New Applications of Lasers to Chemistry
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; ...
39 downloads
1095 Views
4MB Size
Report
This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form
New Applications of Lasers to Chemistry
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
New Applications of Lasers to Chemistry G a r y M . Hieftje,
EDITOR
Indiana University
Based on a symposium sponsored by the ACS Division of Analytical Chemistry at the 175th Meeting of the American Chemical Society, Anaheim, California, March 14-15, 1978.
ACS
SYMPOSIUM
SERIES
AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C.
1978
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
85
Library of Congress CIP Data New applications of lasers to chemistry. (ACS symposium series; 85 ISSN 0097-6156) Includes bibliographies and index. 1. Lasers in chemistry—Congresses. I. Hieftje, Gary M. II. American Chemical Society. Division of Analytical Chemistry. III. Series: American Chemical Society. ACS symposium series; 85. QD715.N48 543 78-22032 ISBN 0-8412-0459-4 ASCMC 8 85 1-242 1978
Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES O F AMERICA
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ACS Symposium Series Robert F . Gould, Editor
Advisory Board Kenneth B. Bischoff
Nina I. McClelland
Donald G. Crosby
John B. Pfeiffer
Jeremiah P. Freeman
Joseph V. Rodricks
E. Desmond Goddard
F. Sherwood Rowland
Jack Halpern
Alan C. Sartorelli
Robert A. Hofstader
Raymond B. Seymour
James P. Lodge
Roy L. Whistler
John L. Margrave
Aaron Wold
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
FOREWORD The A C S S Y M P O S I U a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y S E R I E S except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors i n camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PREFACE *he papers collected in this volume reflect the content of the two-day invited symposium which was organized under the auspices of the Analytical Division of the American Chemical Society; however, because the presentations contained subject matter not ordinarily associated directly with chemical analysis, a more general symposium title was chosen. This same title is born by the present volume. A rather general title seems especially appropriate here. It is clear that the present and futur only be assessed from th cation to chemistry. By its very nature, analytical chemistry relies heavily on advances in measurement technology and, consequently, on developments in physics and engineering. Accordingly, symposium speakers were invited who addressed the subject of laser application from several vantage points. Physicists, engineers, and chemists all participated and represented not only academic laboratories but government agencies and industrial organizations as well. Consequently, subject material ranged from new developments in laser technology and measurement systems to the use of lasers in the detection of single atoms and molecules. This volume is organized in essentially the same way as the Anaheim Symposium. Although no well-delineated separation among topics occurs, thefirstportion of the volume deals principally with the subject of high-resolution spectroscopy, the second with high-sensitivity analysis, the third with time-resolved or kinetic spectroscopy, and the last with new techniques in laser Raman spectrometry. To a large extent, these divisions represent areas of chemistry in which the laser has had greatest impact. The high spectroscopic resolution afforded by modern lasers makes possible the more accurate identification of molecular and atomic energy levels, affords the specificity necessary to spectroscopically resolve components in a mixture, and permits isotope analysis and separation. Similarly, the high power available from some lasers, coupled with new measurement techniques, has extended analytical sensitivities to the single atom and molecule limit, as indicated earlier. Moreover, the brief pulses emitted by mode-locked lasers provides time resolution below the picosecond regime. Finally, new linear and nonlinear Raman methods made possible by high-power, monochromatic laser sources have extended the range of applicability of Raman techniques, their specificity, and sensitivity. ix In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Unfortunately, not all individuals who participated in the Anaheim Symposium have contributed to this volume. However, an excellent cross section of topics and authors appears and serves to represent the scope of current applications of lasers to chemistry and the potential importance of lasers in the future of chemistry and chemical analysis. Indiana University Bloomington, Indiana August 7, 1978
GARY M . HIEFTJE
χ
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1 Selective Excitation of Probe Ion Luminescence (SEPIL) J O H N C. W R I G H T , F R E D E R I C K J. G U S T A F S O N , and L A U R A C. P O R T E R Department of Chemistry, University of Wisconsin, Madison, W I 53706
There have been numerous s t u d i e s t h a t illustrate the e x c e l l e n t d e t e c t i o n l i m i t s obtained when a l a s e r e x c i t a t i o n source generates o p t i c a l emission from a sample (1-7). When working a t such low c o n c e n t r a t i o n l e v e l s , one must b a t t l e the new problems of contamination and i m p u r i t i e s . The l a s e r can provide a potential advantage here as w e l l because o f the very narrow s p e c t r a l bandpasses o f modern l a s e r s . I f the a n a l y t i c a l system has sharp line o p t i c a l t r a n s i t i o n s whose wavelength depends upon the partic u l a r a n a l y t e , the narrow bandwidth can provide a high degree o f selectivity f o r the analyte of i n t e r e s t . A common method o f p r o v i d i n g the narrow t r a n s i t i o n s i s t o convert the a n a l y t i c a l sample t o a gas as is commonly done in atomic a b s o r p t i o n and atomic fluorescence experiments (7). The techniques f o r accomp l i s h i n g t h i s transformation have a long h i s t o r y i n the field and there can be little doubt about the eventual success. We have been studying the feasibility of a very d i f f e r e n t method o f a c h i e v i n g narrow t r a n s i t i o n s t h a t are c h a r a c t e r i s t i c o f an a n a l y t e . The lanthanide ions have narrow l i n e t r a n s i t i o n s , even i n condensed phases, as a r e s u l t o f s h i e l d i n g o f the o p t i c a l l y a c t i v e 4f e l e c t r o n s h e l l by the outer 5s 5p o r b i t a l s (8,9). A small c r y s t a l f i e l d s p l i t t i n g i s produced by the immediate surroundings. T h i s s p l i t t i n g can be used as a s h o r t range s p e c t r o s c o p i c probe o f the condensed phase. In p a r t i c u l a r i f analyte ions are a l s o present i n the condensed phase near a l a n thanide probe i o n , a c r y s t a l f i e l d s p l i t t i n g w i l l be produced that i s c h a r a c t e r i s t i c o f the presence of that a n a l y t e . n
2
6
0-8412-0459-4/78/47-085-001$05.00/0 © 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2
NEW
A P P L I C A T I O N S O F LASERS T O C H E M I S T R Y
In an a r b i t r a r y condensed phase, lanthanide ions w i l l encounter a number o f d i f f e r e n t surroundings e i t h e r because o f d i f f e r e n t ways the ions o f the phase can be ordered around the lanthanide i o n or because other ions (analytes) have a l s o entered the phase. The a b s o r p t i o n and fluorescence spectra can t h e r e f o r e become q u i t e complex because o f the d i f f e r e n t s p l i t t i n g s o f the lanthanide ions i n d i f f e r e n t surroundings. This complexity may be s i m p l i f i e d i f a tuneable l a s e r i s used to e x c i t e a t a wavelength t h a t matches an absorption l i n e o f a lanthanide i o n with a p a r t i c u l a r surroundings (10). Since the lanthanide ions with d i f f e r e n t surroundings have d i f f e r e n t c r y s t a l f i e l d s p l i t t i n g s , these ions w i l l not be e x c i t e d . The fluorescence spectrum then c o n t a i n s only l i n e s from a lanthanide with one type o f surroundings. T h i s process i s c a l l e d s e l e c t i v e l a s e r e x c i t a t i o n o r s i t e s e l e c t i v e spectroscopy o f one s i t e may be obtaine l i n e while scanning the dye l a s e r e x c i t a t i o n wavelength. Thus a high s e l e c t i v i t y f o r a p a r t i c u l a r analyte may be obtained by e i t h e r e x c i t i n g o r monitoring s p e c t r a l t r a n s i t i o n s o f the l a n t h a nide i o n t h a t has the analyte o f i n t e r e s t i n the immediate surroundings . In order t o make t h i s idea p r a c t i c a l , one must solve the chemical problem o f b r i n g i n g about an a s s o c i a t i o n between an analyte i o n and a lanthanide i o n . There are a number o f methods that can be used t o accomplish t h i s . The simplest method i s t o make the condensed phase i t s e l f from lanthanide ions, i . e . a pure lanthanide compound. Any f o r e i g n i o n t h a t enters the c r y s t a l l i n e l a t t i c e o f such a compound w i l l have to perturb a lanthanide i o n . In a second method, the presence o f analyte ions could l e a d to the establishment o f a second c r y s t a l l i n e phase which w i l l p a r t i c i p a t e i n the p a r t i t i o n i n g o f t r a c e amounts o f lanthanide i o n s . I f two phases a r e present, one c o n t a i n i n g the analyte and the other an innocuous d i l u e n t , the lanthanide spectra w i l l r e f l e c t the presence o f both phases. A t h i r d method introduces both the lanthanide and a n a l y t e ions a t t r a c e concentrations i n a host compound and r e l i e s upon i n t e r a c t i o n s between them to form assoc i a t e s o r complexes. The i n t e r a c t i o n s can be the r e s u l t o f chemical bonding o r o f d i f f e r e n c e s i n i o n i c r a d i i or charge s t a t e between analyte and lanthanide. F o r example, s u b s t i t u t i o n o f a t r i v a l e n t lanthanide f o r a d i v a l e n t cadmium i n cadmium molybdate produces a charge imbalance which c o u l d be compensated by r e p l a c i n g t h e hexavalent molybdenum by a quadravalent niobium. The e f f e c t i v e p o s i t i v e charge o f the lanthanide i o n ( r e l a t i v e t o the l a t t i c e ) and the e f f e c t i v e negative charge o f the niobium
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1.
Probe Ion Luminescence
WRIGHT E T AL.
3
w i l l cause Coulombic a t t r a c t i o n s which w i l l favor formation o f a lanthanide-niobium complex i n the l a t t i c e . The p e r t u r b a t i o n s o f the l o c a l c r y s t a l f i e l d s by the niobium w i l l produce c r y s t a l f i e l d s p l i t t i n g s on the lanthanide i o n t h a t are unique and permit the use o f s e l e c t i v e l a s e r techniques to e x c i t e only lanthanide ions that have nearby niobium i o n s . The i n t e n s i t y i s p r o p o r t i o n a l to the number o f niobium i o n s . Any o f the lanthanide ions which are f l u o r e s c e n t can be used f o r these procedures. In a p r a c t i c a l a n a l y s i s , i t would be b e s t to choose the lanthanide i o n and the s p e c i f i c t r a n s i t i o n s t h a t gave the best s e l e c t i v i t y and s e n s i t i v i t y . In order to l i m i t the number o f v a r i a b l e s t h a t need to be optimized, we have chosen to use europium as the lanthanide i o n i n a l l o f our s t u d i e s because of s e v e r a l unique p r o p e r t i e s t h a t make Eu p a r t i c u l a r l y s u i t a b l e f o r p r e l i m i n a r y work. Th F i g u r e 1 (8). ^The groun e x c i t e d s t a t e DQ are both s i n g l e t l e v e l s (a J=0 s t a t e can have^ only an Mj =0) and t h e r e f o r e f l u o r e s c e n t t r a n s i t i o n s from D Q " * F Q o r a b s o r p t i o n t r a n s i t i o n s from ^ F - ^ D Q have only one t r a n s i t i o n . Thus, when one monitors the "^p^ f l u o r e s c e n c e t r a n s i t i o n s with an instrument of s u f f i c i e n t l y l a r g e bandpass to i n c l u d e t r a n s i t i o n s from the Eu ions i n a l l p o s s i b l e c r y s t a l l o g r a p h i c s i t e s as one scans a tuneable l a s e r over the r e g i o n of " ^ F Q - ^ D Q t r a n s i t i o n s , the r e s u l t i n g spectrum w i l l c o n t a i n o n l y one l i n e f o r each type o f c r y s t a l l o g r a p h i c environment t h a t Eu encounters i n the sample. T h i s procedure permits a r a p i d c h a r a c t e r i z a t i o n o f the s i t e s present i n a sample by t a k i n g a s i n g l e scan. The advantage i s p a r t i a l l y o f f s e t because the p o s i t i o n o f the ^ F Q - ^ D Q t r a n s i t i o n r e s u l t s only from a second order p e r t u r b a t i o n and i t s i n t e n s i t y i s lower because i t i s forbidden i n f i r s t order. There i s a l s o a p o s s i b i l i t y o f having the l i n e from a Eu with a nearby analyte a c c i d e n t a l l y overlapping l i n e s from Eu ions i n other s i t e s . These problems can be e l i m i n a t e d by using the other Eu t r a n s i t i o n s such as ^ Q ~ ^ 2 ky using other lanthanide i o n s . Q
F
>
D
o
r
LANTHANIDE ANALYSIS Our research i n t o the f e a s i b i l i t y o f implementing these ideas as an a n a l y t i c a l methodology has been d i v i d e d i n t o two s e c t i o n s development of trace methods f o r lanthanide i o n a n a l y s i s where the a s s o c i a t i o n with an analyte i s not a f a c t o r (the lanthanide i o n i t s e l f i s the analyte) and development of methods f o r other ions where the analyte ion must be a s s o c i a t e d with the l a n t h a n i d e . The o v e r - a l l problem can then be approached i n smaller s e c t i o n s while simultaneously p r o v i d i n g new a n a l y t i c a l methods. The procedure f o r performing a lanthanide a n a l y s i s c o n s i s t s of adding Ca(N0 )2 to the s o l u t i o n c o n t a i n i n g unknown amounts o f lanthanide ions and p r e c i p i t a t i n g with the a d d i t i o n of NH4F (11). The lanthanide ions c o - p r e c i p i t a t e with a very f a v o r a b l e 3
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4
NEW
APPLICATIONS
O F LASERS
T O CHEMISTRY
-,21.61
Energy cm: xio 1
21.60
3
20 21.59 17.34
r
6 5432-
o 1
ô-
MA! Eu
Eu
MA:EU,X
3+
Figure 1. The Eu electronic energy levels of the free ion are shown on the left. The splittings and shifts that occur when the Eu ion is placed within two different crystalfieldsis shown on the right for the D and Ό manifolds. Note the scale expan sion required to see the crystal field splittings. The MA:Eu represents Eu doped in a httice MA and the MA:Eu,X repre sents Eu and an analyte X in association in the MA Ixittice. 3+
5
5
2
0
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1.
