeated in those places where common expressions exist between the techniques. The same equation for efficiency will be employed

VI'Z

N * [4(ltk')/k'(0-1)]~ where N, k' and a have the meanings previously ascribed to them. The analysis time t is given by t = (ltk'l)L/u

where k'l is the capacity ratio of the last eluted peak. L = NH

Furthermore and from equation (1) =

Xdp

or

where

2.

.(a t bk, + ckg d 24(1 + ke)s Dmp ue H = A t Blu + Cu A = 2Xdp B = 27% (a+bk,tcke2) C = 24(ltkz

From equations (6) and ( 1 3 ) L = N[A+B/utCu]

Now from the Darcy Equation L =

where Equating (14) and (15)

2 -

dY2 D,,,

Thus the velocity

u

can be directly calculated.

The Effect of Particle Diameter on Analysis Time in LC Employing equation ( 1 7 ) to calculate the optimum velocity, equation (9A) to calculate the column length and equation (11A) to calculate analysis time, curves can be constructed relating analysis time to the separation ratio of the critical pair; a set of such curves for particle diameters of 3 , 5 and 10 micron are shown in Figure 9 .

The typical values for the pertinent variables were

assumed as shown in Table 3 . TABLE 3

Xs0.5,

q=O.O025P $=35 k;=2.5

7=0.6,

Dm=3.5x10-5 cm2/sec

Inlet Pressure 3000 psi

I micron B 5 micron C 10 micron

A 3

\I

I02

I00

1.06

b04

Separation Ratio Figure 9. Graphs of analysis time against separation ratio for columns packed with particles of different diameters. The rapid increase in analysis time with reduction in the critical pair is no more than would be expected. diameter, however, is not

so

obvious.

CY

value o f the

The effect of particle

It is seen that particles 3 pm in

diameter provide the shortest analysis time for separation ratios down to about 1.03.

Solute pairs having separation ratios between about 1.02 and 1.03 will be

separated in the shortest time by employing particles 5 pm in diameter whereas solute pairs having separation ratios 1 . 0 1 and 1.02 would require particles having diameters of 1 0 pm to achieve the separation in the minimum time.

The

more difficult the separation (the lower the separation ratio) the larger must be the particle diameter for minimum analysis time.

This is a direct result of

having a limited inlet pressure; as the separation becomes more difficult, the more theoretical plates are required to effect the separation and consequently the longer the column must be.

Eo i p s o , if the pressure is limited, then the

particle size must be increased to permit the necessary solvent flow through the longer column. particle

The natural corollary of thisis that there will be an optimum

diameter for any given separation that will permit the analysis to be

completed in the minimum time.

In fact, this is exactly analogous to the

optimum capillary column radius or packed column optimum particle size that is necessary for GC separations. Employing the same equation, curves relating analysis time to particle diameter were constructed for solute pairs having different separation ratios

1.02, 1 . 0 4 , and 1.06 and the results are shown in Figure 10A.

It is seen that

indeed there is an optimum diameter that provides the minimum analysis time for a given solute pair.

It is also apparent that the minimum in the analysis time

curve is much sharper for small particles separating simple mixtures than for larger particles separating more difficult mixtures (~1.02). Consequently, for optimum performance in terms of analysis time the particle diameter is more critical for simple separations than for the more difficult separations.

Again

employing an iterative technique and with the aid of the computer, graphs can be constructed relating optimum particle diameter for minimum analysis time to the separation ratio of the critical solute pair.

The results obtained from such

calculations for three different inlet pressures are shown in Figure 10B.

It is

seen that very small particles of 1 or 2 pm in diameter should only be used for very simple separations whereas the high efficiencies necessary for the separation of difficult mixtures require the use of particles having relatively large diameters. The majority of separations carried out today have separation ratios for the critical pair of 1.1 or even more. It is seen from Figure 10B that for optimum performance the particle diameter should be 4 1 pm.

Particles

1 pm in diameter are not available at present and neither are packing procedures developed for use with them.

It follows that the smallest particles available,

namely 3 pm, will have to be used for the time being which also means that optimum performance cannot be achieved at present for simple separations.

It

should also be noted that raising the inlet pressure from 4000 to 6000 p.s.i. has a relatively small effect on the magnitude of the optimum particle diameter

.

425

A =

I

2000 psi B = 4000 psi C = 6000 psi

Separation Ratio 1.02 10

-

\\\

u

0)

.L?

I

1

0

2

4

6

e

D

I2

1.00

Particle Diameter (micron)

I

I04

1.08

1

1,12

Separation Ratio

Figure 1 0 A and I O B . Graphs of analysis time against particle diameter for the separation of different solute pairs having different separation ratios. Graphs o f optimum particle diameter f o r minimum analysis time against separation ratio. Chromatographic Performance When Operating With Optimum Particle Diameter Curves relating analysis time and separation ratio together with those relating column length with separation ratio for columns packed wit-h particles of optimum diameter are shown in Figure 11A and 1 1 B .

The values were calculated

using the same equations and the same iteration procedure as that described previously.

Curves were constructed for three different inlet pressures 2000,

4000 and 6000 p.s.i.

It is seen that minimum analysis time ranges from 2 o r 3

sec when separating solute pajrs having separation ratios of 1.12 to 3 h for the separation o f a solute pair having a separation ratio o f 1.01.

It is also

interesting to note that increasing the inlet pressure for 4000 to 6000 p.s.i. only reduces the analysis time by about 30%.

Such an improvement may well not

be worthwhile considering the price t o be paid in terms o f both instrument complexity and cost.

At lower pressures less demands would be made on pump

seals, non-return valves, and sample valves, rendering and less costly t o make.

the equipment easier

If particles o f optimum, o r near optimum size are

426

employed, 4000 or even 3000 p.s.i. inlet pressure may prove to be an excellent compromise between that which is theoretically desirable to that which is ically acceptable.

A = 2000 psi B = 4000 psi C = 6000 psi

I

Ibe

1.04

‘0

1.12

Separation Ratio Figure 1 1 A and 1 1 B . Graphs of analysis time and column length against separation ratio for optimum particle diameter It is interesting to calculate the linear mobile phase velocity that is used with the particles of optimum diameter to achieve the minimum analysis. Knox and Saleem (10) suggested that the minimum analysis time could only be obtained using the optimum linear mobile phase velocity but

as

they employed an

empirical equation for H it was not possible to confirm this. Differentiating equation (1) with respect to u and equating to zero it can easily be shown that:

During the iteration procedure by the computer the value of u can be calculated at the optimum particle diameter from equation (17) and the optimum mobile phase velocity calculated from equation ( 1 8 ) .

Values of a ,

II

and

[Iopt

are given in Table 4 . It is seen that the contention by Knox and Saleem (10) that the optimum particle diameter must be employed with the optimum linear velocity to provide the minimum analysis time is indeed correct.

427

TABLE 4 MOBILE PHASE LINEAR VELOCITY BY C0lCpUTF.R ITERATION MID BY DIRECT CALCULATION

0.0586 0.1170 0.1759 0.2345 0.2934 0.3517 0.4097 0.4674 0.5262 0.5842 0.6440 0.7035

1.01 1.02 1.03 1.04

1.05 1.06 1.07

1.08 1.09 1.10 1.11 1.12

0.0586 0.1173 0.1756 0.2341 0.2924 0.3512 0.4104 0.4699 0.5282 0.5874 0.6428 0.7024

___________________-____________________---------------------However, by having an explicit equation for H, an equation for the optimum particle diameter can now be obtained. At the optimum mobile phase velocity the value for H is at a minimum and by substituting for

u

from equation (18) and

(13): Hmin = A + 2(BC)ll2

(19) Substituting for H from equation (19) into (6) and equating to equation (9A), simplifying and solving for dp it can be seen that:

Equation (20) provides a means of calculating the optimum particle diameter in absolute terms without employing an iterative procedure.

In Table 5 values for

the optimum particle diameter calculated by the computer using an iterative procedure are given for a series of values of the separation ratio a , together with the optimum values of dp calculated by equation (20).

It is seen that

excellent agreement is obtained and that equation (20) can be employed with confidence in column design to calculate the optimum particle diameter. Figure 11B shows the relationship between column length and separation ratio for columns packed with particles of optimum diameter.

It is seen that an

optimized column for separating mixtures where the critical pair has a separation ratio of greater than 1.08 is less than 1 cm in length. Once more the practical use of particles 1 pm in diameter packed in columns less than 1 cm long comes into question.

It is true that very short columns are fairly easy to

pack with small particles but the practical value of reducing the particle size to 1 pm and packing them in a column less than 1 cm long remains to be established.

428

TABLE 5 OPTIMUM PARTICLE DIAMETERS BY COMPUTER ITERATION ANII BY DIRECT CALCULATION

Separation Ratio

Particle Diameter (pm)

.................................................. From Computer Iteration

By Direct Calculation

________________________________________-----------------------11.63 5.81 3.88 2.91 2.33 1.94 1.66 1.45 1.29 1.16 1.06 0.97

1.01 1.02 1.03

1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12

11.89 5.95 3.96 2.97 2.38 1.98 1.70 1.49 1.32 1.19 1.08 0.99

It is suggested that 2-pm particles packed in columns 2 cm long, which would be about optimum for separating solute pairs having a separation ratio of about 1.08, might be the practical limit in both particle size and column length. The Peak Capacity of Optimized Chromatographic Column

The peak capacity of a chromatographic system is numerically equivalent to the total number of fully resolved solute peaks that can be fitted into a chromatogram between the dead volume peak and the peak for the last eluted solute.

A number of equations have been developed to calculate the peak

capacity of a chromatographic system such as those of Giddings (36) and Scott (37).

More recently, Davis and Giddings (38) pointed out that the theoretical

peak capacity is an exaggerated value of the true peak capacity due to the statistically irregular distribution of the individual k' values of each solute. Nevertheless, the theoretical value will be given here as a relative measure of the true peak capacity.

The equation that will be used i s that of Scott (37),

namely ,

Q

= log [I-

($l+k' k' +

0.511

-

PI)]

/log P'

(21)

where 41 is the theoretical peak capacity and P ' = [N -2(N)+]/[N + 2(N)*].. In Figure 12 the upper curve shows the relationship between peak capacity and separation ratio for a column packed with particles of optimum diameter. The relationship is exactly that which one would expect for a non-optimized system;

429 the peak capacity is greatest where the a value is least and consequently, the highest efficiency is available.

It is seen clearly from equation ( 2 1 ) that the

peak capacity increases as the efficiency increases. Furthermore, even for a column optimized for the separation of a solute pair having an

c1

value of 1.12

the peak capacity is still quite significant and about 20. >r CI

-d

u

m a m

Inlet Pressure 3000 psi

300-

U

Y

m al

I00

E

v

!.I

4

m

p: 20

!2 cs

102

104 106 108 110

I12

Separation Ratio

Figure 12. Graphs o f peak capacity, column radius and solvent consumption against separation ratio for columns packed with particles of optimum diameter. Column Radius Explicit equations for the calculation of the column radius have been put forward by Reese and Scott ( 3 9 ) and Katz (40) and can be given in the following form r = [ 0 . 0 9 o ~ ( a- l)/dp]j

where

UA

(22)

is the standard deviation of the dispersion due to the instrument and

the other symbols have the meanings previously ascribed to them. In Figure 12 the center curve relates column radius with separation ratio. It is seen that a linear relationship exists and that to cover the range of

CY

values from 1 . 0 1 to 1.08 (bearing in mind that, in practice, a limit of 3 pm is placed on the minimum particle diameter-) a range of column radii between about 0.2 to 3 mm needs to be available.

This is a very practical range of column

diameters for the design of LC columns. Solvent Consumption The solvent consumption can be simply calculated from the column volume and the capacity factor of the last eluted peak

430

V

= nrZLe(1

where V is the volume per analysis,

E

+ k')

is the ratio of mobile phase volume to

total column volume and the other symbols have the meanings previously ascribed to them. The lower graph in Figure 12 shows the solvent consumption per analysis plotted against separation ratio.

It is seen that the solvent consumption

increases slowly with separation difficulty as a decreases to about 1.04. a =

Below

1.04 however, the solvent consumption increases very rapidly and it is

therefore extremely important that for difficult separations the minimum column radius is used, otherwise solvent consumption could be extremely excessive.

It

is also seen that the optimum column radius for a separation ratio of about 1.01 would be about 0.5 mm. Practical Use of an LC Colum Desim Protocol

Explicit equations now being available to calculate all the necessary properties of the optimum column system and consequently, a very simple computer program can be written to provide the chromatographer with the dimensions and properties of the column required and the performance that can be expected. Information is requested in sequence starting with the separation ratio a of the critical pair and ending with the column volume factor as follows: Enter separation ratio of the critical pair? 1.04 Enter capacity factor of first peak of the critical pair? 3 Enter separation ratio of last peak to the first of the critical pair? 2 Enter resolution required (for Rs=l, then 4 ) ? 4 Enter inlet pressure? 3000. Enter solvent viscosity in poise (n-hexane, 0.0026 P; water, 0.01 P)?

0.0026 Enter solute diffusivity (benzyl acetate in n-hexane, is 3 . 5 x 10-5 cm21sec) ? 0.000035. Enter multipath factor ( X ) ( 0 . 5 for a well packed column)? 0.5 Enter longitudinal diffusion factor (y)(0.6 for a well packed column)? 0.6 Enter.instrument dispersion (standard deviation in millilitres)? 0.0015. Enter column factor (€)(mobile phase volumeltotal column volume, normally 0.65)? 0.65. Where appropriate, guidance is given as to the likely values of some of the basic parameters.

For example solvent viscosity in poise is requested and the

user is reminded that hexane has a value of 0.0026 P and water a value of 0.01

P.

After the final entry, the calculations are commenced and the results are

tabulated as follows. First the operator is reminded of the performance required: Adequate resolution

431 Minimum analysis time Maximum mass sensitivity Minimum solvent consumption The program can then provide

the column specifications and operating

conditions. Column length: 10.6 cm Column radius: 0.17 cm Optimum particle diameter: 2 . 1 pm Column flow-rate: 0.88 ml/min Linear mobile phase velocity: 0.247 cm/sec

It is seen from the column specifications that the column length will be 10.6 cm and the column I.D. 3.4 mm.

The optimum particle diameter would be

2.7pm which would require a flow-rate of 0.88 ml/min. Finally, the expected chromatographic performance is listed. Analytical Specifications

Column efficiency in theoretical plates 17,800 Analysis time 301 sec Solvent consumption per analysis 4.43 ml Total peak capacity 65.4 The optimized column would have an efficiency of 17,800 theoretical plates. The analysis would be completed in just over 5 min, the solvent consumption per analysis would be 4.4 ml and the total peak capacity would be 65. A l l these values are still in the range of general practical LC analysis, but it should be noted that the separation of a fairly complex and reasonably difficult mixture could be completed in 5 min.

This performance could only be achieved with

complete optimization of the system where the column design and operating conditions are made completely compatible with the instrument specifications and consequently provide optimum performance. Explicit equations are now available that will permit all the pertinent parameters of either a GC or an LC system to be calculated.

Consequently, with

the aid of a simple computer program the chromatographer can easily and rapidly determine the optimum dimensions of the column necessary to carry out the analysis.

This will include the particle size of the packing or radius of the

capillary column and all the necessary operating conditions to achieve the separation in the most efficient manner. Certain interesting facts have arisen from the theoretical development of the design protocol that are not generally known or understood at the time of writing this review. For any particular separation, whether it be by gas or liquid chromatography, there is a unique particle size if a packed column is to be used o r a unique radius if a capillary column is to be used, that will allow

the separation to be achieved in the most efficient manner.

The magnitude o f

this radius or particle diameter will depend on the physical and chemical properties of the solute and phase system and the mobile phase inlet pressure that is available from the chromatograph.

In a like manner, if the optimum

radius or particle diameter is employed, then there will also be a unique column length and, for a given instrument dispersion, an optimum column radius if a packed column is used. Thus, once the phase system has been chosen for a particular solute mixture that is to be separated on a specific chromatographic system, then there is one, and only one, column o f a particular length and radius or particle diameter that will achieve the analysis in the minimum time. Another aspect of column design that is not generally appreciated is the fact that, with packed columns, small particles are optimum for simple separations whereas large particles are optimum for difficult separations.

In a

similar manner, in capillary gas chromatography small diameter columns are optimum for simple separations and wider bore columns are optimum for more difficult separations where long columns are necessary to achieve the required number of theoretical plates.

This concept is in complete conflict with the

opinion presently held by the majority of workers in the field. This arises from the fact that smaller particle diameters and smaller radius capillary columns give smaller HETP values and thus more theoretical plates per unit length.

What

is not appreciated is that the small particles offer a much greater impedance to flow and thus if the column inlet pressure is limited by chromatographic design then there is a maximum column length that can be used. Consequently, if the separation demands more theoretical plates than can be provided by the maximum column length, then the diameter of the particles has to be increased to reduce the impedance to flow and thus allow a longer column to be employed.

For

example, if the particle diameter is doubled the column impedance is reduced by a factor of four; thus, for the same inlet pressure the column length can be extended by a factor of four. As a result of the increased particle diameter, the variance is also increased but it can be shown theoretically that the net effect is to increase the resolution by about a factor of 1.5.

It follows that

the larger the particle diameter the longer the column that can be used and hence the greater the resolving power. References 1.

J.H. Purnell, Nature (London), 184, Suppl. 26 (1959) 2009.

2. J.H. Purnell and C.P. Quinn, in R.P.W. Scott (Editor), "Gas Chromatography 1960", Butterworths, London, 1960, p. 184. 3. D.H. Desty and A. Goldup, in R.P.W. Scott (Editor), "Gas Chromatography 1960", Butterworths, London, 1960, p. 162.

433 4. R.P.W. Scott and G . S . F . Hazeldean, in R.P.W. Scott (Editor), "Gas Chromatography 1960". Butterworths, London, 1960, p. 144. 5.

L.R. Snyder, J . Chromatogr. Sci., 10 (1972) 364.

M. Martin, G . Blu, C. Eon and G . Guiochon, J. Chromatogr. Sci., 12 ( 1 9 7 4 ) 6. 438. 7.

I . Halasz, H. Schmidt and P. Vogtel, J . Chromatogr., 126 (1976) 19.

8.

G. Guiochon, J . Chromatogr., 1 8 5 ( 1 9 7 9 ) 3 .

9.

J.C. Kraak, H. Poppe and F. Smedes, J. Chromatogr., 122 (1976) 147.

10.

J.H. Knox and M. Saleem, J . Chromatogr. Sci., 7 (1969) 614.

11.

C.E. Reese and R.P.W. Scott, J . Chromatogr. Sci., 18 ( 1 9 8 0 ) 479.

12.

J.H. Knox, J . Chromatogr. Sci., 18 ( 1 9 8 0 ) 453.

13.

J.C. Sternberg, Advan. Chromatogr., 2 ( 1 9 6 6 ) 205.

14.

M. Martin, C. Eon and

G.

Guiochon, J . Chromatogr., 108 (1975) 229.

1 5 . R.P.W. Scott, "Liquid Chromatography Detectors", Elsevier Amsterdam, 1977, p. 46. 16.

G.K.C. Low and P.R. Haddad, J . Chromatogr., 198 (1980) 235.

17. J . J . Van Deemter, F.J. Zuiderweg and A. Klinkenberg, Chem. Eng. Sci., 5 (1956) 271. 18. A.I.M. Keulemans and A. Kwantes, in D.H. Desty and C.L.A. Harbourn (Editors), "Vapor Phase Chromatography", Butterworths, London, 1956, p. A 1 0 . 19.

J.C. Giddings, J. Chromatogr., 5 (1961) 46.

20.

J.F.K. Huber and J.A.R.J. Hulsman, Anal. Chim. Acta, 38 (1967) 305.

21.

G.J. Kennedy and J.H. Knox, J . Chromatogr. Sci., 10 (1972) 549.

22.

J.N. Done and J.H. Knox, J . Chromatogr. Sci., 10 (1972) 606.

23. J . N . Done, G.J. Kennedy and J.H. Knox, in S . G . Perry (Editor), "Gas Chromatography 1972", Applied Sci. Publ. Barking, 1973, p. 145. 24.

Cs. Horvsth and H.-J. Lin, J. Chromatogr., 126 (1976) 401.

25.

Cs. Horvsth and H . - J .

26.

E.D. Katz, K.L. Ogan and R.P.W. Scott, J . Chromatogr., 270 ( 1 9 8 3 ) 51.

Lin, J . Chromatogr., 149 (1978) 43.

27. M.J.E. Golay, in D.H. Desty Butterworths, London, 1958, p. 3.

(Editor),

"Gas Chromatography

28.

K. Ogan and R.P.W. Scott, J . High Res. Chrom., 7 (1984) 382.

29.

A.T. James and A.J.P. Martin, Biochem. J., 50 (1952) 679.

30.

M.J.E. Golay, Anal. Chem., 29 (1957) 928.

1958",

434

31.

D.H. Desty, A. Goldup and B.F. Wyman, J. Int. Petrol., 45 (1959) 287.

32.

R.D. Dandenau and E.M. Zenner, J. High Res. Chromatogr., 2 (1979) 351.

33.

K.L. Ogan, C. Reese and R.P.W. Scott, J. Chromatogr. Sci., 20 (1982) 425.

34.

E.D. Katz and R.P.W. Scott, J. Chromatogr., 270 (1983) 29.

35. A. Kwantes and G.W.A. Rijnders, in D.H. Desty (Editor) "Gas Chromatography 1958", Butterworths, London, 1958, p. 125. 36.

J.C. Giddings, Anal. Chem., 39 (1967) 1027.

37.

R.P.W. Scott, J. Chromatogr. Sci., 9 (1971) 449.

38.

J.M. Davis and J.C. Giddings, Anal. Chem., 55 (1983) 418.

39.

C.E. Reese and R.P.W. Scott, J. Chromatogr. Sci., 18 (1980) 479

40.

E.D. Katz, in R.P.W. Scott (Editor), "Small Bore Liquid Chromatography Columns", John Wiley & Sons, New York, 1984, p . 53.

435

M I N I A T U R I Z A T I O N OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(FIICRO-HPLC)

M. VERZELE and C.DEWAELE Laboratory o f Organic Chemistry, S t a t e U n i v e r s i t y o f Ghent, K r i j g s l a a n , 281 (S.4), 8-9000 GHENT (Belgium)

I. INTRODUCTION Microbore systems, C a p i l l a r y LC, High Speed L i q u i d Chromatography (HSLC) , Low Dispersion L i q u i d Chromatography (LDLC) a r e new developments which have received much a t t e n t i o n i n t h e l a s t years. I t i s probable t h a t m i n i a t u r i z e d forms o f

LC w i l l be much more important i n t h e f u t u r e than t h e y a r e now. I n a

book c o n s i d e r i n g past, present and f u t u r e o f chromatography these trends of chromatography should t h e r e f o r e be present. T h e i r common c h a r a c t e r i s t i c i s t h e m i n i a t u r i z a t i o n o f some aspect o f High Performance L i q u i d Chromatography (HPLC) i n i t s now i n s t i t u t i o n a l i z e d form. A common denominator f o r t h e new development s could t h e r e f o r e be Micro-HPLC. This paper discusses what Micro-HPLC i s , why i t i s o f i n t e r e s t and what could be expected i n t h e f u t u r e o f Micro-HPLC. Two

books were published i n 1984 on t h e m i n i a t u r i z a t i o n o f HPLC (1, 2). An ACS symposium i s s u e devoted a l s o much o f i t s content t o t h e same s u b j e c t ( 3 ) . To our l i k i n g , t h e c o n t r i b u t i o n s o f Guiochon and C o l i n ( l a ) , o f Novotny ( l b ) , of Henion ( l c ) i n Kucera's book ( 1 ) and t h a t o f Janq-in U l t r a h i g h R e s o l u t i o n Chromatography ( 3 ) a r e most r e l e v a n t t o the f u t u r e o f Micro-HPLC. The present c o n t r i b u t i o n cannot be as thorough and e l a b o r a t e as these mentioned. We t r y however t o b r i n g a general overview, more s p e c i f i c a l l y t a k i n g i n t o account t h e impact o f t h e i n t r o d u c t i o n o f 1 and 2

m p a r t i c l e s as we have been d e s c r i b i n g

l a t e l y (4, 5 ) . For a more d e t a i l e d d i s c u s s i o n o f t h e p o s s i b i l i t i e s o f Micro-HPLC we r e f e r t h e reader t o Guiochon and C o l i n ( l a ) .

11.

WHAT I S MICRO-HPLC ? Only t h r e e parameters o f t h e column can be m i n i a t u r i z e d : t h e i n t e r n a l d i a -

meter (ID), t h e l e n g h t ( L ) and t h e p a t i c l e diameter o f t h e packing m a t e r i a l (dp). H o p e f u l l y t h e m i n i a t u r i z a t i o n o f these w i l l l e a d t o an improvement i n

436

t h e b a s i c chromatography parameters o f e f f i c i e n c y , s e n s i t i v i t y , s e l e c t i v i t y and speed w h i l e any economic aspects a r e o f c o u r s e a l s o i m p o r t a n t . The s t a r t i n g p o i n t i s t h e by now c l a s s i c a l column o f 10-30 cm l e n g t h w i t h 4-5 mm I D , packed w i t h e i t h e r 5 o r 10 p m d i a m e t e r s i l i c a g e l based p a r t i c l e s . T h i s i s t h e column m o s t l y used i n c u r r e n t HPLC (6, 7 ) . 11.1.

Impact o f m i n i a t u r i z a t i o n on e f f i c i e n c y

The e f f i c i e n c y o f a chromatographic column i s expressed b y i t s p l a t e num-

(N). The q u a l i t y of t h i s number i s deduced f r o m comparison w i t h g e n e r a l

ber

o b t a i n e d o r e a r l i e r o b t a i n e d f i g u r e s o r f r o m known p o s s i b l e l i m i t v a l u e s . For easy comparison t h e p l a t e number i s o f t e n r e c a l c u l a t e d p e r meter ( a l t h o u g h met e r l o n g columns a r e m o s t l y i m p o s s i b l e t o h a n d l e because o f p e r m e a b i l i t y d i f f i c u l t i e s ) . F o r more general comparison o f d i f f e r e n t p a r t i c l e s i z e s t h e s o - c a l l e d "reduced parameters" such as reduced p l a t e h e i g h t h i n t r o d u c e d by G i d d i n g s ( 8 ) and worked o u t by Knox and coworkers (9, 10) i s o f t e n used. h

=

SETP -

with

HETP

=

L/N

dP l e n g t h o f t h e column

L

:

N

: column p l a t e number

A reduced p l a t e h e i g h t o f 2 i s e x c e l l e n t , 2.5 t o 4 i s good and 5 t o 10 i s reason a b l e and i s sometimes unavoidable. The h i g h e s t p o s s i b l e p l a t e numbers p e r u n i t length

a r e o b t a i n e d on o c t a d e c y l a t e d and endcapped s i l i c a g e l w i t h a h i g h

l o a d i n g o f o r g a n i c m a t e r i a l (complete coverage) w i t h a c e t o n i t r i l e / w a t e r m i x t u r e s as e l u e n t and w i t h p o l y c y c l i c a r o m a t i c hydrocarbons as t e s t m i x t u r e . The p o s s i b l y b e s t e v e r ( w o r l d r e c o r d ) f i g u r e o b t a i n e d a t t h i s l a b o r a t o r y was h = 1.4 dp f o r a p a r t i c u l a r b a t c h o f 1 0 , m RSiL-C,,-HL-D

( a n i r r e g u l a r s i l i c a g e l pack-

i n g m a t e r i a l from Alltech-RSL Europe) i n a 25 x 0.46 cm column f o r pyrene w i t h a k ' v a l u e o f about 5 ( 1 1 ) . I n t h e o r y , t h e column I D does n o t i n f l u e n c e e f f i c i e n c y . M i n i a t u r i z a t i o n o f t h e I D does n o t improve t h i s parameter. Rather t o t h e c o n t r a r y s i n c e i t seems t o be more d i f f i c u l t t o pack e.g. m i c r o b o r e columns o f 1 mm I D as i n t r o d u c e d by S c o t t and Kucera ( 1 2 ) . I t i s p o s s i b l e t o a c h i e v e t h e same e x c e l l e n t e f f i c i e n c y w i t h a 1 mm I D column as w i t h a 4.6 mm I D column, b u t n o t so e a s i l y . S h o r t e n i n g t h e column l e n g t h must reduce t h e e f f i c i e n c y p r o p o r t i o n a l l y . T h i s can however be o f f s e t by u s i n g s m a l l e r p a r t i c l e d i a m e t e r column p a c k i n g m a t e r i -

437

a l s . These two parameters can t h e r e f o r e b e s t be m i n i a t u r i z e d t o g e t h e r . I f t h e l i m i t reduced p l a t e h e i g h t o b t a i n a b l e ( 2 ) i s t h e same f o r d i f f e r e n t p a r t i c l e s i z e s , i t i s o b v i o u s t h a t t h e p l a t e l o s s by h a l v i n g t h e column l e n g t h , can b e compensated by h a l v i n g t h e p a r t i c l e s i z e f o r t h e s h o r t e r column.

11.2.

Impact o f m i n i a t u r i z a t i o n on t h e s e n s i t i v i t y

I t i s now g e n e r a l l y r e c o g n i s e d t h a t t h e c l a i m of h i g h e r s e n s i t i v i t y f o r

m i c r o b o r e columns i s n o t c o r r e c t . Sample s i z e has t o be adapted t o t h e volume sample s i z e i s a p p l i e d t o a

o f s t a t i o n a r y phase i n t h e column. Ift h e same

25 x 0.46 cm u s u a l column and t o a 25 x 0.1 cm m i c r o b o r e column, t h e n t h e m i c r o bore column i s loaded t w e n t y t i m e s as h e a v i l y as t h e w i d e r b o r e column. T h i s g i v e s b e t t e r s e n s i t i v i t y o f c o u r s e b u t maybe a l s o o v e r l o a d i n g . The same h i g h s e n s i t i v i t y w i l l be o b t a i n e d on t h e l a r g e r I D column i f t h e sample s i z e i s i n creased t w e n t y t i m e s . Reducing t h e column I D t o enhance s e n s i t i v i t y c o u l d be o f i n t e r e s t i f t h e a v a i l a b l e sample amount i s v e r y s m a l l . T h i s can o c c u r i n c l i n i c a l a n a l y s i s o r w i t h v e r y v a l u a b l e samples. S e n s i t i v i t y o f a chromatographic system i s v e r y much r e l a t e d t o t h e " d i s D e r s i o n " caused b y t h e i n s t r u m e n t and t h e column. Low d i s p e r s i o n s h o u l d be s t r i v e d for.

D i s p e r s i o n i s e q u i v a l e n t t o band broadening and can t h e r e f o r e be d i s c u s s e d

as f o l l o w s

N

16(VR/B)2

thus B and D

=

=

4VR/'m =

, VR

=

Vo(k' t 1)

,

Vo

= rrr2L.0

4rr*Lp)(k' t l ) / m

4V0/ d a - , r 2 . L /

W i t h VR = r e t e n t i o n volume, B = band w i d t h on t h e base l i n e , r = column r a d i u s ,

Vo = column v o i d volume, k ' = c a p a c i t y f a c t o r ,

0

= column p o r o s i t y ,

D = disper-

sion. The d i s p e r s i o n D as d e f i n e d above i s t h e band w i d t h o f a non r e t a r d e d peak (k'

=

0). D i s p e r s i o n i s determined by r 2 , L and N , b u t as a l r e a d y shown above,

r2 i s r e l a t e d t o t h e sample s i z e . Examples o f t h e impact o f m i n i a t u r i z a t i o n t h r o u g h s h o r t e n i n g L and m a x i m i z i n g N ( o r m i n i m i z i n g p a r t i c l e s i z e ) can be found i n r e f e r e n c e s 4 and 5 (4, 5 ) . 11.3. Speed and m i n i a t u r i z a t i o n Reducing t h e I D o f t h e column has no i n f l u e n c e on t h e chromatographic speed,

438

since the l i n e a r eluent v e l o c i t y must be t h e same regardless ID, f o r optimum r e s u l t s . Reducing the column l e n g t h has however d i r e c t impact on speed. This i s t h e f i e l d o f High Speed L i q u i d Chromatography (HSLC) as developed i n a l a r ge number o f papers. An e x c e l l e n t overview by Dong (13) c i t e s many o f these r e ferences. I n discussing HSLC i n these papers the accent i s mostly on t h e speed f a c t o r . The gain i n s e n s i t i v i t y by reducing the column l e n g t h i s however a l s o a very important p o i n t . 11.4.

S e l e c t i v i t y and m i n i a t u r i z a t i o n

S e l e c t i v i t y has nothinq t o do on f i r s t s i g t h w i t h the m i n i a t u r i z a t i o n of I D , L and dp. The reduced volume o f s t a t i o n a r y phase i n t h e column and of elu-

e n t needed a l l o w however t o use extremely expensive phases and solvents, possib l y leading t o i n t e r e s t i n g s e l e c t i v i t y changes. Novotny c a l l s t h i s t h e use of

'I

" e x o t i c " phases. An e x c e l l e n t i n t r o d u c t i o n t o t h i s p a r t i c u l a r aspect o f Micro-HPLC has been published by McGuffin (14). The explosive expansion of t h e number o f a v a i l a b l e HPLC phase reminds us o f the s i t u a t i o n i n GC about 15-20 years ago. Fortunately t h i s has n o t continued i n GC as i t might be feared. Probably t h e same e v o l u t i o n w i l l occur i n HPLC. 111.

WHY WAS MINIATURIZATION OF HPLC INTRODUCED ?

Although l i q u i d chromatography (LC) i s a much o l d e r d i s c i p l i n e than gas chromatography (GC) i t has o n l y developed i n t o i t s modern HPLC form under t h e impulse o f GC. Very successful aspects o f GC a r e t h e c a p i l l a r y column technology and the coupling o f GC t o mass spectrometry (GC-MS). I t i s o n l y natural therefore t h a t LC research s c i e n t i s t s thought e a r l y o f c a p i l l a r y LC and o f comb i n i n g LC t o MS. I n the e a r l y days o f GC-MS,

l a r g e volume columns were used and

several devices were developed t h a t could remove t h e excess c a r r i e r gas before sample i n t r o d u c t i o n i n t o the MS. The advent o f small bore c a p i l l a r y GC allowed d i r e c t MS i n t r o d u c t i o n which was a decided improverpent. Therefore i t i s obvious i n LC-MS too, t o t r y f o r smaller columns and t o work toward d i r e c t sample and t o t a l column e f f l u e n t i n t r o d u c t i o n . I s h i i (15) s t a r t e d t h e experimental c o n t r i butions toward LC m i n i a t u r i z a t i o n . I s h i i used packed c a p i l l a r y columns as w e l l as open c a p i l l a r y columns (16, 17). Novotny too used both approaches as w e l l as an intermediate technique i n which the packing i s fused i n t o t h e column w a l l ,

439 thus l e a d i n g t o a permeable b u t s t i l l s t a b l e s t r u c t u r e (18, 1 9 ) . On t h e i n t r o d u c t i o n o f f l e x i b l e fused s i l i c a c a p i l l a r y columns a t t h e 1979 Hindelang Symposium (20) most o f t h e c i t e d authors s t a r t e d t o use t h i s m a t e r i a l w i t h s l u r r y packing o f t h e micro- o r packed c a p i l l a r y LC columns (21, 22, 23). None o f these systems has however been commercialized y e t . The r e s u l t s shown proved that

good e f f i c i e n c i e s w i t h f a r l e s s s o l v e n t consumption were p o s s i b l e through

t h i s m i n i a t u r i z a t i o n . This r e d u c t i o n i n s o l v e n t consumption i s a l s o i n t e r e s t i n g o f course. S l i g h t l y l a r g e r systems would s t i l l show these c h a r a c t e r i s t i c s whil e n o t needing completely new equipment. S c o t t and Kucera (12) worked on 1 mm

I D columns and developed t h e v e r y h i g h pressure techniques needed t o pack t h e se s u c c e s s f u l l y . With conventional l e n g t h (25-30 x 0.1 cm) these columns a r e known a l s o as microbore columns. Microbore HPLC indeed consumes much l e s s s o l vent. However, although these columns have been demonstrated f o r h i g h speed and h i g h e f f i c i e n c y chromatography (12, 24, 25, 26) t h e t h r e e major parameters o f chromatography ( e f f i c i e n c y , s e n s i t i v i t y and speed) remain unchanged as a l ready mentioned above. Speed i s t h e r e s u l t o f u s i n g s h o r t e r columns. The l o s s i n e f f i c i e n c y can be countered by u s i n g s m a l l e r p a r t c l e s . Many p u b b l i c a t i o n s i l l u s t r a t e t h i s . A convenient review has a l r e a d y been mentioned (13). Table 1 shows some column types f o r Micro-HPLC. Column t y p e no 1,which i s t h e convent i o n a l column, i s s t i l l by f a r m o s t l y used. Column types 2,3 and 4 a r e commerc i a l l y a v a l a i b l e and a r e g e t t i n g c o n s i d e r a b l e i n t e r e s t from t h e chromatographic comnunity. Types 5 and 6 have been used s u c c e s s f u l l y , b u t t h e y a r e n o t comnerc i a l l y a v a i l a b l e . Column types 7 and 8 have n o t been made t o work experimental-

ly

20 far,

b u t they a r e i n o u r

o p i n i o n v e r y i n t e r e s t i n g as we w i l l t r y t o

show i n t h e n e x t chapter.