WRIGHT E T
5
Probe Ion Luminescence
AL.
d i s t r i b u t i o n c o e f f i c i e n t i n the CaF2. T h i s step serves both as an e x t r a c t i o n , s e p a r a t i o n , and p r e - c o n c e n t r a t i o n . The p r e c i p i t a t e i s f i l t e r e d , washed, and i g n i t e d . The i g n i t i o n step converts the f l u o r i d e i n t e r s t i t i a l charge compensation o f the c o - p r e c i p i t a t e d lanthanide ions to an oxygen charge compensation. T h i s conversion a l s o reduces the number of d i f f e r e n t s i t e s present and i n c r e a s e s the o s c i l l a t o r strengths o f the o p t i c a l t r a n s i t i o n s . The procedure works q u i t e w e l l . No s e p a r a t i o n steps are r e q u i r e d f o r i s o l a t i n g i n d i v i d u a l lanthanide i o n s , l i n e a r c a l i b r a t i o n curves are obtained up t o c o n c e n t r a t i o n s o f 1 ppm, and d e t e c t i o n l i m i t s o f 200 p a r t s i n 1 0 are p o s s i b l e . The l a t t e r l i m i t i s expected to be lowered by a t l e a s t an order of magnitude because o f s e v e r a l improvements i n our d e t e c t i o n e l e c t r o n i c s . 1 5
ANALYTES ASSOCIATED WITH LANTHANIDES The simplest metho the lanthanide i o n and an analyte was by forming an ordered s t r u c ture o f a lanthanide compound i n the presence o f an a n a l y t e . This method i s i l l u s t r a t e d i n F i g u r e 2 f o r E u ( S 0 ) 3 produced by r a p i d evaporation o f s o l u t i o n c o n t a i n i n g 0.5 mole % Na^PO^ r e l a t i v e to SO^. The spectrum i n F i g u r e 2 was obtained by scanning a dye l a s e r over the e x c i t a t i o n r e g i o n o f the ^ F Q ^ ^ D Q t r a n s i t i o n while monitoring the ^ D Q ~ ' ^ 2 fluorescence with a very wide bandpass. Each peak represents a s i n g l e Eu s i t e . The two small peaks to the l e f t o f the main i n t r i n s i c peak are only obtained with PO^~ i n the o r i g i n a l s o l u t i o n and they have an i n t e n s i t y which i s p r o p o r t i o n a l to the P0^~ c o n c e n t r a t i o n . The same_behavior i s obtained i f A s O ^ i s p r e s e n t although the two AsO^ peaks occur a t d i f f e r e n t wavelengths from those of PO^~. Although the analyte peaks do not look very s i g n i f i c a n t i n compari s o n with the main i n t r i n s i c peak and would seem s u s c e p t i b l e to being l o s t i n the main l i n e a t lower c o n c e n t r a t i o n s , t h e i r absol u t e i n t e n s i t y i s c o n s i d e r a b l e . In f a c t , the i n t e r f e r e n c e from the main peak can be e l i m i n a t e d e i t h e r by tuning the l a s e r to e i t h e r of the e x c i t a t i o n wavelengths o f the PO4- peaks and s e l e c t i v e l y e x c i t i n g f l u o r e s c e n c e from only the Eu s i t e s with P04~ nearby or by s e t t i n g a monochromator with a narrower bandpass_to monitor a f l u o r e s c e n c e t r a n s i t i o n o f Eu s i t e s with nearby PO^ and o b t a i n i n g a s i n g l e s i t e e x c i t a t i o n spectrum. E i t h e r method produces s p e c t r a completely f r e e o f the main i n t r i n s i c l i n e s and permits one to f o l l o w the P0^~ peaks to much lower c o n c e n t r a t i o n s . w
2
>
n
i
c
n
n
a
s
b
e
e
n
4
f
=
-
A s s o c i a t i o n between the lanthanide and analyte ions can a l s o be achieved i f the two ions provide a mutual charge compensation f o r each other i n a l a t t i c e o f ions with d i f f e r e n t valences (12). T h i s method i s more complex than the previous example because s e v e r a l a d d i t i o n a l f a c t o r s become important. The Coulombic i n t e r a c t i o n s w i t h i n the charge compensating p a i r t h a t promote t h e i r a s s o c i a t i o n are opposed by the l a t t i c e d i s t o r t i o n s t h a t might
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
r e s u l t i f the p a i r becomes a s s o c i a t e d and by entropy considerat i o n s which favor randomization of the i o n s . I f these a s s o c i a t e s have favorable f r e e energies, they w i l l cause an increase i n the d i s t r i b u t i o n c o e f f i c i e n t s f o r the p a r t i t i o n i n g o f the ions i n t o the c r y s t a l l a t t i c e . A d d i t i o n a l l y , both ions can be compensated by native d e f e c t s that a r e present i n the l a t t i c e g i v i n g r i s e to i n t r i n s i c s i t e s that comprise the s p e c t r a i n the absence o f a n a l y t e s . One can again have a s s o c i a t e s o f e i t h e r lanthanide or analyte ions with the native defects i n a number o f d i f f e r e n t arrangements. A lanthanide i o n w i l l be i n v o l v e d i n a competition between a s s o c i a t i o n with an analyte, a s s o c i a t i o n with a n a t i v e d e f e c t , and d i s s o c i a t i o n . Each o f the p o s s i b l e e q u i l i b r i a w i l l a l s o a f f e c t the native d e f e c t e q u i l i b r i a o f the host l a t t i c e . T h i s s i t u a t i o n can be compared d i r e c t l y with the f a m i l i a r e q u i l i b r i a that a chemist considers f o r complexation o f EDTA with a t r a n s i t i o n metal i n an formation constant can and ammonia l i g a n d c o n c e n t r a t i o n . In the same way, success o f t h i s method r e q u i r e s favorable " c o n d i t i o n a l formation constants" f o r the lanthanide-analyte a s s o c i a t e s . There are d i f f e r e n t l i m i t i n g cases that can be encountered f o r d i f f e r e n t values o f the e q u i l i b r i a constants. 1.
Analyte-lanthanide
a s s o c i a t i o n i s strong.
2.
Lanthanide-native d e f e c t a s s o c i a t i o n i s strong while a n a l y t e native d e f e c t a s s o c i a t i o n and analyte-lanthanide a s s o c i a t i o n are weak.
3.
Both lanthanide-native d e f e c t a s s o c i a t i o n and a n a l y t e - n a t i v e d e f e c t a s s o c i a t i o n a r e strong.
4.
Lanthanide a s s o c i a t i o n with e i t h e r analyte o r native defects i s weak.
Each o f the d i f f e r e n t cases has a d i s t i n c t i v e i n f l u e n c e on the observed s p e c t r a . The f i r s t case i s the a n a l y t i c a l l y u s e f u l one i n which new l i n e s appear i n the s p e c t r a t h a t are d i r e c t l y r e l a t e d to the presence o f an a n a l y t e . T h i s case i s shown i n F i g u r e 3 f o r CdMo0 with Eu and Nb present i n t r a c e q u a n t i t i e s . These s p e c t r a were obtained by monitoring the D Q - * F transition with a very broad bandpass while scanning the wavelength o f a dye l a s e r over the p o s s i b l e F Q - * D Q t r a n s i t i o n s . The l i n e s charact e r i s t i c o f Nb can be s e l e c t i v e l y e x c i t e d or monitored to o b t a i n s p e c t r a o f only those Eu ions with nearby Nb. 4
5
7
2
7
5
For case 2, the a d d i t i o n o f analyte to a m a t e r i a l can cause marked changes i n t h e spectrum of a Eu doped system because o f the disturbance i n native d e f e c t concentrations. The a d d i t i o n o f analyte i s accompanied by an increase i n the n a t i v e d e f e c t concent r a t i o n r e q u i r e d to charge compensate t h a t a n a l y t e . I f there i s l i t t l e a s s o c i a t i o n between the analyte and i t s native d e f e c t , the concentration o f the unassociated n a t i v e d e f e c t w i l l become l a r g e
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1.
7
Probe Ion Luminescence
WRIGHT E T A L .