IV.

HOW FAR WILL OR COULD THE MINIATURIZATION OF HPLC GO ? The m i n i a t u r i z a t i o n o f HPLC w i l l f i n a l l y be l i m i t e d by t h e p o s s i b i l i t i e s

o f d e t e c t i o n . How small t h e d e t e c t o r c e l l s f o r d i f f e r e n t column types should be i s mentioned i n Table 1. I t i s however o u t s i d e t h e scope o f t h e present paper t o discuss d e t e c t i o n .

TABLE 1 : Column types for Micro-HPLC

Column dimensions in cm

Particle size in y.m

Plate number To at Uopt in minutes lo3

Analysis time D in 1 with Gax-10 or 4V0/Vr in minutes

Detector cell volume

1 Normal 10-30 x 0.4-0.5

5-10

10-25

1-4

10-40

h'

2 Narrow bore 10-30 x 0.2-0.3

5-10

10-25

1-4

10-40

2/

20 ,id

5-10 (11

3 Microbore 25-30 x 0.1

5-20

5-25

3-4

30-40

w

10 i d

1-2 Fl

4 High Speed 3-10 x 0.4-0.5

3-10

4-15

0.1-1

1-10

5 Packed capillary 50-200 x 0.02-0.03 200-300

3-5

50-250

6 Open capillary 50-10.000 x 0.001-0.005 10-50,km

none

-1000

(7) High Speed microbore 1-5 x 0.1

1-3

3-1 5

(8) High Speed packed capillary 10-20 x 0.02

1-2

25-100

100 qV.1

h;10-20 G1

1-4 +l

Ll

0.1-0.2

u1

1-2

nl

10-60

100-600

10-2000

100-20.000 dl0 nl

1-10 (seconds) 1-4

- 3 1

Wl pl

0.1-0.2pl

0.03 ,Lil (30 nl)

5-10 nl

0.01-1 10-40

10-20a1

"J

441

IV.l.

Particle size O n l y a few y e a r s ago t h e o p i n i o n was o f t e n expressed t h a t t h e r e was l i t t l e

f u t u r e i n p a r t i c l e s i z e s below 5 um. S i n c e t h e n however 3 ) m

can be s a i d t o be

f i r m l y e s t a b l i s h e d . We have f u r t h e r m o r e shown t h e p o s s i b i l i t y o f u s i n g 2 and even 1 ,urn p a r t i c l e s s u c c e s s f u l l y (4, 5 ) . A v e r y i m p o r t a n t c h a r a c t e r i s t i c o f these e x t r e m e l y s m a l l p a r t i c l e s i s t h a t t h e H/u c u r v e does n o t show an upswing even a t t h e h i g h e s t a t t a i n a b l e e l u e n t speeds. T h i s i s i l l u s t r a t e d by f i g . 1.

1

I

I

F i g . 1 : van Deemter p l o t f o r d i f f e r e n t p a r t i c l e s i z e s : column : 25 x 0.46 cm 15 x 0.46 cm f o r 3 p m ROSiL-C,,-D and f o r 8 and 5 , * m ROSiL-C,,-D, sample : pyrene 4 x 0.46 cm f o r 2 , m , m o b i l e phase : 75 CH,CN/25 H,O, (k Y 6). The C t e r m c o n t r i b u t i o n i n t h e van Deemter e q u a t i o n i s n e g l i g i b l e o r even z e r o o v e r a wide range o f s o l v e n t r a t e s . There i s no c o n t r i b u t i o n f r o m d i f f u s i o n i n t h e e l u e n t l i q u i d o r i n t h e s t a t i o n a r y f i l m phase. To o u r knowledge t h i s p o i n t has n o t been r a i s e d b e f o r e . One i n t e r e s t i n g a s p e c t o f t h e f l a t r e g i o n i n t h e H/u c u r v e w i t h v e r y s m a l l p a r t i c l e s i s t h a t t h e s e n s i t i v t y o f an a n a l y s i s i s independent o f f l o w r a t e . T h i s i s i l l u s t r a t e d i n f i g . 2. The chromatograms o b t a i n e d w i t h 5 Am p a r t i c l e s show a c l e a r r e d u c t i o n i n sens t i v i t y when t h e speed i s i n c r e a s e d t o f o u r t i m e s uopt. c l es.

T h i s i s n o t t h e case f o r t h e chromatograms o b t a i n e d w i t h t h e 2 hm p a r t i -

442

B

A

d ‘=I-‘

0”

30”

60”

B

A

L

F i g . 2 : S e n s i t i v i t y i n f u n c t i o n o f speed and p a r t i c l e s i z e . L e f t : column : 1.5 x 0.4 cm f i l l e d w i t h 5p.m ROSiL-C,,-D; m o b i l e phase : 75 CH,CN/25 H,O, A : 0.8 ml/min, B : 3.5 ml/min. R i g h t : column : 1.5 x 0.4 cm f i l l e d w i t h 2 Mm ROSiL-C18-D; m o b i l e phase : 75 CH,CN/25 .H,O, A : 0.8 ml/min, B : 3.5 ml/min. Sample : naphthalene, anthracene, pyrene. 1 , m i s about t h e l i m i t t h a t can be handled i n p r a c t i c e ( f i l t r a t i o n , s i e ving, d e r i v a t i s a t i o n , v i e w i n g under l i g h t microscope a.s.0.).

Based on 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 Knox p o i n t e d o u t t h a t 2 ~ pma r t i c l e s i s t h e optimum p a r t i c l e s i z e f o r HPLC ( 9 ) . From semi e m p i r i c a l equations, Halasz d e r i v e d t h a t t h e minimum p a r t i c l e s i z e i n HPLC i s between 1 and 3 / I m ( 2 7 ) . F o r even s m a l l e r p a r t i c l e s , new techniques would seem necessary and i n o u r o p i n i o n t h i s i s n o t something f o r t h e near f u t u r e . With such s m a l l p a r t i c l e s t h e m a t t e r o f f r i c t i o n h e a t d i s s i p a t i o n has t o be mentioned. Halasz e t a l . (27), H o r v a t h e t a l . ( 2 8 ) and Poppe e t a l . ( 2 9 ) have d i s c u s s e d f r o m a t h e o r e t i c a l p o i n t o f v i e w t h e i n f l u e n c e o f f r i c t i o n h e a t d i s s i p a t i o n . T h i s e f f e c t must be dependent on t h e u/dp f a c t o r and a l s o on column diameter. I t was suggested t h a t one o f t h e i n t e r e s t i n g p o i n t s o f m i c r o b o r e columns would be t h e reduced i n f l u e n c e o f f r i c t i o n h e a t and t h a t t h e r e f o r e s m a l l e r p a r t i c l e s e.g.

below 5 , ~ mi n d i a m e t e r

c o u l d be contemplated i n such columns. We have however n o t encountered adverse e f f e c t s o f f r i c t i o n heat i n t h e c o n d i t i o n s o f o u r experiments. T h i s i s e v i d e n -

443

ced by fig. 1 for 1 and 2 p m particles in 4-5 mm ID columns. Another experimental result showing that heat friction is not so important is that preparative columns of 25 cm length and 2.2 cm diameter and packed with lO,,wnparticle octadecylated silica gel generally produce higher efficencies ( . ~ 1 0 . 0 0 0 -13.000 plates) than the usual columns packed with the same material (7.000-10.000 pmates). This is also the case with eluents made up of mixtures of methanol/water which are very viscous. Guiochon (la) also comes back to these friction heat effects and calculates that they must be proportional to (ID)4.

A preparative scale column would have to do (22/1)4 or 234256 times worse than a microbore column for this factor ? ! That there is no experimental indication for this shows indeed that friction heat induced eluent velocity differences are not so important. There is therefore no reason not to use 1-2,um particles or even smaller. To the contrary, since there is no drop in sensitivity by increasing the eluent speed with such particles, ways and means to use these at the highest possible eluent velocities must be explored. IV.2. High Speed-Narrow-Microbore HPLC Microbore columns do not produce better chromatographic characteristics than usual columns as already discussed above. They do however consume less solvent. HSLC uses also less solvent per analysis, but solvent consumption per unit of time remains unchanged and is therefore high. It seems obvious that an interesting development that has not yet retained attention currently is to use shorter columns with small particles and with smaller ID. This would indeed be true Micro-HPLC since all three parameters that can be miniaturizpa are involved..A small step into that direction is illustrated by fig. 3. With the same linear speed as for the usual 4.6 mm ID column, the 3 mm ID column used in fig. 3 needs only 0.4 ml/min against 1 m l / m i n . The dimensions o f the column still allow the use of normal instrumentation (detector cell volume : 1.4)11, response time : 20 m s ) . The plate number is 5.000. Compared to a traditional column of 15 x 0.46 cm packed with 10,m particles and producing the excellent plate figure of 6.000, the micro column gives about 10 times the sensitivity. We believe that there is a future for columns between 1 and 5 cm length with 2 to 3 mm ID and packed with 1 to 3,m

particles. We believe that his is

the column that could replace the no 1 type column of table 1. This is possi-

444

Fig. 3 : F a s t s e p a r a t i o n on a Micro-HPLC system : column : 4 x 0.3 cm f i l l e d w i t h 3 PA ROSiL-Ch,-D , m o b i l e phase : 60 CH,CN/40 H,O, 1.8 ml/min, AP = 190 atm, d e t e c t i o n : 254 nm, sample : 1. b e n z y l a l c o h o l , 2. acetophenone, 3. a n i s o l e , 4. b e n z y l c h l o r i d e , 5. t o l u e n e , 6. 1 - n i t r o n a p h t h a l e n e , 7. nap h t h a l ene.

l3

0"

30"

b l e ever, today w i t h many chromatographs i n t h e f i e l d . One a t t e m p t i n t h i s d i r e c t i o n has been p u b l i s h e d by S c o t t w i t h 3 m p a r t i c l e s packed i n t o a 2.5 x 0.26 cm column. T h i s r e s u l t e d i n r a p i d s e p a r a t i o n o f a m u l t i component m i x t u r e i n l e s s t h a n 30 sec by b o t h i s o c r a t i c and g r a d i e n t e l u t i o n ( 3 0 ) . The c u r r e n t comnercial i n s t r u m e n t development w i t h 2 t o 4 ,d d e t e c t o r c e l l volume and 50 ms response t i m e o f t h e d e t e c t o r - r e c o r d e r system does n o t go f a r enough i n t h e d i r e c t i o n o f m i n i a t u r i z a t i o n . Real High Speed-Microbore HPLC e.g. w i t h a column o f 1 mm I D and about 5 cm l e n g t h i s n o t p o s s i b l e w i t h t h e s e chromatograph c h a r a c t e r i s t i c s . I t l o o k s w o r t h w h i l e however t o develop i n s t r u m e n t a t i o n t h a t c o u l d do i t . IV.3.

High Speed Packed C a p i l l a r y

Packed c a p i l l a r y columns o f 50 t o 200 cm l o n g as used by Novotny (18, 19,

31) and Yang (23) show v e r y h i g h p l a t e numbers, b u t t h e y r e q u i r e v e r y l o n g a n a l y s i s t i m e . The remarkable t h i n g about t h e s e columns i s t h a t e.g.

2 m length

i s n o t t o o l o n g f o r a c c p t a b l e p e r m e a b i l i t y w i t h 3 o r 5 p m p a r t i c l e . I n conven-

445

t i o n a l columns packed as t i g h t l y as possible, t h e maximum column l e n g t h f o r a back pressure o f 250-300 Bar f o r 3 and 5 100 cm r e s p e c t i v e l y

. This

Ldn

p a r t i c l e would be about 40 and

can o n l y mean t h a t t h e packed c a p i l l a r y columns

a r e i n f a c t o n l y l o o s e l y packed and t h u s have h i g h p e r m e a b i l i t y . Loose packing i n conventional columns would be unstable. Fig. 4 : Micro-HPLC on 1,hm ROSiL-C,,-D : column : 1.5 x 0.4 cm f i l l e d w i t h 1 m ROSiL-C,,-D, m o b i l e phase : 75 CH,CN/25 HO, 0.8 m l / m i n , d P . , ~ 8 0 atm., d e t e c t i o n : 254 nm (1.4 kl c e l l and 20 ms t i m e c o n s t a n t ) , sample : naphthalene, anthracene, pyrene ( k - 6 ) . F i r s t d i s t u r b a n c e caused by hand i n j e c t i o n . HETP 3 urn !

I 147 OOO/m

J I

Since t h i s i s n o t t h e case f o r packed c a p i l l a r i e s t h i s has t o do w i t h t h e v e r y small I D . To us i t seems acceptable t h a t t h e packing s t r u c t u r e s t a b i l i t y i s h i g h e r i n a v e r y small I D column than f o r a conventional column type. W i t h 1 p m s p h e r i c a l s i l i c a gel p a r t i c l e s ( l,m

ROSiL-C,,-HL-D

from A l l t e c h -

-RSL) t h e l o n g e s t t r a d i t i o n a l column t h a t c o u l d r u n a t a l i n e a r v e l o c i t y o f 1 mm/sec f o r a pressure o f 250-300 Bar would be about 4 cm long. W i t h t h e r e duced p l a t e h e i g h t o f 3 dp t h a t can be obtained on 1 k m octadecylated p a r t i c l e s t h i s would be e q u i v a l e n t t o 13.500 p l a t e s . The t h i n g t o do however i s t o use 1,km p a r t i c l e s i n a m i c r o packed c a p i l l a r y (fused s i l i c a column o f 0.2 mm I D ) . With t h e l o o s e packing p o s s i b l e i n such columns a 10 t o even 20 cm column does seem p o s s i b l e , This would y i e l d 30.000 t o 70.000 p l a t e s w i t h a n a l y s i s times

446

around 1 5 min. Then Micro LC would have e q u a l l e d c a p i l l a r y GC i n e f f i c i e n c y and speed. I f t h i s sounds l i k e science f i c t i o n , we again p o i n t t o t h e papers o f Novot n y (31) and Yang (32) and t o f i g u r e 4 w i t h l p m p a r t i c l e s . Whether t h e combin a t i o n o f these two experimental r e a l i t i e s i s p o s s i b l e w i l l be shown by t h e future. ACKNOWLEDGEMENTS We thank t h e "Nationaal Fonds voot Wetenschappelijk Onderzoek

-

N.F.W.O.",

t h e " I n s t i t u u t t o t Aanmoediging van h e t Wetenschappel ij k Onderzoek i n N i j v e r h e i d en Landbouw

-

I.W.O.N.L."

and t h e " M i n i s t e r i e voor Wetenschapsbeleid" f o r

f i n a n c i a l support t o t h e l a b o r a t o r y .

REFERENCES

1.

Micro column h i g h performance l i q u i d chromatography (Ed. P. Kucera), Elsev i e r 1984.

2.

Small bore l i q u i d chromatography columns : t h e i r p r o p e r t i e s and uses (Chemical Analysis Series, v o l . 42, Ed. R.P.W. S c o t t ) , Wilev, N.Y. 1984.

3.

U1 t r a h i g h r e s o l u t i o n chromatography (Ed. S a t i n d e r Ahuja) American Chemical S o c i e t y Symposium S e r i e s 250, 1984.

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M. Verzele and C. Dewaele, J . Chromatogr.,

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R. Majors, Anal. Chem. Reviews, 56 (1984) 332.

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R. Majors, L.C. Magazine, 2 (1984) 660.

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M. Dong, L.C. Magazine, 2(4) (1984) 294.

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169 (1979) 51.

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R. Daudeneau and E . Zerenner, J . HRC&CC, 2 (1979) 351.

21 *

T . Takeuchi and D. I s h i i , J . Chromatogr.,

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J . Gluckman, A. Hirose, V . McGuffin and M. Novotny, Chromatographia, 17

Takeuchi and

Tsuda and M. Novotrly, Anal. Chem.,

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213 (1981) 25.

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R. Scott, P. Kucera and M. Munroe, J. Chromatogr.,

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H. Poppe, J. Kraak, J . Huber and J . van den Berg, Chromatographia, 14 (1981) 515.

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A. Hirose, D. Wiesler and M. Novotny, Chromatographia, 18 (1984) 239.

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This Page Intentionally Left Blank

449

TRACE GAS CHROMATOGRAPHIC TECHNIQUES BELOW THE PART-PER-BILLION LEVEL ALBERT ZLATKIS, SHARY WEISNER, LABIB GHAOUI AND HENRY SHANFIELD Chemistry Department, U n i v e r s i t y o f Houston, Houston, Texas 1.

77004, U.S.A.

INTRODUCTION D i f f i c u l t i e s a r e f r e q u e n t l y encountered i n a t t e m p t i n g t o analyze d i r e c t l y

o r g a n i c compounds o f i n t e r e s t , which a r e o f t e n below t h e p a r t per b i l l i o n l e v e l . D e s p i t e t h e use o f h i g h l y s e n s i t i v e i n s t r u m e n t s , d e t e c t i o n o f t r a c e amounts of substances i n t h i s range p r e s e n t s a t e c h n i c a l c h a l l e n g e . i s o l a t i o n and

i d e n t i f i c a t i o n o f compounds which

e f f e c t s a t very low concentrations.

has grown

that

have profound p h y s i o l o g i c a l

Another a r e a o f i n t e r e s t i s t h e a n a l y s i s of

v o l a t i l e s from b i o l o g i c a l f l u i d s (e.g., between "normals"

A good example i s t h e

u r i n e ) t o seek d i s t i n c t i v e d i f f e r e n c e s

and those a f f l i c t e d by disease. minute concentrations

of

More r e c e n t l y , t h e awareness

chemical

pollutants

can

have

far-

r e a c h i n g e f f e c t s as h e a l t h hazards, f u r t h e r u n d e r s c o r i n g t h e need f o r r e l i a b l e a n a l y t i c a l techniques.