Figure 2. The excitation spectrum of the F -» Ό transition in Èu (SO^) with PO ~ added obtained by monitoring the fluorescence from D -» F with a broad 7
5
0
0
9
3
u
5
7
0
578
579 Wavelength (nm.)
2
of
h
CdMo0 :Eu 4
CdMo0 !Eu,Nb 4
580.2
580.6
581.0
581.4
Wavelength (nm.) 7
5
Figure 3. The excitation spectra of the F -> F> transition in CdMoO with europium alone and with europium and niobium added in trace quantities. This spectra was obtained by monitoring the D -» F fluorescence with a broad bandpass instrument. The additional line associated with the niobium is readily observed. The niobium concentration is 1 mol%. 0
0
h
5
7
0
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
s
8
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
thus depressing the concentration o f the native defects compensat i n g the Eu i o n . T h i s lowered c o n c e n t r a t i o n can be p a r t i a l l y r e s t o r e d by d i s s o c i a t i o n of Eu-native d e f e c t a s s o c i a t e s which i s r e f l e c t e d by the l o s s of l i n e i n t e n s i t y from these a s s o c i a t e d Eu s i t e s . An example o f t h i s i s shown i n F i g u r e 4 f o r PbMoO^ with Eu and As present i n t r a c e amounts. No new l i n e s appear that can be r e l a t e d to As but there i s a strong i n f l u e n c e o f As on the spectrum. I t should be emphasized at t h i s p o i n t that the explanation given above can only be c l a s s i f i e d as h i g h l y s p e c u l a t i v e a t t h i s p o i n t because of the lack o f research i n t h i s area i n our l a b o r a t o r y and others. The s p e c t r a t h a t are observed i n the t h i r d case are not w e l l d e f i n e d . The a d d i t i o n o f analyte can have no a f f e c t on the Eu spectra i f the a s s o c i a t i o n of analyte and i t s n a t i v e d e f e c t i s strong enough that the defects i s not a f f e c t e d not be t h a t strong and changes are expected although not as marked as case 2. No new l i n e s appear that are c h a r a c t e r i s t i c of the analyte. The f i n a l case corresponds to no changes i n the Eu spectra because o f the a d d i t i o n of an analyte s p e c i e s . The Eu i s i s o l a t e d and not i n t e r a c t i n g with e i t h e r analyte nor n a t i v e d e f e c t compens'ations. The model presented above i s an i d e a l i z e d one i n many respects that i s meant only to focus a t t e n t i o n on the important aspects o f t h i s method o f a n a l y s i s . I t neglects many v a r i a b l e s such as s e v e r a l competing e q u i l i b r i a with other n a t i v e d e f e c t s or i m p u r i t i e s , e q u i l i b r i a with the surrounding atmosphere, and the changes that occur i n d i s t r i b u t i o n c o e f f i c i e n t s because o f the a d d i t i o n of an analyte s p e c i e s . I t does p o i n t out the p o s s i b i l i t y of c o n t r o l l i n g the important d e f e c t e q u i l i b r i a f o r o p t i m i z i n g an a n a l y s i s procedure by analogy to the EDTA e q u i l i b r i a where the pH c o n t r o l s the a n a l y t i c a l l y important e q u i l i b r i a . For c r y s t a l l i n e l a t t i c e s , i t may be p o s s i b l e to c o n t r o l the native d e f e c t concent r a t i o n s to increase " c o n d i t i o n a l formation constants" f o r the lanthanide-analyte a s s o c i a t e s making a p a r t i c u l a r procedure f e a s i b l e or o p t i m a l . CONCLUSIONS These methods represent a departure from conventional f l u o r escence approaches t h a t become f e a s i b l e with the a d d i t i o n o f the l a s e r to the l i n e o f a n a l y t i c a l instruments. The approach i s very promising f o r s e v e r a l reasons. I t i s i n h e r e n t l y a method with very low d e t e c t i o n l i m i t s and can be used over a wide dynamic range of concentrations as demonstrated i n the experiments on t r a c e lanthanide a n a l y s i s . I f the p r e p a r a t i o n step involves a
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1.
9
Probe Ion Luminescence
WRIGHT E T A L .
580
581
Wavelength (nm.)
Figure 4. The excitation spectra of the F -» Ό transition in FhMoO with europium alone and with europium and arsenic added in trace quantities. This spectrum was obtained by monitoring the D —» F fluorescence with a broad bandpass instrument. It can be seen that the addition of the arsenic analyte does not produce any useful lines but it does have a marked effect on the intrinsic europium sites. The arsenic concentra tion is 1 mol%. 7
5
0
0
5
k
7
0
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10
NEW
APPLICATIONS OF
LASERS T O
CHEMISTRY
p r e c i p i t a t i o n , t h i s step w i l l serve as a method of preconcentrat i o n o f the s o l u t i o n i n t o the l a t t i c e . There are two steps i n the method t h a t provide a high s e l e c t i v i t y f o r p a r t i c u l a r analytes thus p o t e n t i a l l y e l i m i n a t i n g the need f o r p r i o r s e p a r a t i o n steps. The p r e p a r a t i o n w i l l exclude o t h e r ions with i o n i c r a d i i and charges incompatible with the c r y s t a l l a t t i c e . The narrow l i n e - w i d t h s o f s p e c t r a l t r a n s i t i o n s permit the s e l e c t i v e e x c i t a t i o n o f only the Eu ions with the analyte o f i n t e r e s t nearby. We have examined a number o f d i f f e r e n t chemical systems to d e t e r mine the range o f a p p l i c a b i l i t y o f such methods. Thus f a r , we have shown a n a l y t i c a l l y u s e f u l l i n e s can be found i n 15 of the elements as w e l l as the lanthanid extended to i n c l u d e a m a j o r i t p e r i o d i c t a b l e . We thus b e l i e v e the method i s widely a p p l i c a b l e and has some powerful advantages but c o n s i d e r a b l y more work i s r e q u i r e d before the method can be c o n s i d e r e d an a d d i t i o n to the a n a l y t i c a l chemist's bag of t r i c k s .
ACKNOWLEDGMENTS We would l i k e to thank the N a t i o n a l Science Foundation f o r the support of t h i s r e s e a r c h under grant number MPS74-24394.
LITERATURE CITED 1.
S t e i n f e l d , J . I . , Tunable Lasers and T h e i r A p p l i c a t i o n in A n a l y t i c a l Chemistry, C.R.C. Crit. Rev. A n a l . Chem. (1975) 5, 225-241.
2.
Fairbank, W.M., Hänsen, R.W. and Schawlow, A.L., Absolute Measurement o f Very Low Sodium-Vapor D e n s i t i e s Using Laser Resonance Fluorescence, J . Opt. Soc. Am. (1975) 65, 199-204.
3.
Hurst, G.S., Nayfeh, M.H. and Young, J.P., A Demonstration o f One-Atom D e t e c t i o n , Appl. Phys. L e t t . (1977) 30, 229-231.
4.
L y t l e , F.E. and Kelsey, M.S., C a v i t y Dumped Argon-Ion Laser as an E x c i t a t i o n Source i n Time-Resolved F l u o r i m e t r y , A n a l . Chem. (1974) 46, 855-860.
5.
Richardson, J.H., W a l l i n , B.W., Johnson, D.C. and Hrubesh, L.W., S u b - P a r t - P e r - T r i l l i o n D e t e c t i o n o f R i b o f l a v i n by Laser Induced Fluorescence, A n a l . Chem. (1976) 98, 620-621.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1.
WRIGHT
ET
AL.
Probe Ion Luminescence
11
6.
Bradley, A.B. and Zare, R.N., Laser F l u o r i m e t r y . Sub-PartPer Trillion D e t e c t i o n o f S o l u t e s , J. Am. Chem. Soc. (1976) 98, 620-621.
7.
F r a s e r , L.M. and Windfordner, J.D., L a s e r - E x c i t e d Atomic Fluorescence Flame Spectrometry, A n a l . Chem. (1971) 43, 1693-1696.
8.
Dieke, G.H., "Spectra and Energy L e v e l s o f Rare Earth Ions i n C r y s t a l s " , I n t e r s c i e n c e P u b l i s h e r s , New York (1968).
9.
Wybourne, B.G., " S p e c t r o s c o p i c P r o p e r t i e s o f Rare E a r t h s " , I n t e r s c i e n c e P u b l i s h e r s , New York (1965).
10.
T a l l a n t , D.R. and Wright, J.C., S e l e c t i v e Laser E x c i t a t i o n of Charge Compensate (1975) 63, 2074-2085
11.
Gustafson, F . J . and Wright, J.C., Ultrat r a c e Method f o r Lanthanide Ion Determination by S e l e c t i v e Laser E x c i t a t i o n , A n a l . Chem. (1977) 49, 1680-1689.
12.
Wright, J.C., Trace A n a l y s i s o f Nonfluorescent Ions by S e l e c t i v e Laser E x c i t a t i o n o f Lanthanide Ions, Anal. Chem. (1977) 49, 1690-1701.
RECEIVED
August 24, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2 Applications of Tunable-Diode-Laser IR Spectroscopy to Chemical Analysis J. F. BUTLER, K. W. NILL, A. W. MANTZ, and R. S. ENG Laser Analytics, Inc., 38 Hartwell Ave., Lexington, MA 02173
Tunable diode l a s e r spectroscopy has become a widely used and important technique f o r u l t r a - h i g h r e s o l u t i o n i n f r a r e d mea surements (1). A r e s o l u t i o n in the order o f 10 cm makes it p o s s i b l e , f o r example, t o study fully r e s o l v e d lineshapes o f Doppler broadened l i n e s o f low pressure molecular gases. Such measurements, which can be made r a p i d l y and e a s i l y with a tunable diode l a s e r spectrometer, are virtually impossible by other t e c h niques. Tunable diode l a s e r s are a l s o being used i n s e n s i t i v e air p o l l u t i o n monitors i n both p o i n t (2) and long-path c o n f i g u r a t i o n s (3). The purpose o f the present paper is t o review and d i s c u s s new a p p l i c a t i o n s o f tunable diode l a s e r spectroscopy in s e v e r a l areas r e l a t i n g specifically t o chemical a n a l y s i s and measurement, i n c l u d i n g n o n l i n e a r spectroscopy and photochemistry. These l a s e r s have a number o f unique f e a t u r e s o f p a r t i c u l a r i n t e r e s t i n chemical a n a l y s i s . Some o f these f e a t u r e s and t h e i r general uses are summarized i n Table I . -4
-1
Low L e v e l Detection Concentrations o f molecular gases can be measured t o excep t i o n a l l y low l e v e l s using tunable diode l a s e r IR absorption spectroscopy. As one example o f t h i s a p p l i c a t i o n , a group a t McMaster U n i v e r s i t y has constructed a h i g h l y s e n s i t i v e a i r p o l l u t i o n monitor using the apparatus diagrammed i n Figure 1 (2^) . The a i r sample flows i n t o the m u l t i - t r a v e r s a l absorption c e l l a t r e duced pressures. An AC modulation superimposed on the DC b i a s current induces a small r e p e t i t i v e scan o f the l a s e r emission frequency, a l l o w i n g the use o f d e r i v a t i v e spectroscopy. The McMaster group was able t o measure absorbance l e v e l s as low as α = 10~ by using the second d e r i v a t i v e . Figure 2 i l l u s t r a t e s the use o f t h i s apparatus f o r the d e t e c t i o n o f atmospheric SO2. The McMaster group reported s e n s i t i v i t y l e v e l s as low as 3 ppb 5
0-8412-0459-4/78/47-085-012$05.00/0
© 1978 American Chemical Society In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Tunable-Diode-Laser
BUTLER E T A L .
IR Spectroscopy
13
TABLE I TUNABLE DIODE LASER FEATURES RELEVANT TO ANALYTICAL INSTRUMENTATION
High S p e c t r a l P u r i t y (10
cm
)
High s p e c i f i c i t y e l i m i n a t e s or g r e a t l y reduces e r r o r s due t o i n t e r f e r i n g absorption l i n e s and the complexity o f a l g o rithms r e q u i r e d t o compensate f o r background absorptions. S p e c t r a l Region 3-30 ym r e g i o n contains absorption s p e c t r a o f n e a r l y a l l molecular substances. Tunability P r e c i s e s p e c t r a l l i n e s or regions may be s e l e c t e d . Information i n the lineshape can be u t i l i z e d . Ease o f Rapid
Modulation
High frequency operation reduces turbulence and p a r t i c u l a t e noise. Novel s i g n a l p r o c e s s i n g techniques can be employed. High Brightness Nearly opaque substances can be analyzed. Transmission over long o p t i c a l path i s f e a s i b l e . —6 Small E m i t t i n g Area (5 χ 10
2 cm )
Small, constrained regions can be probed. Long-path measurements are f a c i l i t a t e d .
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
CHOPPER
DIODE
PUMP
DIODE
LASER
CURRENT MODULATOR REF. L O C K - IN
X-Y
AMPLIFIER
RECORDER
Figure 1. Diagram of a tunable -diode-laser pollution monitoring sys tem. Sensitivity is enhanced by the use of a multi-traversal, low-pressure cell and second-derivative detection.
Figure 2. Typical measurement data for the tunable-diode-laser instrument diagrammed in Figure 1. A calibration measurement of 20 ppb of S0 in Ν is followed by a measurement of air containing 8 ppb of S0 . 2
2
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
BUTLER
ET
AL.