F l a v o r and o d o r a n t s t u d i e s a r e a l s o areas o f i n t e r e s t i n

t h i s a n a l y t i c a l region. A t t h e p a r t per b i l l i o n l e v e l o r l o w e r , i t has almost always been necessary t o use some c u m u l a t i v e o r c o n c e n t r a t i n g t e c h n i q u e t o o b t a i n measurable amounts o f s o u g h t - a f t e r compounds. unwanted "background"

I d e a l l y , we wish t o e l i m i n a t e as much as p o s s i b l e o f

compounds ( u s u a l l y water o r a i r ) whi 1e accumul a t i ng t h e

d e s i r e d substances q u a n t i t a t i v e l y . mise o f these two goals. distillation,

For most t e c h n i q u e s , t h e r e s u l t i s a compro-

Several approaches come t o mind r e a d i l y ;

freeze concentration,

zone m e l t i n g ,

fractional

s o l v e n t e x t r a c t i o n , chroma-

t o g r a p h i c techniques, and a d s o r p t i o n . Another recently,

approach,

which

seemed

to

i s t h e use o f d i r e c t on-column

be

unattainable

until

injection of extraordinarily

analytically

large

sample volumes i n c a p i l l a r y gas chromatographic columns.

Some o f t h e r e c e n t

work i n t h i s a r e a w i l l a l s o be presented i n t h i s survey. 2.

SELECTIVE ADSORPTION USING TENAX-GC POLYMER A wide v a r i e t y o f adsorbents have been i n v e s t i g a t e d as s e l e c t i v e t r a p p i n g

m a t e r i a l s f o r t r a c e organics.

These range from s t r o n g adsorbents l i k e a c t i v a t e d

charcoal t o re1 a t i v e l y weak p o l y m e r i c adsorbents.

D e s o r p t i o n from s t r o n g adsor-

b e n t s i n v o l v e s e i t h e r s o l v e n t e x t r a c t i o n , o r h e a t i n g t o temperatures which o f t e n r e s u l t i n chemical

changes.

Weak adsorbents,

many d e s i r e d substances t o escape.

on t h e o t h e r hand,

w i l l allow

4 50

For most a n a l y t i c a l s i t u a t i o n s i t i s c o n v e n i e n t t o sample a t ambient temperatures, without the necessity f o r c o l d traps. b e n t s which a r e e f f i c i e n t c o l l e c t o r s temperature,

It i s p r e f e r a b l e t o use adsor-

f o r many compounds o f i n t e r e s t a t room

and which d o n o t i n t r o d u c e chemical changes o r a r t i f a c t s d u r i n g

thermal d e s o r p t i o n . Tenax-GC,

a porous polymer o f 2,6-diphenyl -p-phenylene

b e a v e r s a t i l e s e l e c t i v e adsorbent.

o x i d e , has proven t o

It has a modest s u r f a c e area o f 18 m2 g - l

and has a l o w r e t e n t i o n f o r l o w m o l e c u l a r w e i g h t p o l a r substances, water.

especially

Higher m o l e c u l a r w e i g h t compounds h a v i n g r e l a t i v e l y l o w p o l a r i t y a r e

trapped and desorbed t h e r m a l l y w i t h h i g h e f f i c i e n c y .

This i s i l l u s t r a t e d i n

Table 1, which 1 i s t s t h e t r a p p i n g c a p a c i t y and d e s o r p t i o n c h a r a c t e r i s t i c s o f Tenax-GC f o r v a r i o u s o r g a n i c compounds.

T h i s i s e v a l u a t e d by i n j e c t i n g a f i x e d

amount o f each compound on t h e Tenax-GC and e l u t i n g t h e t r a p w i t h 0.5, 5.0

l i t e r s o f pure nitrogen.

c a l l y by d e s o r p t i o n a t 300°C.

1.5,

and

The amount r e t a i n e d i s determined chromatographi-

A t t h i s t e m p e r a t u r e Tenax-GC does n o t c o n t r i b u t e

d e t e c t a b l e a r t i f a c t s , due t o i t s unusual thermal s t a b i l i t y . Tenax-GC i s l i k e any o t h e r chromatographic s t a t i o n a r y phase and must be eval u a t e d from t h e p o i n t o f view o f p a r t i t i o n i n g o f a compound between adsorbent As a consequence, t h e r e s u l t s i n Table 1 a p p l y t o t h e s p e c i f i c

and c a r r i e r gas.

amount o f Tenax-GC employed i n t h e s e expe1,iments "breakthrough"

volume

i s d i r e c t l y proportional

(0.28 9 ) . to

the

That i s

o say, t h e

amount o f adsorbent.

Table 1 shows t h a t f o r m o l e c u l e s w i t h m o l e c u l a r w e i g h t s as h i g h as t h a t o f noctadecane, a d s o r p t i o n and d e s o r p t i o n a r e r e v e r s i b l e and complete. l a r weight

p o l a r compounds (e.g.,

methanol,

ethanol,

r e t e n t i o n and r e l a t i v e l y small b r e a k t h r o u g h volumes.

and

acetone

ow molecuhave l o w

Water i s n o t n o t i c e a b l y

adsorbed. Figure 1 i l l u s t r a t e s a typical

system f o r t r a p p i n g compounds from ambient

a i r o r any o t h e r source of v o l a t i l e o r g a n i c s . drawn t h r o u g h t h e t r a p p i n g tubes, series.

The a i r o r o t h e r c a r r i e r gas i s

which may b e arranged

T y p i c a l l y , a sampling t u b e w i l l be o f about 4 mm i.d.

One-quarter

i n parallel

or i n

and 1 2 cm l e n g t h .

gram o f Tenax-GC o c c u p i e s a l e n g t h o f about 9 cm, and i s h e l d i n

p l a c e by t w o p l u g s o f g l a s s wool.

Flow r a t e s a r e m a i n t a i n e d a t about 30 m l

min-* t o a l l o w s a t i s f a c t o r y c o n t a c t t i m e w i t h t h e Tenax-GC.

For many a p p l i c a -

t i o n s a t o t a l o f 5 l i t e r s o f a i r o r c a r r i e r gas i s adequate f o r 0.25

g o f adsor-

bent, where substances a r e p r e s e n t i n t h e gas below t h e p a r t per m i l l i o n l e v e l . D e s o r p t i o n i s c a r r i e d out a t 300°C b y r a p i d l y h e a t i n g t h e Tenax-GC t r a p w h i l e passing an i n e r t gas t h r o u g h i t , t h e n c o l d - t r a p p i n g t h e v o l a t i l e s ahead o f a chromatographic column.

Subsequently,

t h e c o l d t r a p i s f l a s h - h e a t e d and t h e

c o n t e n t s analyzed by chromatog r a p h i c t e c h n i q u e s . F o r example, Tenax-GC porous polymer adsorbent has been s u c c e s s f u l l y a p p l i e d t o air pollution

s t u d i e s where i t o f f e r s convenience and s i m p l i c i t y .

Figure 2

451 TABLE 1.

Recovery ( X ) o f Organic Compounds from Tenax-GC A f t e r A d s o r p t i o n a t Room Tempe r a t u r e Volume o f N2 passed t h r o u g h t r a p ( l i t e r s )

Compound

0.5

Methanol Ethanol Methyl c h l o r i d e Acetone Chloroform D i e t h y l amine Isobutanol n-Pentane Cycl ohexane n-Hexane Ethyl acetate n-Butanol To1 uene C7-C12 a1 kanes, a1 kenes Styrene, e t h y l benzene Xyl enes Py r id in es Chl orophenol s n-C13,C14,C15,C16,C17,Ci8

1 1 3 68 100 80 100 100 100 100 100 100 100 100 in0 100 100

Source of Organic Volatlles

1.5 0

n 1 2

a4 50 95 50 50 100 100 100 100 100 100 100 100 100 100

1 no

a1 kanes

,,’ ’\

;,>

100

5.0 0 0 0 0

5 1 16 9 9 20 35 35 100 100 100 100 100 100 100

Vacuum Source

TENAX GC SAMPLING TUBES (SERIES OR PARALLEL)

FIG. 1.

Schematic diagram o f t y p i c a l system f o r t r a p p i n g o r g a n i c v o l a t i l e s w i t h Tenax-GC.

i l l u s t r a t e s t h e system employed w i t h r e g u l a t i n g d e v i c e s , and t h e sampling t u b e s c o n t a i n i n g Tenax-GC adsorbent.

Sampling t u b e s were designed t o i n t e r f a c e w i t h

t h e i n j e c t i o n p o r t o f gas chromatographic equipnent.

452

3.

ON-COLUMN, Trace

LARGE VOLUME SAMPLE INJECTION I N CAPILLARY COLUMNS

level

analysis

in

conventional

capillary

gas chromatography

has

g e n e r a l l y been l i m i t e d by t h e sample s i z e which would s t i l l m a i n t a i n s a t i s f a c t o r y resolution. element-specific

I n a r e l a t i v e l y few i n s t a n c e s , i t has been p o s s i b l e t o u t i l i z e d e t e c t i o n devices,

b u t wide spectrum d e t e c t o r s such as t h e

flame i o n i z a t i o n d e t e c t o r ( F I D ) have been l i m i t e d t y p i c a l l y t o t h e p a r t per m i l l i o n range due t o sample s i z e r e s t r i c t i o n s . The

developnent

of

bonded

phase

(nonextractable

or

immobilized)

fused

s i l i c a open t u b u l a r columns (FSOT) has made i t p o s s i b l e t o employ l i q u i d oncolumn i n j e c t i o n t e c h n i q u e s w i t h o u t l o s s o f column performance.

Blomberg e t a l .

(1) have shown t h a t t h e i m m o b i l i z e d s t a t i o n a r y phase i s n o t d i s p l a c e d from t h e

c a p i l l a r y s u r f a c e by s o l v e n t o r even water.

Sandra e t a1

. (2)

have r e p o r t e d

t h a t f i l m t h i c k n e s s , f i l m homogeneity, and r e s o l u t i o n were n o t a f f e c t e d i n such bonded phase FSOT columns b y e x t e n s i v e r i n s i n g w i t h b o t h p o l a r and nonpolar solvents.

Work r e c e n t l y c a r r i e d o u t i n o u r l a b o r a t o r y showed t h a t peak symmetry

o f a r o m a t i c s was preserved i n t h i s column w i t h t h e s o l v e n t n-hexane i n amounts a s l a r g e as 100 uL,

u s i n g an on-column

i n j e c t o r c o n s t r u c t e d f o r t h i s purpose

(3). The successful i n j e c t i o n o f l a r g e samples would s i g n i f i c a n t l y improve t r a c e component a n a l y s i s .

However, t h e s o l v e n t tends t o swamp o u t component peaks.

G r o s s l y d i s t o r t e d chromatograms r e s u l t w i t h sample s i z e s o f t h i s o r d e r even when t h e " t r a c e " components a r e p r e s e n t i n re1 a t i v e l y l a r g e amounts. The work discussed h e r e u t i l i z e s o u r on-column

i n j e c t o r (3) t o introduce

sample volumes o f up t o 100 p L i n t o bonded phase FSOT columns.

The f i r s t s t e p

causes t h e l i q u i d sample t o pass t h r o u g h t h e column i n o r d e r t o adsorb t h e t r a c e components o f i n t e r e s t a t r e l a t i v e l y l o w t e m p e r a t u r e , p e r m i t t i n g t h e s t r i p p i n g s o l v e n t t o l e a v e t h e column.

The second s t e p r e c o v e r s t h e s e t r a c e substances i n

a l i q u i d n i t r o g e n t r a p ; f i n a l l y , a n a l y s i s i s c a r r i e d out i n t h e same column b y operating

i t i n reverse.

"heart-cutt ing" technique

.

I n essence,

A s t o c k s o l u t i o n o f n-nonane,

t h i s procedure m o u n t s t o a m o d i f i e d

n-decane

and n-undecane was prepared i n n-

hexane t o a c o n c e n t r a t i o n o f 5 ng/r.L f o r each component.

T h i s was used t o

o b t a i n r e t e n t i o n t i m e d a t a , and as a s t o c k s o l u t i o n f o r d i l u t i o n t o approximat e l y ppb f o r o t h e r experiments. hexane w r e a1 so prepared

S o l u t i o n s o f t h e same c o n c e n t r a t i o n s i n n-

f o r 2-octanone,

5-nonanoneY and 2-decanone.

bonded phase FSOT column used was a DR-1 60 m x 0.32 mm i.d.,

The

1.0 um f i l m , w h i c h

i s a nonpolar, hydrophobic SE-30 c r o s s - l i n k e d p o l y m e r i c phase. Table 1 summarizes t h e a n a l y t i c a l d a t a f o r t h r e e r u n s w i t h a 1 U L sample o f t h e s t o c k s o l u t i o n o f n-nonane, on-column i n j e c t i o n . components.

n-decane,

and n-undecane ( 5 ng/pL each) u s i n g

T h i s p r o v i d e d r e t e n t i o n t i m e and area c o u n t d a t a f o r t h e s e

453 The tubes c o n t a i n i n g t h e adsorbent were p r e c o n d i t i o n e d a t 350°C i n a stream A heater

o f p u r e n i t r o g e n , t h e n a l l o w e d t o c o o l i n screw-capped t e s t t u b e s . s u r r o u n d i n g t h e Tenax-GC

t u b e was used f o r d e s o r p t i o n o f v o l a t i l e s a t about

3OOOC i n t o a c r y o g e n i c precolumn t r a p . A n a l y s i s was c a r r i e d o u t i n a h i g h - r e s o l u t i o n

c a p i l l a r y column.

Several

hundred compounds were observed, n e a r l y one hundred o f which were i d e n t i f i e d by mass s p e c t r o m e t r y .

The r e p r o d u c i b i l i t y f o r s u c c e s s i v e sampl i n g s i s e x c e l l e n t .

Two o t h e r examples o f t h e a p p l i c a t i o n o f Tenax-GC adsorbent w i l l be n o t e d here.

The f i r s t

of t h e s e i s a s t u d y o f e x h a l e d tobacco smoke.

Specific

r e f e r e n c e c i g a r e t t e s were smoked under t h e s t a n d a r d c o n d i t i o n s used by i n d u s t r y , and 3.5 l i t e r a i r

samples

containing the

exhaled smoke

were

passed

through

f ,

FIG. 2.

Schematic diagram o f t r a p p i n g system components: 1, t r a p s ; 2, r o t a m e t e r s ; 3, n e e d l e v a l v e s ; 4, pump; 5, f l o w e t e r .

Tenax-GC

adsorbent.

cigarettes.

F o r comparison,

smoke samples were t a k e n d i r e c t l y from

The sampling tubes were desorbed i n t h e usual manner, and t h e gases

analyzed by GC-MS.

The r e p r o d u c i b i l i t y o f sample p r o f i l e s was s u r p r i s i n g l y

good. Another

area o f r e s e a r c h t o which Tenax-GC has been a p p l i e d i s t h a t o f

d e t e r m i n i n g t h e t r a c e contaminants i n water f o r a p o l l u t i o n study. t h e h y d r o p h o b i c i t y o f Tenax-GC works t o g r e a t advantage. a d s o r p t i o n o c c u r s f o r compounds above Cg.

Here again

As u s u a l , s i g n i f i c a n t

454

TABLE 1.

A n a l y t i c a l Data f o r 1 V L On-Column I n j e c t i o n o f n-Hexane C o n t a i n i n g 5 ng o f n-Nonane, n-Decane, and n-Undecane n- no n an e

n-undecane

n-decane

area count

retention t i m e , min

area count

retention t i m e , min

area count

retention time, min

3646 3737 3750

21.68 21.71 21.71

3691 3779 3798

29.67 29.69 29.69

3893 3986 401 2

37.84 37.85 37.85

3711 mean s t d dev 56.7 re1 s t d dev ( % ) 1.53

21.70 0.017 0.08

3756 57.1 1.52

29.68 0.012 0.04

3964 62.6 1.56

37.85 0.0058 0.02

T h i s s t o c k s o l u t i o n was t h e n d i l u t e d one h u n d r e d f o l d w i t h n-hexane ng/VL f o r each component) and 100 pL o f t h i s s o l u t i o n i n j e c t e d . o p e r a t i o n , t h e column was n o t connected t o t h e d e t e c t o r .

(0.05

During t h i s

L i q u i d was observed

b u b b l i n g from t h e column o u t l e t a t 6.0 minutes, and i t ceased a t 16.2 min.

At

t h e 15.1 min mark, t h e column o u t l e t f l o w was d i r e c t e d i n t o a s t a i n l e s s s t e e l t r a p immersed i n l i q u i d n i t r o g e n .

Then t h e oven t e m p e r a t u r e was r a i s e d t o 150°C

t o desorb t r a c e substances from t h e column. and t h e column was reversed, i.e.

A f t e r 20 min, t h e oven was c o o l e d ,

t h e o u t l e t o f t h e t r a p was connected t o t h e

i n j e c t o r , and t h e o t h e r end o f t h e column t o t h e d e t e c t o r .

Table 2 summarizes

t h e a n a l y t i c a l d a t a f o r t h r e e runs. TABLE 2.

A n a l y t i c a l Data f o r 100 V L On-Column I n j e c t i o n o f n-Hexane C o n t a i n i n g 5 ng o f n-Nonane, n-Decane, and n-Undecane n- no nan e

n-und ec ane

n-d ec an e

area count

retention t i m e , min

area count

retention time, min

401 7 2 602 3843

21.88 21.65 21.90

4402 4096 3582

29.78 29.73 29.86

4473 3562 3516

37.87 37.89 37.85

mean 3487 s t d dev 772 re1 s t d dev ( % ) 22.1

21.81 0.14 0.64

402 7 41 4 10.3

29.79 0.065 0.22

3850 540 14.0

37.87 0.020 0.053

94.2

107

area r e t e n t i o n c o u n t time, min

97.1

The r e l a t i v e standard d e v i a t i o n s observed i n t h e 100 pL i n j e c t i o n experiments were s u b s t a n t i a l l y l a r g e r than i n t h e 1 p L d a t a ( T a b l e 1 ) .