Tunable-Diode-Laser
15
IR Spectroscopy
f o r SO2. T h e i r p r e d i c t e d s e n s i t i v i t y l e v e l s f o r other p o l l u t a n t s measured by t h i s technique are summarized i n Table I I . As another i l l u s t r a t i o n o f the p o t e n t i a l low l e v e l d e t e c t i o n c a p a b i l i t y of tunable diode l a s e r spectroscopy, consider the mea surement o f CC>2 c o n c e n t r a t i o n . Researchers from Laser A n a l y t i c s and the New York State Department of Health (£,_5) have shown t h a t the i s o t o p i c s h i f t s i n CO2 absorption l i n e s are e a s i l y r e s o l v e d with diode l a s e r s (Figure 3) and have experimentally v e r i f i e d an absorption s t r e n g t h f o r strong l i n e s of about S = 2.1 χ 10"" cm" molecule~ cm . From the AFGL l i n e l i s t i n g s (6), i t i s found t h a t the optimum CC>2 l i n e f o r d e t e c t i o n purposes i s the Ρ (18) l i n e a t 2210.885 cm"" , and t h a t the p r i n c i p a l i n t e r f e r i n g species i n a i r i s N2O. Assume a d e t e c t i o n system i n c o r p o r a t i n g a 6 - l i t e r m u l t i t r a v e r s a l c e l l , a 200 m o p t i c a l path, and α£ = 10" . In a back ground of 7.6 Torr of a i r the minimum d e t e c t a b l e c o n c e n t r a t i o n of C02 i s found to be sponds to a t o t a l mass 1.73 χ 10~ p i c o c u r i e s i n e q u i v a l e n t u n i t s o f r a d i o a c t i v i t y . In pure CO2 without a background of a i r , c o n c e n t r a t i o n as low as 975 molecules/cm corresponding to a t o t a l mass of 4.36 χ 10"^ p i c o grams or 6.48 χ 10""4 p i c o c u r i e s can, i n p r i n c i p l e , be detected. 14
18
1
1
2
li+
1
5
llf
2
3
P a r t i a l Pressure
Determination
Laser s p e c t r o s c o p i c a n a l y s i s o f f e r s a d i r e c t , non-perturbing and general method o f determining p a r t i a l pressures i n gaseous mixtures. As i l l u s t r a t e d i n the H2SO4 study discussed below, p a r t i a l pressures can be measured by t h i s new technique to much lower l e v e l s and under more severe c o n d i t i o n s (e.g., 200°C hot s u l f u r i c acid) than would be p o s s i b l e by other methods. A Laser A n a l y t i c s group has i n v e s t i g a t e d p a r t i a l pressures of H2O, SO3 and H2SO4 vapors over a z e o t r o p i c aqueous s o l u t i o n o f H2SO4 under a program sponsored by the U.S. Environmental P r o t e c t i o n Agency (7_). The p a r t i a l pressures were determined by mea s u r i n g absorption l i n e strengths of SO3 and H2O above the s o l u t i o n and comparing them with l i n e strengths of c a l i b r a t i o n sam p l e s . The H2SO4 pressure was then deduced from a t o t a l pressure measurement using a U-tube manometer f i l l e d with l i q u i d s u l f u r i c acid. F i g u r e 4 shows a diode l a s e r scan of the vapor over an H2SO4 bath near 1416 cm" f o r a sample temperature of 165°C. The broad absorption d i p i n the center i s due t o atmospheric water vapor i n the 2.2m unpurged o p t i c a l path e x t e r n a l to the sample cell. One low pressure water l i n e i s apparent a t the center o f t h i s d i p ; the other l i n e s are due to SO3. The use of diode l a s e r spectroscopy allows s i n g l e l i n e s of SO3 or H2O t o be s e l e c t e d and t h e i r strengths measured. P a r t i a l pressures o f SO3, H2O and H2SO4 obtained i n t h i s study are summarized i n Table I I I . The d i s s o c i a t i o n constant Κ was c a l c u l a t e d from the data of Table I I I and i s i n c l u d e d i n 1
P
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
APPLICATIONS
O F LASERS T O C H E M I S T R Y
TABLE I I MINIMUM DETECTABLE CONCENTRATION FOR A TUNABLE DIODE LASER SYSTEM
Pollutant
Approximate Frequency (cm" )
S0
1140
0
2
1
Sensitivity (ppb) 3
105
3
N0
1150
2
C0
1075
300
H0
1135
50
NH
1050
2
2
2
3
0.05
PAN
1150
-0.3
CH
4
1300
0.03
S0
2
1370
0.3
N0
2
1600
0.02
NO
1880
0.03
CO
2120
0.01
C0„
2350
0.001
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
BUTLER
Tunable-Diode-Laser
ET AL.
17
IR Spectroscopy
Figure 3. Τ unable-diode-laser transmission spec trum near 4.5 ^m showing the C 0 1-0 band R(23) absorption line at 2224.713 cm' , the C 0 ρ s band Τ(62) line at 224.033 cm , the C 0 [ΟΙ ! <- ΟΡΟ] band P(51C) line at 2224.514 cm , and the C 0 v band P(2) line at 2224.257 cm- . The lower figure illustrates that three different, low pressure gas cells in series were used for the measurement. 12
16
2
1
13
16
2
1
13
16
2
1
1
14
16
1
2
3
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 2
4
Figure 4. Tunable-diode-laser transmission spectrum of the vapor above a hot (~165°C) H S 0 solution. A large dip attributable to atmospheric water vapor in the unpurged optical path external to the sample cell is evident.
on
a
w g
S
g
00
•
•ή
Ο
1
Ο
W
h-» 00
2.
BUTLER
ET AL.
Tunable-Diode-Laser
19
IR Spectroscopy
Table I I I . The data i n Table I I I are i n good agreement with an e x t r a p o l a t i o n o f higher temperature data obtained by other methods (8). TABLE I I I PARTIAL VAPOR PRESSURES ABOVE A HOT H SOi+ AZEOTROPIC SOLUTION 2
a. Temp. (°C)
P a r t i a l Pressure H0 S0 2
(Torr)
3
107
0.024 + 0.002
0.022 + 0.002
150
0.23 + 0.02
0.21 + 0.02
180 200
Η 50^ 2
0.034 + 0.02 0.32 + 0.08
Dissociation Constant Κ (Torr) P
0.0155 0.151
0.90 + 0.09 2.3 + 0.2
a. A z e o t r o p i c s o l u t i o n temperature, each case.
c e l l body i s 20°C h o t t e r i n
IR S p e c t r a l A n a l y s i s o f Aqueous S o l u t i o n s I n f r a r e d spectroscopy o f aqueous s o l u t i o n s i s p o t e n t i a l l y a powerful a n a l y t i c a l t o o l i n , f o r example, blood a n a l y s i s , b i o l o g i c a l s t u d i e s and many areas o f chemistry. However, i t has not been widely used i n the past because water samples o f u s e f u l t h i c k n e s s are e s s e n t i a l l y opaque t o IR r a d i a t i o n a t the i n t e n s i t y provided by conventional spectrometers. The a v a i l a b i l i t y o f IR l a s e r s with t h e i r much higher i n t e n s i t y now makes i t p o s s i b l e t o r e a l i s t i c a l l y c o n s i d e r IR spectroscopy o f aqueous s o l u t i o n s . To f u r t h e r pursue t h i s p o s s i b i l i t y , researchers a t Laser A n a l y t i c s have measured the t r a n s m i s s i o n o f l i q u i d water as a f u n c t i o n o f t h i c k n e s s . Laboratory, d e i o n i z e d water was i n s e r t e d i n t o a sample c e l l with microadjustable l e n g t h . A Laser A n a l y t i c s LS-3 spectrometer equipped with a 4.8 ym diode l a s e r was used f o r the measurement. Results are summarized i n F i g u r e 5, which shows absorbance p l o t t e d as a f u n c t i o n o f t h i c k n e s s . S i g n a l - t o - n o i s e r a t i o s i n excess o f two were obtained f o r absorbances as high as 5.2 (a£ = 12), i n samples more than 0.3 mm t h i c k . T h i s i s a s u f f i c i e n t t h i c k n e s s t o allow a v a r i e t y o f important IR a n a l y s i s techniques t o be employed. Nonlinear Spectroscopy and IR Photochemistry Tunable diode l a s e r s have been used i n a number o f r e c e n t experiments i n v o l v i n g n o n l i n e a r absorption and photochemical e x c i t a t i o n . F i g u r e 6, f o r example, i l l u s t r a t e s an arrangement used by a group a t MIT L i n c o l n Laboratory t o study double r e s o nance absorption i n SFg (9). The crossbeam geometry minimized s c a t t e r o f pump beam r a d i a t i o n i n t o the probe beam d e t e c t i o n s y s -
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
LU Ο Ζ < CO ce ο (fi ω <
0-1
0-2
03
THICKNESS (mm)
Figure 5. Tunable-diode-laser measurement of the absorhance of liquid water as a function of thickness at a frequency of 2080 cm . A signal-to-noise ratio greater than two was observed for thickness greater than 0.3 mm. 1
DIODE PROBE BEAM
C0 PUMP BEAM 2
Figure 6. Experimental arrangement for double resonance absorption studies of SF . The crossed-beam geometry was used to minimize scatter of pump radiation into the detection system. G
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
BUTLER E T A L .
Tunable-Diode-Laser
IR Spectroscopy
21
Figure 7. Excitation diagrams for the four DR processes observed in SF Thick arrows represent pump tion. The associated transitions, which are indicated by dashed lines and arrows.
Figure 8. Four representative transient DR signals, each with corresponding and simultaneously-measured, average transmission spectra shown below. Main peaks of DR signals in (a), (b), (c), and (d) occur at 944.91, 945.69, 943.59, and 951.19 cm' , respectively, found using CO pump lines F(18), P(16), P(22), and P(12), respectively. Gas pressure was 0.2 Torr in (a), (b), and (d), and 0.3 Torr in (c). Dotted lines connect DR signals with associated absorption lines. In (d), DR signal is shown with pump laser off, to indicate system noise. 1
z
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
2
tern. The peak i n t e n s i t y of the CO2 l a s e r pump was about 1 kW/cm , while the CW probe beam i n t e n s i t y was l e s s than 5 mW/cm . The CW probe beam was mechanically chopped and, a f t e r d e t e c t i o n and proc e s s i n g by a l o c k - i n a m p l i f i e r , y i e l d e d a s i g n a l p r o p o r t i o n a l to the average diode l a s e r power t r a n s m i t t e d through the gas c e l l . A f a s t component i n the s i g n a l , c o i n c i d e n t with the CO2 l a s e r p u l s e , appeared when the diode was tuned to the double-resonance (DR) c o n d i t i o n ; the f a s t component was recovered with a boxcar integrator. The boxcar output thus represented the t r a n s i e n t change i n the probe beam a b s o r p t i o n caused by the presence o f the pump beam. Figure 7 shows a schematic diagram of the four DR processes observed, and F i g u r e 8 shows the a c t u a l s p e c t r a l data. F i g u r e s 7a and 8a i l l u s t r a t e induced t r a n s i e n t absorption due t o a l a s e r enhanced occupation of the lower l e v e l ( l e v e l 2). F i g u r e s 7b and 8b i l l u s t r a t e induced t r a n s m i s s i o process (CGSP) i n whic F i g u r e s 7c and 8c i l l u s t r a t e induced t r a n s m i s s i o n due to a deplet i o n of the upper l e v e l and strong absorption i n the f i e l d i n duced Rabi sidebands. F i n a l l y , F i g u r e s 7d and 8d i l l u s t r a t e strong induced t r a n s m i s s i o n or b l e a c h i n g caused by the s i m u l t a neous depopulation o f the lower l e v e l and f i l l i n g o f the upper level. Several d i f f e r e n t 1-2 t r a n s i t i o n s are pumped due to power broadening, thus producing the complicated lineshape shown. A d d i t i o n a l recent examples of the use of tunable diode l a s e r s i n r e l a t e d areas i n c l u d e : diode l a s e r induced saturated absorption and Lamb d i p spectroscopy of NH3 (1£) diode l a s e r i n duced photo d i s s o c i a t i o n of dimers i n molecular beams (11); diode l a s e r induced r e a c t i o n between BCL3 and a cryogenic CH^ matrix (12); state s e l e c t i v e e x c i t a t i o n o f molecular beams, and Dopplerf r e e spectroscopy of CO and NO3 (12). 2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
BUTLER
ET
Tunable-Diode-Laser
AL.