Nevertheless, t h e

p e r c e n t r e c o v e r y was o v e r 90% f o r each o f t h e standards. Table 3 l i s t s t h e a n a l y t i c a l d a t a o b t a i n e d f o r t h r e e r u n s u s i n g t h e s t o c k solution

c o n t a i n i n g 5 ng each o f

2-octanone,

5-nonanone,

and 2-decanone ( 1 V L

455

on-column i n j e c t i o n ) .

T h i s was used t o e s t a b l i s h r e t e n t i o n t i m e and area c o u n t

data. TABLE 3.

A n a l y t i c a l Data f o r 1 pL On-Column I n j e c t i o n o f n-Hexane C o n t a i n i n g 5 ng o f 2-0ctanone, 5-Nonanone, and 2-Decanone 2-octanone

5 -no nano ne

2 -decanone

area count

retention t i m e , min

area count

retention t i m e , min

area count

3110 3120 31 34

27.02 27.02 26.99

3372 341 5 341 6

33.93 33.93 33.90

3023 3036 3051

43.43 43.41 43.38

mean 3121 s t d dev 12.1 re1 s t d dev ( % ) 0.39

27.01 0.017 0.063

3401 25.1 0.74

33.92 0.017 0.050

303 7 14.0 0.46

43.41 0.025 0.058

Table 4 l i s t s t h e a n a l y t i c a l

data

retention time, min

f o r t h r e e r u n s o f t h e same s o l u t i o n

d i l u t e d 1 0 0 - f o l d , w i t h 100 pL i n j e c t e d ( 5 ng o f each component t o t a l ) .

Recovery

exceeded 90% f o r each o f t h e standards used. TABLE 4.

A n a l y t i c a l Data f o r 100 uL On-Column I n j e c t i o n o f n-Hexane C o n t a i n i n g 5 ng o f 2-0ctanone, 5-NonanoneY and 2-Oecanone 2-octanone

mean s t d dev r e 1 s t d dev ( X ) recovery ( % )

5-nonanone

2-decanone

area count

retention t i m e , min

area count

retention t i m e , min

4521 4103 3359

27.22 27.17 27.08

383 6 3140 3223

34.10 34.06 33.99

3251 271 4 2 786

43.57 43.54 43.47

3994 589 14.7

27.16 0.071 0.26

3400 380 11.2

34.05 0.056 0.16

291 7 291 9.98

43.53 0.051 0.12

128

area r e t e n t i o n c o u n t time, m i n

100

96

The d a t a r e p o r t e d f o r t h e hydrocarbons and ketones a r e a t a c o n c e n t r a t i o n level

o f approximately

50 ppb.

However,

by o p e r a t i n g t h e flame i o n i z a t i o n

d e t e c t o r a t i t s maximum s e n s i t i v i t y ( w i t h two t o one s i g n a l t o n o i s e r a t i o ) , i t would be f e a s i b l e t o analyze f o r t h e s e substances below t h e p a r t per b i l l i o n level.

With t h i s technique,

cumbersome (and o f t e n n o n q u a n t i t a t i v e )

c e n t r a t i o n steps can be avoided i n many analyses, e.g., pollutants.

Alternatively,

t h e use o f c o n c e n t r a t i o n steps can achieve sen-

s i t i v i t y a t t h e p a r t per t r i l l i o n l e v e l o r even l o w e r . this

method c o u l d

also be

precon-

analysis o f p r i o r i t y

enhanced

by t h e

use o f more

The s e n s i t i v i t y o f selective

detection

456 systems i n c l u d i n g e l e c t r o n c a p t u r e ,

f l a m e photometry, o r p h o t o i o n i z a t i o n .

We

a l s o f e l t t h a t t h i s work e s t a b l i s h e d t h e p o t e n t i a l f e a s i b i l i t y o f a n a l y z i n g t h e t r a c e i m p u r i t i e s f o r one substance d i s s o l v e d i n another s o l v e n t .

T h i s would

i n v o l v e a dual e l i m i n a t i o n ; one f o r t h e s o l v e n t , and t h e o t h e r f o r t h e p r i n c i p a l s o l Ute.

I n a second s e r i e s o f experiments, on-column i n j e c t i o n o f up t o 250 p L o f npentane s o l u t i o n s o f halogenated hydrocarbons was s u c c e s s f u l l y c a r r i e d o u t , u t i l i z i n g an e l e c t r o n c a p t u r e d e t e c t o r (ECD). an ECO,

Since n-pentane does n o t respond t o

i t was e l i m i n a t e d from t h e chromatogram ( e x c e p t f o r s o l v e n t i m p u r i t i e s

r e s p o n s i v e t o ECD).

For t h i s work we sought t o analyze b o t h l o w - b o i l i n g and

h i g h - b o i l i n g halogenated hydrocarbons u s i n g two s t u c k s o l u t i o n s .

The f i r s t con-

t a i n e d t h e l o w - b o i l i n g canpounds d i c h l o r o m e t h a n e ( 6 0 ppm), c h l o r o f o r m (1.0 ppm), and t h e h i g h - b o i l i n g compounds l i n d a n e ( 4 x and e n d r i n ( 4 x 10-3 ppm),

ppm), h e p t a c h l o r ( 4 x 10-3 ppm)

T h i s was used t o f u r t h e r p r e p a r e t h r e e s o l u t i o n s

d i l u t e d w i t h n-pentane by f a c t o r s o f 50,

100 and 250.

A second n-pentane s t o c k

s o l u t i o n , c o n t a i n i n g o n l y d i c h l o r o m e t h a n e ( 6 0 ppm) and c h l o r o f o r m (1.0 ppm) was prepared, from which t h r e e a d d i t i o n a l s o l u t i o n s were prepared, d i l u t e d w i t h n-

lo2, l o 4

pentane by f a c t o r s o f Two bonded

phase,

fused

and 106. s i l i c a c a p i l l a r y columns (FSOT) were j o i n e d

s e r i e s w i t h a s t a i n l e s s s t e e l t r a p u s i n g low dead-volume unions. column (upstream) was 22 m x 0.32

film. pm

5

mm i.d.,

pm

cross-linked

in

The f i r s t

methylsilicone

The second column, a l s o FSOT, (downstream) was 25 m x 0.32 mm i.d.,

0.30

c r o s s - l i n k e d OV-17 f i l m . One s e t o f experiments was conducted based on t h e f i r s t s t o c k s o l u t i o n , t o

observe t h e e f f e c t o f sample volume i n j e c t e d . 250 p L o f t h e t h r e e d i l u t e d s o l u t i o n s (50-, were i n j e c t e d . d i t i o n s were: 2'C/min

For t h i s purpose,

loo-,

T h i s p r o v i d e d a c o n s t a n t amount o f s o l u t i o n s . 40'C

50,

100, and

and 2 5 0 - f o l d y r e s p e c t i v e l y ) I n s t r u m e n t con-

oven t e m p e r a t u r e f o r 5 min, f o l l o w e d by programming up a t

( f o r 1 7 min i n t h e case o f t h e 50 and 100 p L samples, f o r 27 min i n t h e

case o f t h e 250 uL sample).

The helium c a r r i e r gas f l o w r a t e was 1.5 ml/min.

F o l l o w i n g these r e s p e c t i v e p e r i o d s , t h e s t a i n 1 ess s t e e l i n t e r c o n n e c t i n g t r a p was immersed i n l i q u i d n i t r o g e n , and t h e t e m p e r a t u r e r a i s e d t o 190°C f o r a p e r i o d o f 27 min t o t h e r m a l l y desorb (and t r a p ) t h e h i g h - b o i l i n g compounds from t h e f i r s t column ( t h i c k e r bonded phase). was reduced t o 70°C,

A t t h e end o f t h a t p e r i o d , t h e oven t e m p e r a t u r e

t h e l i q u i d n i t r o g e n removed and t h e a n a l y s i s resumed,

programming t h e temperature up a t 5'C/min

t o a maximum o f 220'C.

C a r r i e r gas

f l o w r a t e was m a i n t a i n e d a t 1.5 ml/min.

A

second

set

of

experiment's

was

based

on

the

second

stock

i n j e c t i n g 100 p L o f t h e s o l u t i o n s d i l u t e d by f a c t o r s o f 102,

104,

solution, and 106.

I n s t r u m e n t c o n d i t i o n s were as p r e v i o u s l y noted, except t h a t t h e c a r r i e r gas f l o w

457 r a t e was i n c r e a s e d t o 7 ml/min.

These e x p e r i m e n t s were r u n t o p r o v i d e d i f f e r i n g

c o n c e n t r a t i o n s and i n f o r m a t i o n on s o l v e n t i m p u r i t i e s .

A t h i r d s e t o f e x p e r i m e n t s c o n s i s t e d o f t h e a n a l y s i s o f 1.0 u L o f t h e f i r s t s t o c k s o l u t i o n (1.0 uL on-column

i n j e c t i o n o f undiluted solution),

which were

used t o e s t a b l i s h r e t e n t i o n t i m e s and area c o u n t s as shown i n Table 5.

Elution

o f n-pentane was m o n i t o r e d t h r o u g h t h e use o f t h e flame i o n i z a t i o n d e t e c t o r . TABLE 5.

A n a l y t i c a l d a t a f o r t h r e e runs: s o l u t i o n (area counts)

D i c h l oromethane Me an Std. Dev. Re1 Std. Dev. %

.

S o l u t i o n ppm

1.0 u L o f u n d i l u t e d n-pentane s t o c k

Chloroform

Lindane

4841 123 2.5

10158 644 6.3

3525 52.7 1.5

3 728 130 3.5

60

1.0

10-3

4 x 10-3

4

Heptachlor

Endrin

4166 46.5 1.1 4

10-3

T a b l e 6 summarizes d a t a f o r t h e 5 0 - f o l d d i l u t e d s t o c k s o l u t i o n ( o f Table 5 ) . Here,

50 u L were i n j e c t e d ,

p r o v i d i n g t h e same a b s o l u t e m o u n t s o f

on-column,

each compound. TABLE 6.

Analytical data f o r three runs: sol u t i o n (area counts)

Dichloromethane

50 pL o f 5 0 - f o l d d i l u t e d s t o c k

Chl o r o f o r m

Lindane

Heptachlor

2 752 103 3.7

Me an Std. Dev. Re1 Std Dev.

3 756 42 1 11

9340 257 2.7

4107 20.0 0.49

S o l u t e ppb

1200

20

0.08

.

0.08

Endrin

2378 187 7.8 0.08

T a b l e 7 slmmarizes d a t a f o r a 100 uL on-column i n j e c t i o n o f 1 0 0 - f o l d d i l u t e d stock s o l u t i o n .

4 58

TABLE 7.

A n a l y t i c a l d a t a f o r t h r e e runs: s o l u t i o n (area counts)

Dichl ormethane Mean Std. Dev. R e l . Std. Dev. % S o l u t e ppb

100 pL o f 1 0 0 - f o l d d i l u t e d s t o c k

Chl oroform

Lindane

Heptachl o r

Endrin

4117 182 4.4

8510 175 2.1

4241 145 3.4

2579 245 9.5

3163 70.5 2.2

600

10

0.04

0.04

0.04

Table 8 summarizes d a t a f o r a 250 pL on-column i n j e c t i o n o f 2 5 0 - f o l d d i l u t e d stock solution.

This d i l u t i o n b r i n g s t h e concentration o f lindane, heptachlor

and e n d r i n i n t o t h e p a r t per t r i l l i o n range. TABLE 8.

A n a l y t i c a l d a t a f o r t h r e e runs: s o l u t i o n (area counts)

D i c h l oromethane Mean Std. Dev. Rel. Std. Dev. X Solute ppt

4312 1043 24 240,000

250 UL o f 2 5 0 - f o l d d i l u t e d s t o c k

Chloroform

Lindane

Heptachlor

Endrin

890 3 753 8.4

3411 149 4.4

2570 114 4.4

2175 98.8 4.5

4000

16

16

16

Chromatograms were o b t a i n e d f o r 100 pL on-column

i n j e c t e d samples o f t h e

d i l u t e d second s t o c k s o l u t i o n (102, 104, and 106 f o l d ) which c o n t a i n e d t h e l o w b o i l i n g dichloromethane and c h l o r o f o r m o n l y i n t h e n-pentane s o l v e n t ( s e e Table 9): TABLE 9.

C o n c e n t r a t i o n s and a b s o l u t e amounts o f D i c h l oromethane and Chloroform (100 U L ) .

Dilution ratio 102 104 106

Dichl ormethane Conc Abs. Amt.

.

600 ppb 6.0 ppb 60 PPt

60 ng 600 P9 6 P9

Chloroform Conc Abs. ht.

.

10 PPb 0.10 ppb 1.0 p p t

1.0 ng 10 P9 0.10 pg

4 59

We concluded t h a t t h e a n a l y s i s o f b o t h l o w - b o i l i n g and h i g h - b o i l i n g halogenated hydrocarbons i s f e a s i b l e a t t h e p a r t per t r i l l i o n l e v e l u s i n g t h i s procedure.

The use o f n-pentane as s o l v e n t p e r m i t s d e t e c t i o n o f t h e s e s o l u t e s w i t h

ECD.

We noted, however, t h a t s o l v e n t p u r i t y becomes h i g h l y i m p o r t a n t a t such

trace levels,

and f o r more e x a c t i n g work,

i t would be necessary t o improve t h e

p u r i t y o f t h e s o l v e n t , p r o b a b l y t h r o u g h a chromatographic procedure. REFERENCES

1

L. Blomberg,

J. B u i t j e n , K. Markides, and T. Wannman, J. Chromatogr.,

239

(1982) 51-60. 2 3

Sandra, G. Redant, E. Schacht, and M. Verzele, J. High Resolut. Chromatogr., 4 (1981) 411-412. F.-S. Wang, H. S h a n f i e l d , and A. Z l a t k i s , Anal. Chem., 54 (1982) 1886-1888.

P.

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461

EARTH'S CHANGING ATMOSPHERE F . SHERWOOD ROWLAND Department of C h e m i s t r y , U n i v e r s i t y of C a l i f o r n i a , I r v i n e (U.S.A.

)

ATMOSPHERIC COMPOSITION N i t r o g e n a n d oxygen have been known f o r a b o u t two c e n t u r i e s t o compose a b o u t 4 / 5 and 1 / 5 , r e s p e c t i v e l y , of t h e e a r t h ' s p r e s e n t a t m o s p h e r e .

S l o w l y and pro-

g r e s s i v e l y d u r i n g t h e 1800s and t h e 1 9 0 0 s , o t h e r c h e m i c a l s p e c i e s h a v e b e e n i d e n tified--the

n o b l e g a s a r g o n , c a r b o n d i o x i d e , methane, h y d r o g e n , n i t r o u s o x i d e ,

o z o n e , ammonia, n i t r i c o x i d e and n i t r o g e n d i o x i d e , c a r b o n monoxide, h e l i u m and t h e r e m a i n i n g n o b l e g a s e s , and of c o u r s e , water v a p o r .

A l i s t of a t m o s p h e r i c

components p r e s e n t i n c o n c e n t r a t i o n s e x c e e d i n g one p a r t p e r m i l l i o n by volume (ppmv) c o n t a i n s n i n e m o l e c u l e s , a s g i v e n i n T a b l e 1 .

The t o t a l number of compo-

n e n t s l i s t e d i n t h e 1976 S t a n d a r d Atmosphere ( r e f . 1 ) and i n Table 1 is 1 9 , w i t h 17 h a v i n g c o n c e n t r a t i o n s of 1 p a r t p e r b i l l i o n by volume ( p p b v , E x t e n s i o n t o p a r t s p e r b i l l i o n by volume ( p p b v , lo-')

or g r e a t e r .

b r i n g s t h e number of chem-

i c a l s p e c i e s i n T a b l e 1 t o 1 7 , p l u s two more w i t h c o n c e n t r a t i o n s less t h a n 1 ppbv. A l t h o u g h s t r o n g e v i d e n c e e x i s t s t o show t h a t t h e a t m o s p h e r e had a v e r y d i f f e r e n t c o m p o s i t i o n two or t h r e e b i l l i o n y e a r s a g o ( r e f . 2 1 , t h e components o f t h e 1976 S t a n d a r d Atmosphere were a l l a s s i g n e d f i x e d t r o p o s p h e r i c c o n c e n t r a t i o n s w i t h t h e e x c e p t i o n of C 0 2 which was acknowledged t o be i n c r e a s i n g i n c o n t e m p o r a r y times. However, t h e c o n c e p t s l B p u r i t y t ' and l'compositionlT a r e a l w a y s f u n c t i o n s of t h e a v a i l a b l e a n a l y t i c a l t e c h n i q u e s , and t h o s e a v a i l a b l e f o r a p p l i c a t i o n t o t h e a t m o s p h e r e h a v e improved g r e a t l y i n b o t h p r e c i s i o n and s e n s i t i v i t y i n t h e p a s t 15 y e a r s .

C o n c u r r e n t l y , an enormous e x p l o s i o n o f i n t e r e s t i n and knowledge o f

a t m o s p h e r i c c h e m i s t r y h a s a l s o o c c u r r e d , t r i g g e r e d by s e v e r a l r e l a t e d d e v e l o p ments.

F i r s t , t h e a v a i l a b i l i t y of g a s chromatography a s a s e p a r a t i o n t e c h n i q u e

h a s p e r m i t t e d t h e c o n v e n i e n t i s o l a t i o n of a m u l t i t u d e of t r a c e components. S e c o n d , t h e development of some s p e c i f i c d e t e c t i o n t e c h n i q u e s c a p a b l e o f g r e a t s e n s i t i v i t y h a s made p o s s i b l e t h e q u a n t i t a t i v e a s s a y o f t h e s e t r a c e components a t l e v e l s f a r below t h e ppmv o r ppbv limits p r e v i o u s l y a p p l i c a b l e .

The demonstra-

t i o n by Lovelock ( r e f . 3 ) t h a t e l e c t r o n - c a p t u r e d e t e c t o r s p e r m i t t e d t h e d i r e c t a s s a y of many h a l o g e n a t e d h y d r o c a r b o n s and f l u o r o c a r b o n s a t t h e p a r t s p e r t r i l l i o n (pptv,

l e v e l i n t h e a m b i e n t a t m o s p h e r e l e d t h e way i n t o t h e p r e v i -

ously inaccessible s e n s i t i v i t y ranges.