IR Spectroscopy
23
Literature Cited 1.
Nill,
Κ. W.,
Laser Focus (1977)
13,
32
2.
Reid, J . , Garside, Β. Κ., Shewchun, J . , El-Sherbiny, M., and Ballik, Ε. Α., Appl. O p t i c s (1978) 17, 1806
3.
Hinkley, E. D., 155
4.
Eng, R. S., 16, 3072
Nill,
5.
Wahlen, M., 16, 2350
Eng,
6.
McClatchey, R. Α. D. E., C a l f e e , R. F., Fox, K., Rothman, L. S. and Garing, J . C., U.S. A i r Force Report, AFCRL-TR-73-0096 (1973)
7.
Eng., R. S., Nill, U.S. Environmental 2482 (1978)
8.
Gmitro, J . I. and Vermeulen, Am. (1964) 10, 740
9.
Moulton, P. F., Larsen, D. Μ., Walpole, J . Ν., Mooradian, Α., O p t i c s L e t t . (1977) 1, 51
O p t i c a l and Quantum E l e c t r o n i c s
K. W.
(1976) 8,
and Wahlen, Μ., Appl. O p t i c s (1977)
R. S. and
Nill,
K. W.,
Appl O p t i c s (1977)
K. W. and B u t l e r , J . F., F i n a l Report, P r o t e c t i o n Agency, Contract No. 68-02-
I n s t . Chem. Eng. J o u r n a l
10.
Jennings, D. Ε., Appl. Phys. L e t t .
11.
Gough, T. E. and Leroy, R., Tenth I n t e r n a t i o n a l Conference on Quantum E l e c t r o n i c s , (May 29-June 1, 1978) A t l a n t a , Georgia
12.
Catalano, E. and B a r l e t t a , R. E., American Chemical Meeting, (March 12-17, 1978) Anaheim, C a l i f o r n i a
13.
Gough, T. E., M i l l e r , R. E., Scoles, G. Appl. Phys. L e t t . (1977) 30, 338
RECEIVED
August 23,
(1978) 33,
493
1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Soc.
3 Two-Photon Excited Molecular Fluorescence M. J. W I R T H and F. E. 1
LYTLE
Department of Chemistry, Purdue University, West Lafayette, IN 47907
A two-fold a p p r o a c h is t a k e n in the investigation of the ana lytical a s p e c t s of t w o - p h o t o is introduced a n d characterized two-photon absorption; and two-photon excited fluorescence spec troscopy is d e m o n s t r a t e d a s a promising method for c h e m i c a l a n a l y sis, with a diversity of applications. The h i g h p e a k power a n d c o n t i n u o u s n a t u r e of the synchronous ly pumped cw dye laser allows t h e generation of a sensitive a n d precise two-photon excitation response, and the wide tunability r a n g e allows acquisition of t w o - p h o t o n excitation spectra. I t has b e e n d e t e r m i n e d that t h e cavity dumped version of the laser yields t h e b e s t p e r f o r m a n c e for t w o - p h o t o n s p e c t r o s c o p y a n d offers t h e most versatile o u t p u t method. The difference in selection rules for o n e - a n d t w o - p h o t o n s p e c t r o s c o p y g i v e s rise to a c o m p l e m e n t a r y s e t of excitation spec tra. Additionally, polarization information is retained in the t w o - p h o t o n excitation of r a n d o m l y oriented m o l e c u l e s , therefore the excitation r e s p o n s e c a n b e selective with r e s p e c t to the sym metry of the excited state. These f e a t u r e s s u g g e s t t h e a p p l i c a b i l i t y o f two-photon s p e c t r o s c o p y as a unique t o o l f o r q u a l i t a t i v e analysis. Since the wavelength o f the i n c i d e n t r a d i a t i o n i s twice t h a t which corresponds t o the t r a n s i t i o n , the two-photon e x c i t a t i o n m e t h o d c a n b e u s e d t o p r o b e s p e c i e s i n o p t i c a l l y dense m e d i a . I t i s demonstrated t h a t f l u o r o p h o r s can be q u a n t i f i e d i n m a t r i c e s h a v i n g l a r g e and v a r y i n g absorbances, w i t h d e t e c t i o n l i m i t s supe r i o r t o those o f one-photon s p e c t r o s c o p y . A l s o , t h e e r r o r due t o r e a b s o r p t i o n o f e m i s s i o n can be m i n i m i z e d by u t i l i z i n g t h e s p a t i a l s e l e c t i v i t y o f the e x c i t a t i o n process. G e n e r a l C o n s i d e r a t i o n s . The t e r m n o n l i n e a r o p t i c s r e f e r s t o t h o s e phenomena i n v o l v i n g l i g h t where t h e i n d u c e d p o l a r i z a t i o n , P, o f a n a t o m i c o r m o l e c u l a r e l e c t r o n c l o u d i s n o t a t t h e same 'Current address: Department of Chemistry, University of Wisconsin, Madison. W I 53706 0-8412-0459-4/78/47-085-024$06.50/0 © 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
WERTH AND
25
Two-Photon Excited Molecular Fluorescence
LYTLE
f r e q u e n c y as t h a t o f t h e d r i v i n g e l e c t r i c f i e l d s t r e n g t h , E. For a centrosymmetric m o l e c u l e , Ρ i s r e l a t e d t o Ε by t h e e x p r e s s i o n
ο Ρ = ε (χ Ε + χ Ε ο
1
+ ···}
3
Eq.
1
w h e r e ε i s t h e p e r m i t t i v i t y o f f r e e s p a c e and t h e χ. a r e t h e s u s c e p t i b i l i t y t e n s o r s o f o r d e r i and r a n k ( i + l ) ( l ) . L i n e a r o p t i c s o c c u r s when t h e s u s c e p t i b i l i t i e s a r e s p a c e and t i m e i n d e p e n d e n t , and when χ » χ^···. F o r t h i s s i t u a t i o n Eq. 1 c a n be r e w r i t t e n as 1
Ρ
1
(
ω
)
=
ε
χ 0
ι
(
ω
)
Ε
(
ω
)
E q
2
*
where ω i s t h e f r e q u e n c y o f t h e d r i v i n g r a d i a t i o n . To d e s c r i b e t h e b e h a v i o r o f Eq. 2 a t a l l f r e q u e n c i e s t h susceptibilit t be c o n s i d e r e d a c o m p l e
X
±
= χ' 1
+ ίχ^'.
Eq.
3
A t f r e q u e n c i e s f a r removed f r o m a t r a n s i t i o n r e s o n a n c e , t h e r e a l p a r t o f Eq. 3 p r e d o m i n a t e s . The r e s u l t a n t r e l a t i o n s h i p b e t w e e n Ρ and Ε c o n t a i n s a p h a s e l a g w h i c h i s e x p e r i m e n t a l l y o b s e r v e d as t h e r e f r a c t i v e i n d e x , where n^ = 1 + χ^ . At frequencies near reso nance the imaginary p a r t predominates. The e l e c t r i c f i e l d i s t h e n a b s o r b e d t o an e x t e n t d e t e r m i n e d by t h e e x t i n c t i o n c o e f f i c i e n t , k w h e r e 2nk = X-^" · ^ "behavior o f t h e s y s t e m t o w a r d s any g i v e n ω w i l l depend upon t h e r e l a t i v e m a g n i t u d e s o f Χ-^ (ω) and χ^"(ω). 1
r
ie
!
be
N o n - l i n e a r O p t i c s . The expanded i n t o the form
t h i r d o r d e r p o l a r i z a t i o n o f Eq.
Ρ ( ω ) = ε χ ( ω , ω ^ , ω , ω ) Ε ( ω )Ε(ω^)Ε(ω ) 3
1
0
3
1
1ε
1
1
1
Eq.
can
k
w h e r e f o u r waves a r e b e i n g m i x e d b y χ^. S e v e r a l common c o m b i n a t i o n s o f f r e q u e n c i e s a r e shown i n T a b l e I . L i k e t h e f i r s t o r d e r p o l a r i z a t i o n , the t h i r d o r d e r case e x h i b i t s important subcombina t i o n s t h a t a r e i d e n t i f i e d b y v a r i o u s r e s o n a n c e enhancements. Since these correspond t o atomic or molecular t r a n s i t i o n s , the n o n l i n e a r e f f e c t c a n be u s e d t o o b t a i n s t r u c t u r a l i n f o r m a t i o n . The p o s s i b i l i t y o f o b s e r v i n g o p t i c a l f r e q u e n c y n o n l i n e a r p h e nomena has l o n g b e e n e s t a b l i s h e d . An u p p e r b o u n d f o r t h e r e q u i r e d f i e l d s t r e n g t h can be e s t i m a t e d b y t h e f i e l d , E , b i n d i n g an e l e c t r o n t o t h e n u c l e i and t h e a p p r o x i m a t e g r u l e t h a t Χη/Χο ^ (2). A t y p i c a l v a l u e f o r E Q w o u l d be 3 x 10 Vcmwhich corresponds to an i n t e n s i t y o f ^ 1 0 l ? Wcm~ . The e x p e r i m e n t a l d i l e m m a was a l l e v i a t e d when i n l a t e I 9 6 0 , T. Maiman p u b l i s h e d t h e d e s i g n o f t h e f i r s t l a s e r (3.). A f t e r t h i s d a t e n o n l i n e a r p r o c e s s e s were d i s c o v e r e d and/or v e r i f i e d i n r a p i d s u c c e s s i o n - second harmonic g e n e r a t i o n (k) and t w o - p h o t o n a b s o r p t i o n (5.) i n 1 9 6 l ; s t i m u l a t e d Raman s c a t t e r i n g (6_), sum and d i f f e r e n c e f r e q u e n c y g e n e r a t i o n (£), n
1
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ω
1 9
±9
ω
ι
±
9
ι
, ω
ω
±
2
ω
ω
ω
ι
0
Ί
= ω.
- ω = ω 2
2 -
η
1
ΐ " 2 = ω
ω
+ ω = ω < 2
1
0
e
1
dependent r e f r a c t i v e
e
transition;
index.
= a vibrational transition.
field
strength
C o h e r e n t a n t i - S t o k e s Raman e m i s s i o n .
F o u r wave m i x i n g .
I n v e r s e Raman a b s o r p t i o n .
S t i m u l a t e d Raman e m i s s i o n .
Two-photon a b s o r p t i o n .
Intensity
Self-induced
transparence.
tripling.
Self-focusing.
Frequency
0) = a n e l e c t r o n i c
ω
χ
ω , ω
Phenomenon
(b)
ω
V
ω
Resonance Frequency
The s u p e r s c r i p t s t a r n o t a t i o n d e n o t e s t h a t t h e c o m p l e x c o n j u g a t e o f t h e e l e c t r i c i s u t i l i z e d i n the p o l a r i z a t i o n equation.
ΐ " 2
π
Mixed Frequencies
Examples o f F o u r Wave M i x i n g
(a)
2 ω
3 ω
Resultant Frequency
Table I .
3.
wiRTH
Two-Photon Excited Molecular Fluorescence
AND LYTLE
27
o p t i c a l r e c t i f i c a t i o n (8_), a n d f r e q u e n c y t r i p l i n g (£) i n 1962; s e l f - f o c u s i n g i n 196k (10); a n d c o h e r e n t a n t i S t o k e s Raman (CARS) g e n e r a t i o n i n 1965 ( l l ) . T h u s , much o f t h e e a r l y w o r k was i n v o l v e d w i t h t h e u n d e r s t a n d i n g a n d p r e d i c t i o n o f t h e s e f o r m e r l y unob served e f f e c t s . Two-Photon A b s o r p t i o n . The t h i r d o r d e r s u s c e p t i b i l i t y g i v i n g r i s e t o t w o - p h o t o n a b s o r p t i o n c a n b e w r i t t e n a s χ^ω^,ω,,-ω,^,ω^). For t h i s case t h e f i e l d s t r e n g t h product i s w r i t t e n as Ε ( ω ) Ε * ( ω ) Ε ( ω ) o r Ι Ε ( ω ) , where L^, i s t h e c y c l e a v e r a g e d i n tensity o f ω^. The t h i r d o r d e r s u s c e p t i b i l i t y c a n t h e n b e w r i t t e n a s a c o r r e c t i o n t e r m , δχ, t o t h e f i r s t o r d e r , where 1
1
2
1
2
Eq. 5
δχ (ω ) = χ (ω ,ω ,-ω ,ω ) ï . 1
The
2
2
1
1
2
±
polarization atω Ρ(ω ) = ε { (ω )Ε(ω ) + δ χ ^ ω ^ Ε ^ ) } 2
0
Χ ι
2
2
Eq. 6
As l o n g a s χ^" a n d ό χ " a r e n e g l i g i b l e , ω w i l l n o t b e a b s o r b e d . T h i s g i v e s r i s e t o a n i n t e n s i t y dependent r e f r a c t i v e i n d e x a s t h e δχ t e r m a p p r o a c h e s t h e m a g n i t u d e o f χ ^ . "Whenever χ " i s l a r g e and p o s i t i v e , d i r e c t a b s o r p t i o n o f ω o c c u r s . Whenever, δχ" i s l a r g e a n d p o s i t i v e , a n i n t e n s i t y dependent a b s o r p t i o n o f ω occurs. F i n a l l y whenever δ χ ^ i s l a r g e and n e g a t i v e , an i n t e n s i t y dependent s t i m u l a t e d e m i s s i o n o f ω o c c u r s . In t h e two-photon a b s o r p t i o n p r o c e s s , an e x c i t e d s t a t e i s c r e a t e d w i t h a n e n e r g y t w i c e t h a t o f t h e i n c i d e n t p h o t o n s . The a b s o r p t i o n l a w t h a t t h e p r o c e s s obeys i s s i m i l a r t o t h a t f o r o n e photon except t h a t t h e a b s o r p t i o n i s i n t e n s i t y dependent, as p r e dicted i n the previous section. For small values, the f r a c t i o n o f l i g h t a b s o r b e d (12) c a n be d e t e r m i n e d b y 1
2
1
!
2
2
π
2
Eq. 7
ΔΡ/Ρ = [δΡ/Α] IC
where Ρ i s t h e i n c i d e n t p o w e r , ΔΡ i s t h e change i n power due t o a b s o r p t i o n , δ i s t h e two-photon a b s o r p t i v i t y , C i s t h e concen t r a t i o n , I i s t h e path l e n g t h , and A i s t h e t r a n s v e r s e a r e a o f t h e i n c i d e n t beam. From e q u a t i o n 7 i t i s s e e n t h a t t h e t w o photon absorbance i s p r o p o r t i o n a l t o c o n c e n t r a t i o n and path l e n g t h as i n B e e r ' s l a w , and i n a d d i t i o n i t i s p r o p o r t i o n a l t o t h e l i g h t i n t e n s i t y , P/A. T h u s , t h e a b s o r p t i o n c a n b e e n h a n c e d b y i n c r e a s i n g t h e power o r b y f o c u s i n g t h e beam more t i g h t l y . The l a s e r p a r a m e t e r dependence makes measurements o f a b s o l u t e absorptions d i f f i c u l t . _Q ^ _ _^ A t y p i c a l v a l u e f o r δ i s 10 cm s p h o t o n molecule , w h i c h becomes 1 0 " L cm m o l e " W a t t " f o r m o l a r c o n c e n t r a t i o n and 333 nm r a d i a t i o n . The l a s e r s y s t e m u s e d i n t h i s s t u d y c o u l d g e n e r a t e a 1 kW p u l s e a n d f o c u s i t t o a 10 ym s p o t . Thus, t h e t w o - p h o t o n c r o s s - s e c t i o n , δΡ/Α, i s t y p i c a l l y 10~ L m o l " c m " , 1
1 1
1
1
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1
1
28
NEW
APPLICATIONS
O F LASERS T O C H E M I S T R Y
i . e . , s i x orders o f magnitude s m a l l e r than t h e c o r r e s p o n d i n g onephoton c r o s s - s e c t i o n . The s m a l l m a g n i t u d e o f t h e c r o s s - s e c t i o n i s t h e b a s i s o f a n e x p e r i m e n t a l c h a l l e n g e i n two-photon spectroscopy. Two-photon a b sorbance can be d e t e c t e d e i t h e r by measuring t h e a t t e n u a t i o n o f t h e beam p o w e r , w h i c h i s a r e l a t i v e l y i n s e n s i t i v e means, o r b y monitoring thermal, a c o u s t i c a l , i o n i z a t i o n , o r luminescence e f f e c t s i n t h e sample. I n t h e r e p o r t e d work t h e response i s m o n i t o r e d e x c l u s i v e l y by f l u o r e s c e n c e because o f i t s s e n s i t i v i t y . From e q u a t i o n 7 i t c a n be s e e n t h a t t h e f l u o r e s c e n c e 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 ΔΡ, a n d t h e r e f o r e t o P . 2
The S y n c h r o n o u s l y Spectroscopy
Pumped CW Dye L a s e r As A S o u r c e f o r Two-Photon
The s m a l l m a g n i t u d non-linear nature o f th sources be employed i n two-photon s p e c t r o s c o p y i n o r d e r t o g e n e r a t e a m e a s u r a b l e number o f e x c i t e d s t a t e s . The u s u a l d i f f i c u l t y w i t h t h i s method i s t h a t t h e e x c i t a t i o n p u l s e s v a r y u n p r e d i c t a b l y i n a m p l i t u d e , n e c e s s i t a t i n g some means o f c o r r e c t i n g t h e m e a s u r e d response f o rl a s e r pulse c h a r a c t e r i s t i c s . Pulse height v a r i a t i o n s present a p a r t i c u l a r l y severe problem i n two-photon spectroscopy b e c a u s e t h e e x c i t a t i o n depends upon t h e s q u a r e o f t h e i n c i d e n t power. T h i s p r o b l e m m i g h t be m i n i m i z e d b y t h e u s e o f c o n t i n u o u s , mode-locked l a s e r s because p u l s e f l u c t u a t i o n s w i t h these devices are s m a l l . The s y n c h r o n o u s l y pumped dye l a s e r (SPDL) i s a c o n t i n u o u s , mode-locked l a s e r w i t h demonstrated a b i l i t y t o e x c i t e two-photon t r a n s i t i o n s (13). Pulse generation i s achieved by matching t h e c a v i t y l e n g t h o f t h e dye l a s e r t o t h a t o f t h e c o n t i n u o u s l y model o c k e d a r g o n i o n l a s e r (lh 15., l 6 ) . Since mode-locking produces a t r a i n o f p u l s e s h i g h l y r e p r o d u c i b l e i n amplitude and temporal p r o f i l e , t h e p e a k power o f e a c h p u l s e i s p r o p o r t i o n a l t o t h e a v e r age power o f t h e t r a i n . Measurement o f t h e a v e r a g e power o f a c o n t i n u o u s l a s e r i s s t r a i g h t f o r w a r d a n d c a n be u s e d t o c o r r e c t the measured two-photon e x c i t a t i o n response f o r t h e i n c i d e n t peak power. The L a s e r . The o p t i c a l a n d e l e c t r o n i c l a y o u t o f t h e s y s t e m i s shown i n F i g u r e 1. A d e t a i l e d d e s c r i p t i o n o f t h e l a s e r has b e e n g i v e n p r e v i o u s l y b y H a r r i s e t a l . (ΐ]+. ΐχ) · A C o h e r e n t R a d i a t i o n CR-6 a r g o n - i o n l a s e r i s m o d e - l o c k e d b y a C o h e r e n t R a d i a t i o n M o d e l k6k a c o u s t o - o p t i c m o d u l a t o r d r i v e n a t kk MHz, p r o d u c i n g a c o n t i n u o u s t r a i n o f O.k ns w i d e p u l s e s s p a c e d 11 ns a p a r t . This t r a i n i s u s e d t o pump a C o h e r e n t R a d i a t i o n M o d e l h90 j e t s t r e a m dye l a s e r , t h e c a v i t y l e n g t h o f w h i c h i s a d j u s t e d t o e q u a l t h e pulse spacing. A S p e c t r a - P h y s i c s M o d e l 365 a c o u s t o - o p t i c d e f l e c t o r i s i n c o r p o r a t e d i n t o t h e dye l a s e r i n o r d e r t o dump p u l s e s a t a c o n t r o l l e d r a t e . The s y n c h r o n i z a t i o n b e t w e e n t h e m o d e - l o c k e d o p t i c a l p u l s e c i r c u l a t i n g i n t h e c a v i t y and t h e d e f l e c t o r i s 9
5
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. MODE
DUMPER
CAVITY
LOCKER
SAMPLE
C. W.
DYE
ARGON - ION
PM
REFERENCE
LASER
LASER
POWER METER
Figure 1. Optical and electronic block diagram of the two-photon fluorimeter. The components are described in the text.