Lists of known a t m o s p h e r i c components can

now e a s i l y c o n t a i n 100 or more c h e m i c a l s p e c i e s , i n c l u d i n g many p r e s e n t i n t h e

46 2

range of 10-12-10-15p a r t s by volume.

Third, and not n e c e s s a r i l y least--although

obviously dependent upon t h e f i r s t two f a c t o r s - - t h e chemical and photochemical i n t e r a c t i o n s of t r a c e s p e c i e s i n concentrations l e s s than 1 ppbv have proven t o be of g r e a t s i g n i f i c a n c e t o t h e o v e r a l l chemistry of t h e atmosphere d e s p i t e t h e i r minuscule concentrations ( r e f s . 4-6).

F i n a l l y , t h e a p p l i c a t i o n of s e n s i t i v e , pre-

c i s e a n a l y t i c a l techniques has l e d a f t e r a few years of measurements t o t h e conf i r m a t i o n t h a t t h e t r a c e composition of t h e atmosphere has been changing s t e a d i l y over r e c e n t decades, and w i l l c e r t a i n l y continue t o change i n t h e f u t u r e ( r e f . 6). Continually i n c r e a s i n g concentrations have been demonstrated f o r many gaseous species:

C H 4 , N20, HF, C C 1 F , CC12F2, CH CC13, C C 1 4 , CC12FCC1F2, probably C O , CF4

and SF,-, e t c .

3 3 Changes have a l s o been reported i n aerosol concentrations and i n

t h e a c i d i t y of p r e c i p i t a t i o n a s i t leaves t h e atmosphere.

This perception of

steady a l t e r a t i o n i n atmospheric composition has forced c o n s i d e r a t i o n of s e v e r a l important p o t e n t i a l problems which may e a s i l y worsen i n the f u t u r e :

t h e warming

of the atmosphere through t h e greenhouse e f f e c t ( r e f . 7 ) ; a l t e r a t i o n of the biosphere through increased a c i d i t y of p r e c i p i t a t i o n ( r e f . 8 ) ; d e p l e t i o n of s t r a t o s p h e r i c ozone ( r e f s . 9,lO); and extension of t h e world's d e s e r t s . TABLE 1

Composition of t h e E a r t h ' s atmosphere ( r e f . 1). Gas s p e c i e s Nitrogen, N2 Oxygen, O2 Argon, Ar Carbon Dioxide, c02 Neon, Ne Helium, He Krypton, Kr Xenon, Xe Methane, CH4 Hydrogen, H 2 Water, H20

F r a c t i o n a l Volume

0.7808 0.2095 0.00934 0.000314 0.0000182 0.00000524 0.000001 14 0.000000087 Variable from

0.000002 0.0000005 t o 0.035

Gas s p e c i e s

ppbv (lo-')

Carbon Dioxide, C02 Methane, CH4 Hydrogen, H Nitrous O x i g e , N20 Carbon Monoxide, CO Ozone, 0 Ammonia, 3NH S u l f u r Diox?de, SO Nitrogen Dioxide, N i t r i c Oxide, NO Hydrogen S u l f i d e , H2S

322,000 1500 500 270

190 40 4 1

1 0.5 0.05

463 The original explanation by Chapman (ref. 11) in 1930 of the qualitative existence and quantitative amounts of stratospheric ozone relied solely upon reactions involving species of oxygen:

02, 0 and 0. The description of stratospheric

3

chemistry became slowly more complicated for the next four decades, and has been accelerating rapidly in its complexity for the past 15 years. Dutsch's 1970 Outline of the chemistry of stratospheric ozone (ref. 12) included nine components (02, 0 3 , 0, 0*,H, HO, H02, H 0, H 0 ) and 17 chemical reactions, and was labeled 2 2 2 by him as "very much complicated'' by the necessity for including five hydrogenous species as well as four varieties of oxygen.

In contrast, a typical current at-

mospheric model applied to stratospheric ozone will now contain about 50 chemical species and 200 chemical and photochemical reactions, even while excluding many species known to be present in the stratosphere (e.g. all of the sulfur-containing compounds; all but a few of the chlorofluorocarbons).

Some of the complicating

features of the recent advances in atmospheric chemistry are already illustrated in Dutsch's list of components: the presence of highly reactive free radicals such of HO and H02; and the existence in the atmosphere of molecules such as H202,

which are not released directly in that form but rather were created in situ by the chemical reactions of other species. In current atmospheric modeling, the expanded rosters of important chemical species now include other related free CH 0, CH 0 ) and other molecules 3 3 2 formed by cross-termination or other interactions of free radical chains (HOC1, radical families (NO, NO2, NO3; C1, C10; CH

3'

HON02, H02N02, C10N02, CH20, CH ON02, CH 0 H, etc.) 3 3 2 Our primary concern here will, however, be with those reasonably stable molecules which have been released directly to the atmosphere, usually at the surface, and which have survived long enough for meteorological transport to most remote surface sites of the earth.

These gases have generally been identified and meas-

ured through gas chromatographic investigation of air samples collected in the troposphere (surface to 10-15 km) or lower stratosphere (10-15 to 30 km).

Such

molecular specles are often decomposed in subsequent photochemical or chemical steps and become source molecules for the free radicals, and therefore indirectly also for the molecules formed in situ in the atmosphere. For example, the longlived (estimated as 100 to 150 years) source molecule supplying NO and NO2 to the stratosphere is N20 released at the earth's surface by bacterial action, transported widely in the ensuing decades, and eventually decomposed near 30 km 1

either by direct solar photolysis or indirectly through reaction with O( D) atoms released by photolysis of ozone.

Similarly, the most important immediate source 1

is the decomposition of H20 by reaction with O ( D) atoms. A major 2 carrier of hydrogen atoms to the stratosphere is CH,,, which is decomposed there for HO and HO

by reaction with HO or O ( 1 D), releasing the H atoms to appear as HO, H02 and then H20.

At the beginning of the 20th century, the major source for stratospheric C1

and C10 was CH C 1 released a t t h e s u r f a c e i n various b i o l o g i c a l processes. Most 3 molecules of CH C 1 a r e decomposed i n t h e troposphere, b u t some p e n e t r a t e t o t h e 3 s t r a t o s p h e r e and a r e destroyed t h e r e with t h e r e l e a s e of atomic c h l o r i n e . The r e a c t i o n of C 1 w i t h 0

then formed C10, and long r e a c t i o n chains followed. 3 Many of these longer-lived molecular s p e c i e s i n t h e troposphere a r e carbon-

containing organic compounds, including:

( a ) hydrocarbons: a l k a n e s , a l k e n e s ,

alkynes, benzene and terpenes; ( b ) halocarbons such a s CC12F2, C C 1 F, CH3C1, 3 CH B r , CF,,, C H C 1 - C C l 2 and C C 1 2 = C C 1 2 ; ( c ) s u l f u r - c o n t a i n i n g mole-

C C 1 4 , CH3CC13,

3

cules such a s O=C=S, CS2, CH SH, CH SCH and CH SSCH3; and ( d ) various o t h e r 3 3 3 3 organic s p e c i e s such a s a c e t o n i t r i l e (CH CN), acetone ( C H COCH ) , formaldehyde 3 3 3 (CH20) and acetaldehyde (CH C H O ) . The r e l e a s e of C 1 and C10 i n t h e s t r a t o s p h e r e 3 i s now dominated by t h e decomposition t h e r e of gases r e l e a s e d a t t h e s u r f a c e by t h e technological a c t i v i t i e s of man, c h i e f l y involving CC12F2, C C 1 F , C C I Q and 3 The f i r s t t h r e e of t h e s e halocarbon molecules do not contain C-H bonds 3 3' and a r e e s s e n t i a l l y i n e r t i n t h e troposphere, s o t h a t t h e i r only important f a t e s

CH C C 1

involve s t r a t o s p h e r i c decomposition i n i t i a t e d by s o l a r r a d i a t i o n . SOURCES, SINKS AND ATMOSPHERIC TRANSPORT The atmospheric concentration of any chemical s p e c i e s is determined by t h r e e general processes--the p r o d u c t i v i t y of i t s s o u r c e s , t h e s t r e n g t h of its s i n k s , and t h e r a p i d i t y of t r a n s p o r t from source t o s i n k .

Most of t h e removal processes

which a f f e c t t r a c e chemicals i n t h e atmosphere f a l l i n t o one of s e v e r a l c l a s s e s : ( a ) d i r e c t s o l a r p h o t o l y s i s ; ( b ) heterogeneous i n t e r a c t i o n s , such a s attachment t o d u s t p a r t i c l e s or d i s s o l u t i o n i n t o r a i n ; and ( c ) r e a c t i o n with a c t i v e chemical

s p e c i e s , o f t e n those c r e a t e d by t h e r e a c t i o n s i n i t i a t e d by s o l a r p h o t o l y s i s .

The

daylight i n t e n s i t y of v i s i b l e r a d i a t i o n is s o g r e a t throughout t h e atmosphere t h a t e s s e n t i a l l y a l l s p e c i e s which absorb i n t h e v i s i b l e region ( i . e . a r e c o l o r e d ) a r e r a p i d l y decomposed, and play no s i g n i f i c a n t r o l e i n t h e chemistry of t h e atmosphere.

The only important p a r t i a l exception t o t h i s conclusion i s t h e brown NO

observed i n photochemical smog.

2 The s o l a r photodecomposition of NO2 t o NO p l u s 0

is indeed very r a p i d , r e q u i r i n g only a few minutes a t ground l e v e l during d a y l i g h t

However, t h e product NO r e a c t s almost immediately with 0 t o form NO2 3 again, preventing t h e complete disappearance of NO2 even i n b r i g h t s u n l i g h t . An

hours.

appreciable s t e a d y - s t a t e concentration of both NO and NO2 is maintained throughout t h e s u n l i t hours u n t i l s u n s e t terminates s o l a r p h o t o l y t i c processes and t h e NOX molecules accumulate overnight a s NO2 ( p l u s some NO

3

and N205).

Many molecules t r a n s p a r e n t t o v i s i b l e l i g h t a r e n e v e r t h e l e s s s h o r t - l i v e d i n t h e atmosphere because of absorption of r a d i a t i o n i n t h e near u l t r a v i o l e t .

The c r i t i -

c a l wavelengths f o r u l t r a v i o l e t photolysis i n t h e troposphere a r e those between 400 nm and 295 nm, t h e l a t t e r being t h e "ozone c u t o f f " f o r s o l a r

near t h e s u r f a c e .

U.V.

radiation

Wavelengths s h o r t e r than 295 nm a r e e s s e n t i a l l y completely

465

o r by absorption i n e i t h e r 0 or 3' 3 Almost any measurable absorption c r o s s s e c t i o n

removed i n t h e s t r a t o s p h e r e by absorption i n 0

f o r wavelengths below 242 nm. 2 f o r wavelengths longer than 295 nm i s s u f f i c i e n t t o l i m i t t h e atmospheric l i f e t i m e

0

f o r a molecule t o a year or two, and most absorbing chemical s p e c i e s have s u r v i v a l times measured i n days, hours o r minutes.

Solar u l t r a v i o l e t photolysis i n the

lower s t r a t o s p h e r e is a l s o l i m i t e d e s s e n t i a l l y t o wavelengths longer than 295 nm because t h e major f r a c t i o n of atmospheric ozone i s present a t s t i l l higher a l t i t u d e s , and a c t s a s an e f f e c t i v e absorber of r a d i a t i o n of s h o r t e r wavelengths, i n t e r c e p t i n g most of i t i n t h e upper s t r a t o s p h e r e .

U l t r a v i o l e t p h o t o l y s i s of

t r a c e components can occur r e a d i l y i n t h e upper s t r a t o s p h e r e above most of t h e atmospheric ozone, and is important i n t h e 195-235 nm band f o r a l t i t u d e s above 30 km.

However, molecules which a r e vulnerable i n t h e 195-235 nm region, b u t

a r e t r a n s p a r e n t i n t h e near u l t r a v i o l e t and t h e v i s i b l e , have very much longer tropospheric l i f e t i m e s because >98%of t h e e a r t h ' s atmosphere l i e s below 30 kin a l t i t u d e , and t h e r e f o r e only a Small f r a c t i o n of t h e molecules of a well-mixed gaseous component is exposed t o 220 nm r a d i a t i o n a t any given time.

These t r a n s -

parent molecules s p e n d most of t h e i r time i n t h e atmosphere a t a l t i t u d e s i n which they a r e p r o t e c t e d a g a i n s t photochemical decomposition by t h e absence of s h o r t wavelength u l t r a v i o l e t r a d i a t i o n , absorbed a t higher a l t i t u d e s by ozone. The chlorofluoromethanes C C 1 F and C C 1 F a r e e x c e l l e n t examples of chemical 3 2 2 s p e c i e s which a r e b a s i c a l l y i n e r t toward chemical a n d photochemical d e s t r u c t i o n processes i n t h e troposphere ( r e f . 13).

Neither molecule absorbs v i s i b l e r a d i a -

t i o n o r s o l a r u l t r a v i o l e t a t wavelengths longer than 240 nm, and consequently each photolyzes only very slowly below 25 km a l t i t u d e .

The p h o t o l y s i s r a t e f o r

C C 1 F a t 17 km was estimated t o be no more than 0.3% per year ( r e f . 131, and i t s

3 average l i f e t i m e i n t h e e n t i r e atmosphere with removal only by s t r a t o s p h e r i c photolysis has been c a l c u l a t e d t o be 40 t o 80 years ( r e f s . 9 , 1 0 , 1 3 ) .

The l e s s

c h l o r i n a t e d C C 1 F absorbs u l t r a v i o l e t r a d i a t i o n even more weakly than C C 1 F and 2 2 3 hence has a c a l c u l a t e d average l i f e t i m e before s o l a r photolysis of 80 t o 150 years.

Carbon t e t r a c h l o r i d e is a more r e a c t i v e chemical than t h e chlorofluoro-

methanes under many experimental c o n d i t i o n s , b u t i t , t o o , is t r a n s p a r e n t f o r wavelengths longer than 295 nm and has an atmospheric l i f e t i m e of s e v e r a l decades or more ( r e f s . 9 , l O ) . About 85% of t h e world population is concentrated i n t h e northern hemisphere, and an even l a r g e r percentage of technological a c t i v i t y occurs t h e r e , with about 75% concentrated i n t h e q u a r t e r of t h e e a r t h ' s s u r f a c e n o r t h of 30°N l a t i t u d e . Consequently, t h e major r e l e a s e s of e s s e n t i a l l y a l l molecules of anthropogenic o r i g i n occur predominantly i n t h e northern hemisphere.

Tropospheric mixing occurs

within a few weeks w i t h i n t h e northern and southern hemispheres s e p a r a t e l y , b u t inter-mixing a c r o s s t h e equator is a process r e q u i r i n g 12 t o 15 months on t h e average.

W i t h r e l e a s e i n t h e n o r t h temperate zone and moderately slow t r a n s p o r t

across t h e equator, a permanent concentration g r a d i e n t e x i s t s along l o n g i t u d i n a l l i n e s with higher concentrations i n t h e north.

Th e measured concentration r a t i o s

between t h e n o r t h and south temperate zones i n 1984 were about 1.08 f o r t h e longl i v e d molecules C C 1 F and C C 1 F , whose major r e l e a s e s have occurred i n t h e p a s t 2 2 3 two decades, and only about 1.05 f o r C C 1 4 , whose widespread i n d u s t r i a l applicat i o n s began about 50 years ago.

On t h e o t h e r hand, methylchloroform ( CH C C 1 ) , 3 3 w i d e l y used a s an i n d u s t r i a l degreasing a g e n t , e x h i b i t s a much l a r g e r north/south

concentration r a t i o of about 1 . 4 , even thou;h

a l l four of t h e s e chlorocarbons a r e

released overwhelmingly a t l a t i t u d e s north of 30°N.

T h i s l a r g e r g r a d i e n t is a

c l e a r i n d i c a t i o n of one or more r a p i d tropospheric l o s s processes f o r CH C C 1 3 3’ f a s t enough f o r some competition between t h e time s c a l e s f o r tropospheric chemical r e a c t i o n and f o r interhemispheric t r a n s p o r t .

With t h i s molecule and w i t h many

o t h e r s , t h e chemical removal process i s a t t a c k by tropospheric HO r a d i c a l s , a very important r e a c t i o n f o r cleansing t h e atmosphere of organic chemical s p e c i e s . The atmospheric l i f e t i m e f o r CH C C 1 has been estimated from t h e magnitude of 3 3 t h e interhemispheric g r a d i e n t . t o be about s i x years ( r e f . 1 4 ) . The average time spent i n t h e atmosphere by t h e CH C C 1 molecules now t h e r e can a l s o be estimated 3 3 by comparison of t h e p r e s e n t l y observed CH C C 1 concentrations versus t h e t o t a l 3 3 amounts already r e l e a s e d t o t h e atmosphere: t h e observed 50% s u r v i v a l corresponds t o about a 7 year atmospheric l i f e t i m e ( r e f . 151, i n e x c e l l e n t agreement w i t h t h a t determined from t h e north/south concentration g r a d i e n t .

The hemispheric

concentration r a t i o s a r e e s p e c i a l l y s t e e p f o r molecules such a s C C 12=CC12 and CHC1=CC12, whose atmospheric l i f e t i m e s a r e only a few months and a few weeks,

respectively.

Southern hemispheric a i r i n remote l o c a t i o n s normally has l e v e l s

of these two o l e f i n i c compounds a t t h e 2 pptv l e v e l or lower, a f a c t o r of 10 o r more below t h a t found i n t h e north. These organic compounds a r e almost i n s o l u b l e i n water and a r e not removed by r a i n f a l l or washout.