DRIVER
DUMPER
GENERATOR
PULSE
PRESCALER
DRIVER
LOCKER
COUNTER
FREQUENCY
Ci CO
(S co Ci Co
8 Ο -*
CO Cl
?—J
Ο
a,
Co
Ο
ο
I
ο
H F W
*<
F
ϋ
>
30
NEW
APPLICATIONS O F LASERS
TO
CHEMISTRY
p r o v i d e d by a h i g h speed p r e s c a l e r o b t a i n e d from E l e c t r o n i c D i g i t a l Systems, L a f a y e t t e , Indiana. This u n i t provides a v a r i a b l e output r a t e 10 MHz t o ^ 5 KHz) b y a c t i v a t i n g a H e w l e t t - P a c k a r d M o d e l 8013B p u l s e g e n e r a t o r , w h i c h s u b s e q u e n t l y g a t e s t h e 365 r f power. The dye l a s e r was c a v i t y dumped a t r e p e t i t i o n r a t e s n e a r 2 MHz t o o p t i m i z e t h e f l u o r e s c e n c e i n t e n s i t y . The w a v e l e n g t h was m o n i t o r e d w i t h a p r i s m s p e c t r o s c o p e h a v i n g a p r e c i s i o n o f V ) . 2 nm. S p e c t r a l C o v e r a g e . T h r e e d i f f e r e n t dyes were u s e d t o o b t a i n t h e f l u o r e s c e n c e d a t a , s o d i u m f l u o r e s c e i n , r h o d a m i n e 6G, a n d r h o damine B. F o r t h e r h o d a m i n e d y e s , a l l f o u r m i r r o r s i n t h e dye l a s e r c a v i t y were c o a t e d f o r r h o d a m i n e 6G. F o r s o d i u m f l u o r e s c e i n t h e p a i r o f m i r r o r s i n t h e dye l a s e r h e a d were c o u m a r i n 6 m i r r o r s and t h e dumper m i r r o r s were c o a t e d f o r t h e a r g o n - i o n l a s e r . The t u n i n g c u r v e s f o r t h e dyes a r e g i v e n i n F i g u r e 2. From t h e f i g u r e i t c a n be s e e n t h a t t h e x t e n d s f r o m 530 t o 67 s t a t e s a r e o n e - h a l f t h e w a v e l e n g t h o f t h e i n c i d e n t beam, t h e e f f e c t i v e t u n a b i l i t y r a n g e e x t e n d s f r o m 265 t o 325 nm. A w i d e v a r i e t y o f m o l e c u l e s have t r a n s i t i o n s i n t h i s s p e c t r a l r e g i o n , thus i t i s a u s e f u l working range f o r e x p l o r i n g t h e 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 two-photon e x c i t e d f l u o r e s c e n c e spectroscopy. I n a d d i t i o n t o t h e above dyes o t h e r g r o u p s have c o n s t r u c t e d s o u r c e s b a s e d on a K r y p t o n - i o n l a s e r and t h e dyes c o u m a r i n - 1 0 2 , - 3 0 and -7 (l8_), a n d o x a z i n e - 1 , DOTC a n d HITC (l£). T h i s g i v e s a p o t e n t i a l t u n i n g r a n g e o f ^70-920 nm t o a c c e s s t w o - p h o t o n s t a t e s b e t w e e n t h e w a v e l e n g t h l i m i t s o f 235-^+60 nm. T e m p o r a l P r o p e r t i e s . The s i m p l e s t e x p e r i m e n t a l a p p r o a c h t o o b t a i n i n g two-photon s p e c t r a i s t o a d j u s t t h e wavelength by r o t a t i o n o f t h e L y o t f i l t e r a n d t o measure t h e r e s u l t i n g f l u o r e s c e n c e i n t e n s i t y a n d l a s e r power. A f t e r s u b t r a c t i o n o f a b l a n k r e a d i n g , t h e f l u o r e s c e n c e i n t e n s i t y i s normalized f o r t h e square o f t h e i n c i d e n t power. C o r r e c t i o n s o f t h i s t y p e r e q u i r e t h a t t h e shape o f t h e p i c o s e c o n d p u l s e s r e m a i n c o n s t a n t t h r o u g h o u t t h e range o f wavelengths. I f t h e s y n c h r o n o u s l y pumped l a s e r i s t r u l y m o d e - l o c k e d , t h e n t h e p u l s e shape w i l l a l w a y s r e m a i n c o n s t a n t . Mode-locked p u l s e s a r e t r a n s f o r m l i m i t e d , meaning t h a t t h e p u l s e width i s the i n v e r s e o f t h e s p e c t r a l bandwidth o f t h e l a s i n g t r a n s ition. Since the bandwidth i s constant, t h e pulse width o f a w e l l mode-locked l a s e r should remain f i x e d throughout t h e t u n i n g range. I n p r a c t i c e , h o w e v e r , t h e m o d u l a t i o n i n g a i n t h a t c a u s e s mode l o c k i n g i s n o t p e r f e c t i n t h a t i t i s d e p e n d e n t upon t h e m a g n i t u d e o f the g a i n . Since t h e g a i n i t s e l f i s wavelength dependent, i t i s e x p e c t e d t h a t t h e p u l s e shapes w i l l be a l t e r e d a t d i f f e r e n t p o i n t s i n t h e e x c i t a t i o n s p e c t r a . S u c h changes i n t h e o u t p u t must be taken i n t o account t o o b t a i n a c c u r a t e two-photon e x c i t a t i o n s p e c t r a T h e r e a r e two p o s s i b l e means o f c h a r a c t e r i z i n g t h e t w o - p h o t o n r e s p o n s e o f t h e s y n c h r o n o u s l y pumped l a s e r : m e a s u r i n g t h e p u l s e w i d t h as a f u n c t i o n o f w a v e l e n g t h i n o r d e r t o c a l c u l a t e t h e a c t u a l
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Figure 2. Tuning curves for the synchronously pumped dye laser. Fl,fluorescein;Rh-6, rhodamine 6G; and Rh-B, rhodamine B.
4
CO ι—·
Ci
Go 05 (S
ο
a
Si" ».
Γ2.
H
ta
Ο
Ο
I
Ο
κί
F
ο
>
I
ço
32
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
p e a k power; and g e n e r a t i n g t h e e x c i t a t i o n s p e c t r u m o f a known com pound t o o b t a i n c o r r e c t i o n f a c t o r s a p p l i c a b l e t o a l l s u c c e s s i v e s p e c t r a . B o t h methods w e r e a p p l i e d i n t h e w o r k t o d e t e r m i n e t h e i r relative merits. The p u l s e w i d t h o f t h e s y n c h r o n o u s l y pumped dye l a s e r h a s b e e n r e p o r t e d a s t o b e 10 p s o r l e s s (15,20.,21,22). Thus i t c a n n o t be m e a s u r e d b y any d e t e c t o r , w i t h t h e p o s s i b l e e x c e p t i o n o f a s t r e a k camera. The u s u a l method e m p l o y e d t o m e a s u r e such, s h o r t pulse durations i s an o p t i c a l a u t o c o r r e l a t i o n o f the pulse t r a i n u s i n g a M i c h e l s o n i n t e r f e r o m e t e r . The p u l s e p r o f i l e s w e r e m e a s u r e d i n t h i s w o r k , u n d e r s e v e r a l l a s e r c o n d i t i o n s . The r e s u l t s o f t h e i n t e r f e r o m e t r y showed t h a t t h e p u l s e s were t r a n s f o r m l i m i t e d o n l y a t v e r y l o w g a i n . A t h i g h g a i n s t h e p u l s e s were not s i m p l y b r o a d e n e d , b u t a c t u a l l y w e r e o r g a n i z e d i n t o two p u l s e s . A s a r e s u l t an i n t e r f e r o g r a m w i l l be o f l i m i t e d u t i l i t y i n c o r r e c t i n g e x c i t a t i o n s p e c t r a becaus be r e l i a b l e f o r d e t e r m i n i n g a i n e d f r o m t h i s measurement s u p p o r t s t h e c o n t e n t i o n t h a t t h e p u l s e shapes a r e not g u a r a n t e e d t o b e u n i f o r m t h r o u g h o u t t h e t u n i n g r a n g e and t h e r e i s d e f i n i t e l y a n e e d t o c o r r e c t f o r l a s e r p u l s e shape v a r i a t i o n s i n o r d e r t o o b t a i n t h e t r u e e x c i t a t i o n spectra. The s e c o n d a p p r o a c h f o r o b t a i n i n g c o r r e c t e d e x c i t a t i o n s p e c t r a i s t o g e n e r a t e a known s p e c t r u m t o b e u s e d a s a r e f e r e n c e f o r a l l s u b s e q u e n t s p e c t r a . T h i s method i s l i m i t e d b y t h e a b i l i t y t o p r e c i s e l y d e t e r m i n e t h e s p e c t r u m o f t h e r e f e r e n c e compound and can t h e r e f o r e i n t r o d u c e l a r g e u n c e r t a i n t i e s i n t o t h e measurement. A t p r e s e n t , h o w e v e r , i t i s a more p r a c t i c a l means o f c o r r e c t i o n t h a n t h e p u l s e w i d t h measurement. I t w o u l d b e most c o n v e n i e n t t o u s e a r e f e r e n c e compound w i t h a f a i r l y b r o a d , s t r u c t u r e l e s s s p e c t r u m i n order to maintain constant p r e c i s i o n . In preliminary s t u d i e s , s e v e r a l compounds w e r e o b s e r v e d t o h a v e f e a t u r e l e s s s p e c t r a , a n d one compound i n p a r t i c u l a r , b i s - m e t h y l s t y r y l b e n z e n e (bisMSB), a p p e a r e d t o b e r e l a t i v e l y f l a t t h r o u g h o u t t h e r h o d a m i n e 6G r a n g e . The s p e c t r u m f o r t h e compound i s shown i n F i g u r e 3a. Points that d e v i a t e d f r o m t h e c u r v e w e r e n o t r e p r o d u c i b l e f r o m one s p e c t r u m t o t h e n e x t and w e r e a t t r i b u t e d t o m i s m a t c h e s i n t h e s y n c h r o n o u s cavity length. Rhodamine 6G has s u f f i c i e n t l y l a r g e g a i n t o a l l o w c a v i t y dumped l a s e r o p e r a t i o n w i t h o u t m o d e - l o c k i n g , t h u s a f f o r d i n g t h e c a p a b i l i t y t o scan the e x c i t a t i o n spectrum w i t h a w e l l d e f i n e d p e a k power. A b i s M S B s p e c t r u m was c o r r e c t e d b y t h i s method and i s shown i n F i g u r e 3b. The s p e c t r u m was m e a s u r e d t o b e f l a t t o w i t h i n 15%, i n d i c a t i n g t h a t t h e compound c a n s e r v e a s a u s e f u l r e f e r e n c e . A c o m p a r i s o n o f t h e two s p e c t r a s u g g e s t s t h a t t h e p u l s e shape i m p r o v e s a t t h e w i n g s o f t h e t u n i n g r a n g e b e c a u s e t h e s y n c h r o n o u s l y pumped l a s e r g e n e r a t e d s p e c t r u m e x h i b i t s o v e r - c o r r e c t ions i n t h i s region.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
wiRTH A N D LYTLE
Two-Photon Excited Molecular Fluorescence
Q u a l i t a t i v e A n a l y s i s V i a Two-Photon
33
Spectroscopy
The t w o - p h o t o n a b s o r p t i o n p r o c e s s was f i r s t p r e d i c t e d i n 1931 by G e o p p a r t - M a y e r (23) f r o m r e c o g n i z i n g t h a t t h e D i r a c t h e o r y f o r Raman s c a t t e r i n g c o u l d be m o d i f i e d t o d e s c r i b e t h e s i m u l t a n e o u s a b s o r p t i o n o f two p h o t o n s . S i n c e t h e e a r l y I960 s , g r o u p t h e o r y and quantum m e c h a n i c s have b e e n e x c l u s i v e l y u s e d as t o o l s t o r e l a t e t h e measured a b s o r p t i v i t y t o t h e t r a n s i t i o n t e n s o r i n o r d e r t o e x t r a c t the c h a r a c t e r i s t i c molecular i n f o r m a t i o n . F o r e l e c t r i c d i p o l e t r a n s i t i o n s i n molecules p o s s e s s i n g a c e n t e r o f symmetry i t h a s b e e n shown t h a t t h e p a r i t y o f t h e e x c i t e d s t a t e r e s u l t i n g from two-photon a b s o r p t i o n i s o p p o s i t e t o t h a t from one-photon a b s o r p t i o n ( 5) . A t w o - p h o t o n s p e c t r u m w o u l d t h e r e f o r e r e v e a l t r a n s i t i o n s due t o g e r a d e ( e v e n ) s t a t e s t h a t a r e c o n v e n t i o n a l l y considered forbidden i n e l e c t r o n i c spectroscopy. This i s analo gous t o t h e complementar spectra. Another unique f e a t u r e o f two-photon s p e c t r a i s the presence o f a b s o r p t i o n peaks due t o two e l e c t r o n t r a n s i t i o n s (2^). I n o n e p h o t o n s p e c t r o s c o p y , t h e s t a t e s c o n n e c t e d b y a t r a n s i t i o n may d i f f e r b y o n l y one o r b i t a l . I n two-photon s p e c t r o s c o p y , s i n c e t h e i n t e r m e d i a t e s t a t e d i f f e r s f r o m t h e i n i t i a l s t a t e b y one o r b i t a l , t h e n t h e f i n a l s t a t e may d i f f e r b y two o r b i t a l s . The f i n a l s t a t e can t h e r e f o r e r e s u l t f r o m two p r o m o t i o n s o f one e l e c t r o n o r one p r o m o t i o n o f two e l e c t r o n s . The l a t t e r i s s t r i c t l y f o r b i d d e n i n one-photon spectroscopy. A r e l a t e d area w i t h a n a l y t i c a l a p p l i c a b i l i t y i s m u l t i p l e photon i o n i z a t i o n . S u c h methods t y p i c a l l y employ a t w o - p h o t o n i n d u c e d i o n i z a t i o n , where s e l e c t i v i t y i s b r o u g h t a b o u t b y t h e p r e s e n c e o f a r e s o n a n t i n t e r m e d i a t e s t a t e t o enhance t h e c r o s s s e c t i o n , a n d good d e t e c t a b i l i t y i s a c h i e v e d f r o m t h e a v a i l a b i l i t y o f s e n s i t i v e d e t e c t i o n methods f o r i o n s (25.)· A s i m i l a r method i s i n v o l v e d i n the p u r s u i t o f isotope s e p a r a t i o n v i a multi-photon ionization. E f f o r t s a r e c o n c e n t r a t e d i n t h e i n f r a r e d r e g i o n where t y p i c a l l y 30 o r more p h o t o n s a r e n e c e s s a r y f o r i o n i z a t i o n . S t u d i e s are p r e s e n t l y c e n t e r e d about t h e p h y s i c s o f t h e e x c i t a t i o n process i n order t o understand the f a c t o r s t h a t determine t h e s e l e c t i v i t y (26). 1