Furthermore, they do not r e a c t w i t h r a d i c a l s such a s HO, H02

W i t h t h e usual atmospheric r e 3’ moval processes i n e f f e c t i v e , many o t h e r l e s s f a m i l i a r removal processes have a l s o

or CH

e t c . , or w i t h ground-state O ( 3 P ) atoms.

been considered, such a s c a t a l y t i c decomposition on hot d e s e r t sand or a d s o r p t i o n trapping i n A n t a r c t i c snow. None of t h e s e s p e c i a l mechanisms has upon examination proven t o be an important s i n k f o r e i t h e r C C 1 F o r CC12F2, and a c t u a l measurements 3 of t h e i r s t e a d i l y increasing concentrations i n t h e atmosphere i t s e l f have l e d t o l i f e t i m e e s t i m a t e s of 60 t o 80 years f o r C C 1 F , and even longer f o r CC12F2 ( r e f s . 3 These measured atmospheric l i f e t i m e s a r e t h e same w i t h i n t h e e r r o r limits

9,101.

a s thoge c a l c u l a t e d f o r s t r a t o s p h e r i c loss a l o n e , and i t is apparent t h a t t h e f i n a l f a t e f o r almost a l l such molecules is decomposition above 25 km w i t h t h e r e l e a s e of atomic chlorine. The concentrations of s e v e r a l of t h e s e halocarbon gases have been r e g u l a r l y monitored f o r t h e p a s t decade, and a l l of those w i t h concentrations i n excess of

467

100 pptv have shown c o n s i s t e n t , s t e a d y i n c r e a s e s over t h e e n t i r e period.

The con-

c e n t r a t i o n s of C C 1 F and C C 1 F a r e now approaching 400 p p t v and 250 p p t v , respec2 2 3 t i v e l y , while those of C C 1 4 and CH C C 1 a r e i n t h e 120-140 p p t v range. These f o u r 3 3 anthopogenic gases together can account f o r a t o t a l tropospheric c h l o r i n e concent r a t i o n of almost 2500 p p t v (e.g. 400 p p t v CC12F2

=

800 p p t v C l ) , and t o g e t h e r

with 600 p p t v of CH C 1 provide a t o t a l C 1 l e v e l exceeding 3000 pptv. The o t h e r 3 c h l o r i n a t e d molecules provide smaller c o n c e n t r a t i o n s , b u t t h e i r summed contribut i o n s c e r t a i n l y amount t o another s e v e r a l hundred p p t v of C 1 i n t h e troposphere. Only CH C 1 of t h e s e f i v e major organochlorine compounds has any s i g n i f i c a n t 3 n a t u r a l source, and i t s 600 p p t v of C 1 was presumably t h e dominant source of C 1 i n t h e atmosphere p r i o r t o t h e 20th century.

Carbon t e t r a c h l o r i d e was t h e f i r s t

widely used chlorocarbon w i t h a long atmospheric l i f e t i m e , and was followed i n t o major usages by CC12F2, C C 1 F and then CH CC1

The t o t a l organochlorine content 3 3 3' probably r o s e from i t s 600+ p p t v from CH C 1 i n 1900 or 1930 t o about 1500 p p t v i n 3 1970. The cumulative tropospheric C 1 concentration probably reached 3000 pptv e a r l y i n t h e 1980s and appears t o b e i n c r e a s i n g now a t a r a t e of about 1000 pptv per decade. This tropospheric accumulation of organochlorine compounds l e a d s inexorably t o higher concentrations of C 1 and C10 i n t h e upper s t r a t o s p h e r e , where they then p a r t i c i p a t e i n t h e C I O x chain r e a c t i o n which converts 0

molecules of 02.

and 0 back i n t o two 3 The long-term consequences f o r s t r a t o s p h e r i c ozone concentra-

t i o n s which w i l l r e s u l t from i n c r e a s i n g amounts of C1 and C10 have been discussed r e g u l a r l y and i n d e t a i l over t h e p a s t decade i n a s e r i e s of r e p o r t s from t h e National Academy of Sciences ( r e f s . 9,101 and o t h e r s . I N F R A R E D R A D I A T I O N A N D THE GREENHOUSE EFFECT

The i n t e n s i t y of s o l a r r a d i a t i o n is g r e a t e s t i n t h e wavelength range of v i s i b l e l i g h t between 400 and 700 nm, corresponding t o quanta with e n e r g i e s ranging from about 2 t o 3.5 e l e c t r o n v o l t s .

U l t r a v i o l e t r a d i a t i o n c a r r i e s more energy per

quantum, enough t o cause chemical r e a c t i o n and photodecomposition i f absorbed, b u t t h e numbers of u l t r a v i o l e t quanta f a l l r a p i d l y a s t h e wavelength s h o r t e n s below 350 nm.

A l l of t h e major atmospheric components a r e t r a n s p a r e n t i n t h e v i s i b l e

and 295 t o 400 nm u l t r a v i o l e t r e g i o n s , a s a r e minor s p e c i e s such as C02, N20 and CH4.

The numbers of quanta a l s o decrease r a p i d l y on t h e long wavelength s i d e of

t h e v i s i b l e a s well, and only a small f r a c t i o n of t h e s o l a r photochemical energy a r r i v i n g a t t h e top of t h e atmosphere is c a r r i e d by i n f r a r e d wavelengths.

Fu r -

thermore, s i n g l e quanta of l i g h t i n t h e i n f r a r e d range c a r r y too l i t t l e energy t o cause molecular decomposition o r rearrangement f o r most molecular s p e c i e s , and a r e g e n e r a l l y not important a s i n i t i a t o r s of atmospheric chemical r e a c t i o n s .

Never-

t h e l e s s , i n f r a r e d r a d i a t i o n i s r e a d i l y absorbed i n t o t h e complex v i b r a t i o n s of chemical s p e c i e s containing t h r e e o r more atoms (C02, H20, 03, N20. C H q , CC13F, CC12F2, e t c . ) , and much of t h e incoming s o l a r i n f r a r e d f l u x i s absorbed i n t h e

atmosphere.

While t h e absorption of incoming i n f r a r e d r a d i a t i o n r e p r e s e n t s only a

minor energy c o n t r i b u t i o n t o t h e atmosphere, t h e absorption of outgoing i n f r a r e d r a d i a t i o n is of major importance. The amount of s o l a r energy reaching t h e e a r t h is s u f f i c i e n t t o cause s t e a d i l y i n c r e a s i n g average temperatures unless balanced by t h e emission back i n t o space of an equivalent amount of thermal energy.

The peak wavelengths f o r thermal energy

emission from hot bodies a r e i n v e r s e l y proportional t o t h e i r s u r f a c e temperatures ( i . e . about 5600 K f o r t h e s u n and 280 K fot t h e e a r t h ) , and t h e r e f o r e occur a t wavelengths about 20 times longer f o r t h e outgoing t e r r e s t r i a l r a d i a t i o n than f o r t h e incoming s o l a r quanta.

The maximum i n t e n s i t y of t h e r a d i a t i o n emitted by t h e

e a r t h thus f a l l s i n t o t h e i n f r a r e d with wavelengths i n t h e 8-14 micron range, and can be s t r o n g l y a f f e c t e d by any absorbers f o r t h i s r a d i a t i o n i n t h e atmosphere, including a l l of t h e polyatomic s p e c i e s l i s t e d above.

This i n f r a r e d absorption by

t h e s e t r a c e polyatomic s p e c i e s f u r n i s h e s t h e b a s i s f o r t h e "greenhouse e f f e c t " i n t h e e a r t h ' s atmosphere. The thermal balance between s o l a r input and t e r r e s t r i a l output of r a d i a n t energy is disturbed i f a l a r g e f r a c t i o n of t h e i n f r a r e d r a d i a t i o n is absorbed by t h i s atmospheric blanket, and t h e atmosphere a d j u s t s i t s e l f i n response t o t h i s perturbation.

I n e f f e c t , i f some of the avenues f o r escape of r a d i a t i o n a r e

blocked by i n f r a r e d absorption i n c e r t a i n wavelength bands, then t h e only r o u t e toward energy e q u i l i b r a t i o n f ? r t h e e a r t h is through increased emission i n t h e i n f r a r e d ranges not blocked by t h e s e polyatomic absorbers.

The process by which

increased emission occurs i n t o t h e s e t r a n s p a r e n t bands is simply an i n c r e a s e i n t h e average t e r r e s t r i a l temperature, r a i s i n g t h e p r o b a b i l i t y of energy escape u n t i l t h e emissions t o space from t h e e a r t h come i n t o energy balance with t h e continuing s o l a r i n p u t again.

The atmosphere a t t h e beginning of t h e 20th cen-

t u r y already was a b l e t o t r a p s u f f i c i e n t i n f r a r e d r a d i a t i o n i n t o H20, C02 and 0

3

t o r a i s e t h e average s u r f a c e temperature of t h e e a r t h from about 250 K t o about 280 K through t h i s process.

A major concern f o r t h e world population a s we

approach t h e 2 1 s t century is t h a t i n c r e a s i n g concentrations of t h e s e and o t h e r i n f r a r e d absorbing s p e c i e s i n t h e atmosphere might i n c r e a s e t h e average s u r f a c e temperature by an a d d i t i o n a l s e v e r a l degrees K , with a l l of t h e c o r o l l a r y implications f o r world c l i m a t i c change ( r e f . 7 ) . The e s s e n t i a l problem of t h e greenhouse e f f e c t i n t h e f u t u r e i s t h e p o s s i b i l i t y of continuing changes i n t h e concentrations of t h e important i n f r a r e d absorbing s p e c i e s i n t h e atmosphere.

C l e a r l y , molecules such a s C02, 0

3

and H20 a r e present

i n abundance and a r e q u i t e e f f i c i e n t i n t h e absorption of i n f r a r e d r a d i a t i o n .

In

those i n f r a r e d absorbing regions corresponding t o t h e v i b r a t i o n a l frequencies of these t h r e e molecules, r e l a t i v e l y l i t t l e energy is t r a n s m i t t e d through t h e atmosphere without s e v e r e a t t e n u a t i o n ,

However, incremental i n c r e a s e s i n t h e concen-

t r a t i o n s o f , f o r example C02, a r e not too e f f e c t i v e i n causing increased attenua-

t i o n of t h e i n f r a r e d r a d i a t i o n because each a d d i t o n a l molecule of CO i n t h e 2 atmosphere brings w i t h i t t h e c a p a b i l i t y of absorption f o r p r e c i s e l y those wavel e n g t h s of i n f r a r e d r a d i a t i o n which have been almost completely removed by t h e C02 already present. CO

The i n f r a r e d absorption c a p a b i l i t y of an a d d i t i o n a l 1 ppmv of

added t o t h e 340 ppmv already present is f a r l e s s than t h a t of t h e f i r s t ppmv

2 of C02 added t o t h e atmosphere, or of t h e average a b s o r p t i o n f o r t h e f i r s t 340

ppmv.

T h i s same kind of s a t u r a t i o n e f f e c t i n t h e i n f r a r e d holds t r u e f o r H20 and

0 --these s p e c i e s a r e already present i n s u b s t a n t i a l q u a n t i t i e s and t h e incremen-

3

t a l e f f e c t on i n f r a r e d absorption of i n c r e a s e s i n t h e i r concentration is not very large.

Nevertheless, t h e s t e a d y i n c r e a s e i n concentration of CO

by 1 ppmv per 2 year from about 315 ppmv i n 1958 t o 340 ppmv is s t i l l t h e major component i n

c a l c u l a t i o n s of the t o t a l c o n t r i b u t i o n s of a l l s p e c i e s t o t h e greenhouse e f f e c t . T h e c o n t r i b u t i o n t o enhanced absorption of i n f r a r e d r a d i a t i o n t o be made by

o t h e r t r a c e s p e c i e s is simply then a question of whether t h e i n f r a r e d a b s o r p t i o n c a p a b i l i t i e s of each p a r t i c u l a r s p e c i e s happen t o o v e r l a p w i t h t h o s e of o t h e r more abundant chemical s p e c i e s .

Molecules whose v i b r a t i o n a l frequencies o v e r l a p w i t h

or H 0 o r 0 w i l l cause r e l a t i v e l y l i t t l e incremental i n c r e a s e i n 2 3 2 absorption of i n f r a r e d r a d i a t i o n ; t h o s e whose frequencies happen t o f a l l i n t o t h e those of CO

t r a n s p a r e n t gaps l e f t by t h e more abundant s p e c i e s can be enormously more e f f e c t i v e i n t h e a b s o r p t i o n of i n f r a r e d r a d i a t i o n on a molecule f o r molecule b a s i s . The v i b r a t i o n a l frequencies of halocarbon molecules g e n e r a l l y include s e v e r a l which f a l l i n t o a n e a r l y t r a n s p a r e n t gap, with t h e consequence t h a t f l u o r i n a t e d molecules such a s C C 1 F and C C 1 F a r e roughly l o 4 times more e f f i c i e n t i n absorp3 2 2 t i o n of i n f r a r e d r a d i a t i o n per molecule than an incremental C02 molecule added t o t h e e x i s t i n g atmosphere.

I n t h i s way, t h e t9greenhousettc o n t r i b u t i o n from yearly

i n c r e a s e s of 0.020 ppbv i n t h e c o n c e n t r a t i o n of C C 1 F o r 0.010 ppbv f o r C C 1 F a r e 2 2 3 not n e g l i g i b l e even i n comparison t o t h e y e a r l y i n c r e a s e of about 1000 ppbv which

i s found f o r C02.

Some of t h e important v i b r a t i o n a l frequencies of CH4 a r e a l s o

not screened by t h e abundant t r i a t o m i c s p e c i e s , b u t CH i t s e l f is a l r e a d y p a r t i 4 a l l y s a t u r a t e d and t h e incremental e f f e c t from an a d d i t i o n a l CH4 molecule is about molecule. 2 C a l c u l a t i o n s of t h e cumulative f u t u r e greenhouse e f f e c t t o be expected from

20 times s t r o n g e r than f o r an a d d i t i o n a l CO

changes i n t h e c o n c e n t r a t i o n s of many t r a c e gaseous s p e c i e s a r e somewhat dependent upon t h e p r e c i s e approximations made i n d e s c r i b i n g the r a d i a t i o n balance, and a r e probably even more dependent upon t h e assumptions made i n t h e attempted e x t r a p o l a t i o n i n t o t h e f u t u r e of t h e concentration i n c r e a s e s observed f o r s e v e r a l t r a c e s p e c i e s over t h e past 5 or 1 0 years.

One s e t of such c a l c u l a t e d e f f e c t s f o r

a v a r i e t y of t r a c e s p e c i e s i s given i n Table 2 ( r e f . 7 ) . The e f f e c t i n t h e same atmospheric model f o r a doubling of C02 from 300 ppmv t o 600 ppmv is a s u r f a c e temperature i n c r e a s e of 2 t o 3OC.

The general conclusion from t h i s t a b l e and

o t h e r s i m i l a r s t u d i e s ( r e f . 16) is t h a t , w i t h i n t h e l a r g e u n c e r t a i n t i e s involved,

470

t h e greenhouse e f f e c t from incremental i n c r e a s e s i n a l l other t r a c e gaseous s p e c i e s w i l l be roughly comparable t o , o r even l a r g e r than, t h e e f f e c t a t t r i b u t a b l e t o t h e well-documented i n c r e a s e i n C02 over t h e past 25 y e a r s . TABLE 2.

Some e s t i m a t e s of s u r f a c e temperature change caused by changes i n atmospheric c o n s t i t u e n t s o t h e r than C02 ( r e f . 7 ) . Change i n mixing r a t i o ( i n ppbv) From To

Const i t u e n t Nitrous Oxide, N20 Methane, CH4 Fluorocarbon-11, CC13F Fluorocarbon-12, CC1 F2 Fluorocarbon-22, CHC.?F Carbon t e t r a c h l o r i d e , E C l , Carbon t e t r a f l u o r i d e , CF4 Methylchloroform, CH C C 1 Methylene c h l o r i d e , H!i C 2 Methyl c h l o r i d e , CH C 1 2 3 Chloroform, C H C l 3 Ethylene, H4 S u l f u r dioxlde, SO2 Ammonia, NH TropospheriJ ozone, 0 S t r a t o s p h e r i c water, A20

300 1500 0 0 0 0

Surface temperature change, OC 0.3-0.4 0.3 0.15 0.13 0.04 0.14 0.07 0.02 0.05 0.013 0.1 0.01

600 3000 1 1 1 1 1 1 1

0 0 0 0 0

1

1

0.2 0.4 2 4 6 12 Doubled 3000 6000

0.02 0.09 0.9 0.6

TROPOSPHERIC REMOVAL BY REACTION WITH HO RADICALS The i n t r o d u c t i o n i n t o t h e 20th century atmosphere of newly synthesized complex carbon-containing chemical s p e c i e s has included many o t h e r s which a r e much more r e a c t i v e than t h e nearly i n e r t chlorofluorocarbons.

Many of t h e s e compounds a r e

t r a n s p a r e n t i n t h e v i s i b l e and near u l t r a v i o l e t , and not s u s c e p t i b l e t o d i r e c t s o l a r photodissociation i n t h e troposphere.

The highly water-soluble compounds

can be e n t r a i n e d i n r a i n and p r e c i p i t a t e d out t o t h e s u r f a c e .

Adsorption onto

aerosol p a r t i c l e s or d u s t near t h e s u r f a c e can be very e f f e c t i v e f o r t h e removal of non-volatile s p e c i e s .

The v o l a t i l e gases, even when water s o l u b l e , can s u r v i v e

f o r a remarkably long time i n t h e atmosphere.

A useful i l l u s t r a t i o n of t h e

longevity i n t h e atmosphere of such chemical s p e c i e s is given by CO

2

itself.

Carbon dioxide has two major s i n k s a v a i l a b l e t o i t - - d i s s o l u t i o n i n t h e ocean t o form bicarbonates and carbonates, and photosynthesis i n t o a l l of t h e green p l a n t s of t h e world.

Nevertheless, i t s atmosphere l i f e t i m e is s t i l l between 5 and 10

y e a r s , a s demonstrated by t h e slow diminution i n atmospheric "CO

concentrations 2 following t h e c e s s a t i o n 20 years ago of most atmospheric t e s t i n g of nuclear e x p l o s i v e s , t h e source of t h e "CO

2

excess beyond t h a t caused by cosmic r a d i a t i o n .

471 The key t o t h e question of removal versus s u r v i v a l f o r t h e s e compounds then i s t h e i r i n t e r a c t i o n toward highly r e a c t i v e t r a c e components.

One of t h e most impor-

t a n t of t h e s e p o t e n t i a l r e a c t a n t s is t h e hydroxyl r a d i c a l , whose average d a y l i g h t tropospheric concentration is of t h e order of

lo6

molecules

The source of

tropospheric HO l i e s i n t h e u l t r a v i o l e t p h o t o l y s i s (295-314 nm) of 0 1

3

with the

formation of O ( D ) atoms which then r e a c t with H 0 vapor t o form two HO r a d i c a l s . 2

The r e a c t i o n r a t e c o n s t a n t s f o r HO with CO and CH4 a t 288 K a r e 2.4 x

6.3 x

cm3 molecule-’ sec-’,

respectively.

removal r a t e s f o r CO and CHq a t 288 K a r e 2 . 4 x

With 106 HO r a d i c a l s s e e-1 and 6.3 x

and the see-’,

corresponding t o average l i f e t i m e s of 48 days f o r CO and 5 years f o r CH4.

Our

more p r e c i s e e s t i m a t e s of t h e atmospheric l i f e t i m e of methane p l a c e i t a s (10k2) years ( r e f . 1 7 ) and t h e 24-hour average HO concentration t h e r e f o r e a s about 5 x The mixing of CH4 within t h e atmosphere is r a p i d enough--about

lo5

years--that

1 to 2

i t s l i f e t i m e is e s s e n t i a l l y independent of t h e geographic l o c a t i o n of

any s u r f a c e source.

The l i f e t i m e of CO i s s h o r t enough, however, t h a t t h e season

and l a t i t u d e of i t s emission determine t h e average HO concentration t o which i t w i l l be exposed during t h e next few months, and t h e r e f o r e t h e l i f e t i m e of t h e

i n d i v i d u a l molecule.

The concentration of cosmic-ray induced 14C0 a t 51°N is

t h r e e times g r e a t e r i n winter than i n summer, i n i n v e r s e r a t i o t o t h e seasonal f l u c t u a t i o n i n t h e average HO concentration a t t h a t l a t i t u d e ( r e f . 18). ATMOSPHERIC METHANE The major sources f o r t h e methane found i n t h e atmosphere a r e b i o l o g i c a l - emissions from r i c e paddies, swamps, c a t t l e , e t c . CH

i s i t s r e a c t i o n with HO r a d i c a l s .

The major removal process f o r

While i n t h e atmosphere, i t p a r t i c i p a t e s

4 chemically i n t h e urban smog c y c l e and i s important i n t h e s t r a t o s p h e r e because i t

can d i v e r t atomic C 1 away from t h e ozone-depleting C I O x chain r e a c t i o n s i n t o t h e temporary s i n k of HC1.

The physical c h a r a c t e r i s t i c s of methane a r e a l s o s i g n i f i -

cant i n t h e atmosphere through i t s absorption of i n f r a r e d r a d i a t i o n and contribut i o n t o t h e greenhouse e f f e c t .

F i n a l l y , i t s r e l a t i v e l y long l i f e t i m e tends t o

smooth out t h e magnitude of i t s c o n c e n t r a t i o n changes i n any p a r t i c u l a r l o c a t i o n , permitting a c c u r a t e measurement of i t s world-wide average concentration.

For

t h e s e reasons, t h e gas chromatographic measurement of methane i n t h e troposphere provides a very u s e f u l example of c u r r e n t atmospheric procedures ( r e f s . 17,19-21). The a n a l y s i s f o r methane i n t h e atmosphere i s a gas chromatographic problem

i n which t h e prime experimental need i s p r e c i s i o n of a n a l y s i s .

The c o n c e n t r a t i o n

of CH4 is l i s t e d i n Table 1 a s 1500 p p b v , b u t t h e accuracy and p r e c i s i o n of such e s t i m a t e s made a s r e c e n t l y a s a decade ago have been s t r o n g l y questioned during t h e 198Os, with unexplained e x i s t i n g d i s c r e p a n c i e s a s l a r g e a s 400 ppbv o r 25%. The amounts of methane i n t r o p o s p h e r i c a i r away from any l o c a l sources have c e r t a i n l y exceeded 1 ppmv a t a l l l a t i t u d e s i n t h e past decade, and have probably done s o throughout t h e 20th century.

Such concentrations a r e e a s i l y measured with

a flame i o n i z a t i o n d e t e c t o r on 5 m l STP samples of ambient a i r , w i t h l i t t l e speci a l precaution o t h e r than removal of water vapor t o permit s t a n d a r d i z a t i o n r e l a t i v e t o t h e concentrations i n d r y a i r .

I n our work over t h e past s e v e r a l y e a r s ,

we have taken advantage of t h e e x i s t e n c e of an NBS standard c e r t i f i e d t o contain 0.97 ppmv of CH4 w i t h an accuracy of ? l % , and t h e r e is no obvious need a t present f o r absolute measurements of higher accuracy.

The most important conclusion t o

be drawn from r e c e n t methane observations has l e s s t o do w i t h t h e a b s o l u t e accuracy than w i t h t h e f a c t t h a t t h e precision of measurement and broad spread of coverage have improved s u f f i c i e n t l y t o be a b l e t o document a s t e a d y i n c r e a s e i n t h e world-wide average concentration of CH4 i n t h e e a r t h ' s atmosphere.

The

o r i g i n s of t h i s i n c r e a s e , and s p e c u l a t i o n s about p o s s i b l e changes i n t h e yearly r a t e of increase have been abundant i n t h e past two years. Our e s t i m a t e s of t h e world-wide average t r o p o s p h e r i c concentrations of CH 4 at various times s i n c e e a r l y 1978 a r e given i n Table 3, and a r e based upon our measurements of CH4 concentrations i n a i r samples c o l l e c t e d a t remote s u r f a c e locat i o n s between t h e l a t i t u d e s of 71°N and 53's.

Each of t h e s e i n d i v i d u a l data

points has been assigned t o one of s i x t e e n equal a r e a l a t i t u d i n a l bands (e.g. Barrow, Alaska, a t 71°N l a t i t u d e i s i n Band One, 61°-900N; t h e P a c i f i c i s l a n d of Nauru a t 0.5's

i s i n Band Nine, Oo-7OS; e t c . ) and band averages have then been

c a l c u l a t e d f o r each major c o l l e c t i o n period.

The world-wide averages l i s t e d i n

Table 3 a r e equal t o 1/16 of t h e sum of t h e band averages. t h e concentration of CH

4

The observed growth i n

versus time can r e a d i l y be f i t t e d w i t h a s t r a i g h t l i n e

i n c r e a s e of 17 ppbv per year over t h e seven year period of r e c o r d , corresponding t o a r a t e of i n c r e a s e of about 1 . 1 % per year.

The standard d e v i a t i o n f o r t h e

i n d i v i d u a l values of t h e world-wide averages is 2.3 ppbv, which i s c e r t a i n l y within t h e p r e c i s i o n of our a b i l i t y t o measure a world-wide average w i t h a number

of samples fewer than 100 i n each period.

Our measurements do not support w i t h

any s t a t i s t i c a l s i g n i f i c a n c e any f l u c t u a t i o n s from a l i n e a r i n c r e a s e over t h a t seven year p e r i o d , although d e v i a t i o n s of i 3 ppbv t o t h e observed y e a r l y CH4 increment of 17 ppbv could r e a d i l y be concealed i n t h e data. The measurements of CH 4 concentrations a t any individual l o c a t i o n n a t u r a l l y show d e v i a t i o n s on a day-to-day b a s i s , b u t have a l s o shown c o n s i s t e n t changes w i t h season t o g e t h e r w i t h t h e o v e r a l l i n c r e a s e s i l l u s t r a t e d by Table 3.

One of t h e

most comprehensive s e t of individual measurements of tropospheric CH4 concentrat i o n s is t h a t of Khalil and Rasmussen a t Cape Meares on t h e Oregon c o a s t ( r e f . 21 ).

Data have been c o l l e c t e d by them w i t h a gas chromatograph i n continual

o p e r a t i o n , providing m u l t i p l e d a i l y measurements f o r methane a t t h a t l o c a t i o n s i n c e 1979.

The monthly averages of t h e d a i l y averages a t Cape Meares varied by

about 3% i n each year from 1979 through 1981, a s shown i n Table 4 .

Such l a r g e

v a r i a t i o n s a t a s i n g l e s i t e or l a t i t u d e can tend t o mask any long-term change i n t h e annual average concentration, and make t h e e v a l u a t i o n of its r a t e of change

473 TABLE 3

World-wide average t r o p o s p h e r i c CH,, c o n c e n t r a t i o n s during t h e period 1978-1984.

Date (Mid-Point i n Collection Period)

Average Tropospheric Concentration of CH,,, ppmv

January 1978 June 1979 February 1980 August 1980 November 1980 June 1981 September 1982 A p r i l 1983 August 1983 December 1983 February 1984 June 1984 September 1984 December 1984 difficult.

1.516 1.547 1.560 1.564 1.569 1.580 1.602 1 .615 1.623 1.625 1.625 1.628 1.636 1.639

However, t h e y e a r l y average taken from t h e monthly averages i n Table 4

shows a s u b s t a n t i a l i n c r e a s e from year t o y e a r , and t h e annual t r e n d s shown i n Tables 3 and 4 a r e reasonably c o n s i s t e n t with one another. TABLE 4

Monthly average concentrations of CH4 a t Cape Meares. Oregon ( r e f . 2 1 ) Month

/Year

1979

1980

1981

January February March April May June July August September October November December

1.619(2) 1.619(5) 1.608(4) 1.615(2) 1.614(2) 1.609(1) 1.597(1) 1.618(2) 1.632(1) 1.650(1 ) 1.647(3) 1.636(1

1.639(1) 1.621 ( 1 ) 1.643(0) 1.650(1) 1.651 ( 1 ) 1.633(1) 1.627(1) 1.647(2) 1.665(3) 1.676(1 1.664(1) 1.662(1

1.656(1) 1.670(1) 1.675(1) 1.661 ( 1 ) 1.658(1) 1.638(1 1.626(1) 1.622(1) 1.673(1) 1.692(1) 1.682( 1 ) 1.681 ( 1

Yearly average

1.622

1.648

1.661

Figures i n parentheses give t h e 90% confidence l e v e l f o r t h e mean.

474

Our a n a l y t i c a l p r o c e d u r e i s based upon a l t e r n a t e measurement of f i v e 5 m l STP

a l i q u o t s each of a n a i r sample and of t h e NBS methane s t a n d a r d .

The p r e c i s i o n ob-

t a i n e d i n our r o u t i n e a n a l y s i s is i l l u s t r a t e d i n T a b l e by f i f t y s e q u e n t i a l measurements of t h e NBS s t a n d a r d , made o v e r t h e p e r i o d of one day i n a l t e r n a t i o n w i t h f i v e a l i q u o t s each from t e n t r o p o s p h e r i c a i r s a m p l e s . The d a t a a r e c o n s i s t e n t w i t h random f l u c t u a t i o n s of less t h a n 4 ppbv i n measurements of t h e s t a n d a r d con-

t a i n i n g 970 ppbv, and were i n t e r s p e r s e d w i t h comparable v a r i a b i l i t y i n t h e measurement of a s e t of a i r samples w i t h a b o u t 1600-1700 ppbv c o n c e n t r a t i o n s . T h i s r o u t i n e p r e c i s i o n is s u f f i c i e n t l y h i g h t h a t t h e a c t u a l a s s a y of methane in a n a i r sample n o r m a l l y i n t r o d u c e s c o n s i d e r a b l y smaller errors t h a n does t h e i n t e r - s a m p l e v a r i a b i l i t y , or t h e r e p r e s e n t a t i v e n e s s by t h e i n d i v i d u a l samples of t h e e n t i r e l a t i t u d e band i n which t h e y are found. A v e r y l a r g e number of c o n c e n t r a t i o n measurements a t a s i n g l e s i t e s u c h a s Cape Meares g r e a t l y r e d u c e s t h e i m p o r t a n c e of t h e chance v a r i a b i l i t y of a s i n g l e measurement, b u t d o e s r a i s e t h e problem of whether t h e p a r t i c u l a r l o c a t i o n might have a s p e c i a l bias b e c a u s e of local s o u r c e s

or unusual p r e v a i l i n g wind p a t t e r n s . On t h e o t h e r hand, t h e l o g i s t i c a l limitat i o n s t o a p p r o x i m a t e l y 100 s a m p l e s or fewer from d i f f e r e n t remote l o c a t i o n s i n one c o n c e n t r a t e d time p e r i o d i n t r o d u c e s t h e problem of whether t h e s e v e r a l samples i n each l a t i t u d e band are r e a l l y r e p r e s e n t a t i v e of a l l of t h e a i r i n t h a t band a t t h a t time. F o r t u n a t e l y , t h e g e n e r a l upward t r e n d s i n methane c o n c e n t r a t i o n s from y e a r t o y e a r as i n d i c a t e d by b o t h s e t s of data are i n r e a s o n a b l e agreement w i t h a n a n n u a l increment of about 17 ppbv of CH4. TABLE 5

S u c c e s s i v e measurements of NBS s t a n d a r d f o r CH4 ( p e a k areas i n mm 2 ) Average for 5 2250

2230

2181

2177

2198

2207 f 3 2

21 98

21 76

2230

21 93

2214

2202 f21

2208

2225

2200

21 98

2221

2210 f 1 2

2221

221 8

21 96

2219

221 5

2214 f10

2233

2265

2207

2223

223 1

2232 f21

2200

2235

2221

2220

221 4

2218 f 1 3

2221

2206

2208

2235

21 96

2213 f 1 5

2204

2223

2250

221 6

2202

2219 f19

2220

2223

2220

21 92

2206

2212 i 1 3

2204

2223

2242

221 2

2232

2223 f i 5

Average f o r 50 measurements:

2215.0

S t a n d a r d d e v i a t i o n f o r 50 measurements: f 1 7 . 2 S t a n d a r d d e v i a t i o n f o r 10 measurements:

f

8.5

475 One i n t e r e s t i n g aspect of our d a t a on t r o p o s p h e r i c CHq c o n c e n t r a t i o n s has been t h e homogeneity of t h e d i s t r i b u t i o n o f t e n found i n t h e southern hemisphere.

The

d a t a on CH4 a s measured i n samples c o l l e c t e d a t remote s u r f a c e l o c a t i o n s over a l a t i t u d e range of 47'

i n a period of 25 days i n l a t e 1982 a r e given i n Table 6.

The standard d e v i a t i o n f o r t h e s e 19 s e p a r a t e a i r samples i s not too much l a r g e r than t h e standard d e v i a t i o n observed with r e p e t i t i v e measurements of t h e same a i r sample, and i n d i c a t e s t h a t t h e southern hemispheric a i r is remarkably well-mixed w i t h r e s p e c t t o methane.

P a r t of t h e explanation f o r t h i s l a c k of v a r i a t i o n i n

t h e southern hemisphere is t h a t t h e major sources f o r methane a r e b i o l o g i c a l i n o r i g i n , and l i e predominantly i n t h e northern hemisphere.

These sampling s i t e s i n

t h e P a c i f i c a r e a l s o comparatively d i s t a n t from t h e methane sources which do e x i s t on t h e southern hemispheric land masses of A f r i c a and South America.

The observa-

t i o n s shown i n Table 6 a r e c o n s i s t e n t w i t h our d a t a on t h e c o n c e n t r a t i o n s of C C 1 F 3 and C C 1 F i n t h e same samples, which a l s o demonstrate t h e homogeneity of t h e 2 2 southern hemispheric a i r mass a t t h a t time. S i m i l a r constancy of c o n c e n t r a t i o n s over a l a t i t u d e band of 40'

o r more has been observed more o f t e n than not i n t h e

southern hemisphere. TABLE 6

Methane mixing r a t i o s i n t r o p o s p h e r i c a i r samples c o l l e c t e d i n remote l o c a t i o n s

i n t h e southern hemisphere during t h e period S e p t . 13 t o Oct. 7, 1982.

Location

Nauru Nauru Guadalcanal Cuadalcanal Port Douglas, A u s t r a l i a Tahiti Tahiti Alva Beach, A u s t r a l i a Bloomsbury , A u s t r a l i a Sarina, Australia N . of Yeppon, A u s t r a l i a S. of Yeppon, A u s t r a l i a Teal Bay, New Zealand Whatipu, New Zealand Cape Egmont , New Zealand Hokitika, New Zealand Hawea, New Zealand Gore, New Zealand Bluff, New Zealand Average

-

1.567;

latitude

month/day(l982)

mixing ratio,CHq,ppmv

0.5 0.5 9.5 9.5 16.3 17.5 17.5 19.3

10103 10103 9/28 9/28 9/13 10/07 10/07 9/14 911 4 9/15 9/16 9/16 9/25 9/26 9/23 9/21 9/20 9/19 9/19

1.560 1.567 1.554 1.557 1.569 1.568 1.562 1.565 1.579 1.584 1.565 1.570 1.559 1.569 1.563 1.571 1.573 1.575 1.561

21 .o

21.7 22.9 23.5 35.4 36.5 39.3 42.6 44.7 46.2 47.2

Standard d e v i a t i o n 0.008

_ I _ _ _

__-

476

SUMMARY

The a v a i l a b i l i t y of h i g h p r e c i s i o n , h i g h s e n s i t i v i t y g a s c h r o m a t o g r a p h i c t e c h n i q u e s h a s p l a y e d a n i m p o r t a n t r o l e i n t h e p a s t 15 y e a r s i n making a c c e s s i b l e t o i n v e s t i g a t i o n much more d e t a i l e d measurements of t h e t r a c e components of t h e atmosphere.

M o n i t o r i n g of t h e abundance of many trace s p e c i e s by gas chromato-

graphy h a s t h e n shown t h e e x i s t e n c e of s e v e r a l l o n g - t e r m problems i n a t m o s p h e r i c c h e m i s t r y , problems of p o t e n t i a l major s i g n i f i c a n c e t o mankind.

Continued

development and a p p l i c a t i o n of g a s c h r o m a t o g r a p h i c t e c h n i q u e s w i l l u n d o u b t e d l y c o n t i n u e t o p l a y a c r i t i c a l r o l e i n a t m o s p h e r i c c h e m i s t r y i n t h e coming d e c a d e . ACKNOWLEDGMENTS The a t m o s p h e r i c measurements of methane h a v e been s u p p o r t e d by N.A.S.A. G r a n t NACW-452, and have been d e s c r i b e d i n d e t a i l i n t h e Ph. D . t h e s i s of D .

R. B l a k e , U n i v e r s i t y of C a l i f o r n i a , I r v i n e , 1984. REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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