5
Two-Photon T r a n s i t i o n Moment. F o r c e n t r o s y m m e t r i c molecules the one-photon t r a n s i t i o n o n l y has s i g n i f i c a n t o s c i l l a t o r s t r e n g t h when t h e p a r i t y o f t h e e l e c t r o n i c wave f u n c t i o n changes upon a b sorption. This i s because the e l e c t r i c d i p o l e o p e r a t o r i s an odd f u n c t i o n , t h u s , i n o r d e r f o r t h e t r a n s i t i o n moment t o b e n o n z e r o , t h e s t a t e s c o n n e c t e d b y t h e t r a n s i t i o n must have o p p o s i t e p a r i t y (27). I n most c a s e s t h e g r o u n d s t a t e i s t h e t o t a l l y s y m m e t r i c s t a t e and the energy l e v e l s d e t e c t a b l e by one-photon s p e c t r o s c o p y c o r r e s p o n d t o t h e odd, o r u n g e r a d e , s t a t e s . The t w o - p h o t o n a b s o r p t i o n p r o c e s s can b e t h o u g h t o f a s two simultaneous one-photon a b s o r p t i o n s , w i t h the f i r s t photon
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
34
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
a c h i e v i n g an i n t e r m e d i a t e s t a t e and t h e second photon t a k i n g t h e m o l e c u l e t o t h e f i n a l s t a t e as shown i n F i g u r e k. From t h i s p i c t u r e i t c a n be a s c e r t a i n e d t h a t t h e t w o - p h o t o n e x c i t e d s t a t e w i l l be o p p o s i t e i n p a r i t y t o t h e o n e - p h o t o n e x c i t e d s t a t e . The t r a n s i t i o n moment i s n o n z e r o i f t h e i n t e r m e d i a t e l e v e l h a s u n g e r a d e c h a r a c t e r and t h e f i n a l s t a t e i s gerade. The t w o - p h o t o n s p e c t r u m w i l l t h e r e f o r e be c h a r a c t e r i s t i c o f t h e l o c a t i o n s o f t h e c o n v e n t i o n a l l y f o r b i d d e n g e r a d e s t a t e s ( 5.). The i n t e r m e d i a t e s t a t e , | i > c a n b e c o n s i d e r e d an o f f - r e s o n a n c e l e v e l o f a l l |υ^>. T h i s i s p o s s i b l e s i n c e e a c h | u > c o u l d b e d e s c r i b e d by a L o r e n t z i a n curve and thus e x t e n d t o a l l f r e q u e n c i e s . The t r a n s i e n t n a t u r e o f | i > c a n b e e x p l a i n e d v i a t h e H e i s e n b e r g U n c e r t a i n t y P r i n c i p l e , i . e . , i f E ( \ L ) - E ( i ) = 10,000 c m " , A t i s computed t o b e ^ 1 f s e c . T h i s s h o r t l i f e t i m e r e q u i r e s t h a t t h e photons i n t e r a c t s i m u l t a n e o u s l y t o produce t h e s t a t e | g j · The two p h o t o n t r a n s i t i o be w r i t t e n a s 1
>
L
[e *M(g ,u )][e *M(u ,g )] 1
M(g ,g ) = Q
1
o
k
2
k
1
Σ Ε(ΐ^)
- Πω
1
[e *M(g ,u )][e *M(u ,g )] 2
o
k
1
k
1
Eq. 8 E
(
u
k
)
"
where M ( g , u ) a n d M i u ^ g ^ a r e t h e o n e - p h o t o n t r a n s i t ia o n moments connecting the states U » | s a n d | ^ > , e a n d e~ a r e t h e p o l a r i z a t i o n v e c t o r s o f t h e two p h o t o n s . A t w o - p h o t o n s p e c t r u m w o u l d c o n s i s t o f a l l j moments t o e v e n p a r i t y s t a t e s . S e v e r a l c h a r a c t e r i s t i c s o f two-photon a b s o r p t i o n can be i n f e r r e d f r o m E q u a t i o n 8. F i r s t , as t h e e n e r g y o f e i t h e r p h o t o n a p p r o a c h e s t h a t o f one o f t h e |u^> s t a t e s , t h e m a g n i t u d e o f t h e t r a n s i t i o n moment i n c r e a s e s . S i n c e t h e o s c i l l a t o r s t r e n g t h depends upon t h e s q u a r e o f t h e t r a n s i t i o n moment, t h i s c a n p r o v i d e a r a t h e r d r a m a t i c enhancement i n t h e a b s o r p t i o n c r o s s - s e c t i o n . A g a i n a n a n a l o g y c a n be made w i t h t h e Raman s p e c t r o s c o p y . Second, t h e summation i s p e r f o r m e d o v e r a l l k u n g e r a d e s t a t e s s i n c e | i > i s c o n s i d e r e d t o be a n o f f - r e s o n a n c e s u p e r p o s i t i o n i n g o f e a c h o f them. However, t h o s e s t a t e s c l o s e s t t o | i > w i l l d o m i n a t e t h e t r a n s i t i o n moment e x p r e s s i o n b e c a u s e o f t h e r e s o n a n c e e f f e c t m e n t i o n e d above. F i n a l l y , t h e o r i e n t a t i o n o f t h e two p h o t o n p o l a r i z a t i o n v e c t o r s are independent o f each o t h e r . Both o f these factors play a r o l e i n t h e m a g n i t u d e o f t h e t r a n s i t i o n moment. Q
k
>
0
>
1
1
Symmetry C o n s i d e r a t i o n s . E x p e r i m e n t a l l y , s p e c t r a c a n b e gen e r a t e d b y t h e a b s o r p t i o n o f two p h o t o n s o f t h e same f r e q u e n c y , o r one e a c h o f two d i f f e r e n t f r e q u e n c i e s . W i t h one f r e q u e n c y t h e o p t i c a l p o l a r i z a t i o n can be l i n e a r o r c i r c u l a r , w h i l e t h e a d d i t i o n a l p o s s i b i l i t i e s o f p e r p e n d i c u l a r l i n e a r and c o n t r a r o t a t i n g
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
wiRTH
AND LYTLE
Two-Photon Excited Molecular Fluorescence
CM > OP
580
600
620
640
Wavelength (nm)
Figure 3. Excitation spectrum of bis-methylstyrylbenzene (bisMSB). ( ) Dye laser synchronously pumped (peak power > - kW) and (' ' ') cavity dumped (peak power > 5 W).
Figure 4. Schematic of the twophoton absorption process. Ger ade and ungerade states are denoted |g> and |w>, respec tively. |i> represents the inter mediate level while ω and ω are the frequencies of the two photons. 2
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
36
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
c i r c u l a r e x i s t s w i t h two f r e q u e n c i e s . B e c a u s e t h e two p h o t o n s a r e a b s o r b e d s i m u l t a n e o u s l y , t h e i r r e l a t i v e p o l a r i z a t i o n s map d i r e c t l y i n t o t h e symmetry o f t h e e l e c t r i c s u s c e p t i b i l i t y , a n d i t i s t h e r e f o r e p o s s i b l e t o p e r f o r m p o l a r i z a t i o n measurements i n f l u i d s o l u tion. The m a t h e m a t i c a l d e r i v a t i o n b y M c C l a i n (28) p r o v e s r i g o r o u s l y t h a t t h e p o l a r i z a t i o n t e r m s do n o t v a n i s h when t h e m o l e c u l e s a r e r a n d o m l y o r i e n t e d , a n d t h e r e f o r e t h e symmetry i n f o r m a t i o n i s retained. B y c o n t r a s t , i n o n e - p h o t o n s p e c t r o s c o p y , symmetry a s s i g n m e n t s c a n o n l y b e made f r o m s i n g l e c r y s t a l s t u d i e s . Again, t h e a n a l o g y b e t w e e n i n f r a r e d a n d Raman c a n b e made. i Raman s p e c t r o s c o p y t h e p o l a r i z a t i o n i n f o r m a t i o n i s r e t a i n e d because t h e e x c i t a t i o n and e m i s s i o n a r e simultaneous. I n o r d e r t o r e l a t e t h e m e a s u r e d c r o s s - s e c t i o n s t o t h e symm e t r y o f t h e t r a n s i t i o n , i t i s necessary t o determine t h e spectrum u n d e r t h r e e d i f f e r e n t p o l a r i z a t i o n c o n d i t i o n s a n d compare t h e r e s u l t s t o t h e known p a t t e r n p r a c t i c e t a b l e s have bee I t i s n e c e s s a r y t o have p r i o r knowledge o f t h e p o i n t group o f t h e m o l e c u l e b u t i t s h o u l d be n o t e d t h a t t h e two-photon experiment can be u s e f u l i n u n c o v e r i n g e n v i r o n m e n t a l e f f e c t s w h i c h l o w e r t h e symmetry o f t h e m o l e c u l e . Two-photon s p e c t r o s c o p y i s p a r t i c u l a r l y u s e f u l i n i d e n t i f y i n g t h e s y m m e t r i e s o f v i b r a t i o n a l modes w h i c h a r e c o u p l e d t o e l e c t r o n ic transitions. The s t a t e symmetry i s c a l c u l a t e d b y t h e d i r e c t p r o d u c t o f t h e e l e c t r o n i c t e r m w i t h t h e v i b r a t i o n a l t e r m (27) a n d c o m p a r e d w i t h t h e e x p e r i m e n t a l l y d e t e r m i n e d s t a t e symmetry. The a b i l i t y t o make symmetry a s s i g n m e n t s f o r e l e c t r o n i c a n d v i b r o n i c t r a n s i t i o n s i s a p r i m a r y advantage o f two-photon s p e c t r o s c o p y . The most s i m p l e t w o - p h o t o n e x p e r i m e n t s a r e p e r f o r m e d u s i n g o n l y one l a s e r f o r t h e e x c i t a t i o n p r o c e s s . I n t h i s c a s e t h e o n l y p o s s i b l e p o l a r i z a t i o n s a r e p a r a l l e l l i n e a r and c o r o t a t i n g c i r c u l a r . S i n c e t h r e e i n d e p e n d e n t p o l a r i z a t i o n measurements a r e r e q u i r e d t o u n i q u e l y s p e c i f y t h e symmetry o f t h e f i n a l s t a t e , t h e one l a s e r m e t h o d i s i n a d e q u a t e f o r t h i s p u r p o s e . W i t h two l a s e r s , t h e a d d i t i o n a l p o s s i b i l i t i e s o f p e r p e n d i c u l a r l i n e a r and c o n t r a - r o t a t i n g c i r c u l a r p e r m i t complete p o l a r i z a t i o n s t u d i e s . I t i s therefore n e c e s s a r y t o u t i l i z e two s e p a r a t e l y t u n a b l e l a s e r s i n o r d e r t o d e t e r m i n e m o l e c u l a r symmetry f r o m t w o - p h o t o n e x c i t a t i o n m e a s u r e ments . n
The N a p h t h a l e n e S p e c t r u m . The t w o - p h o t o n e x c i t a t i o n s p e c t r u m o f n a p h t h a l e n e s e r v e s t o d e m o n s t r a t e t h e c o n c e p t s d i s c u s s e d above. As a n e x a m p l e , t h e p a r t i a l e n e r g y l e v e l d i a g r a m o f F i g u r e 5 c a n be u s e d t o p r e d i c t d i f f e r e n c e s b a s e d on p a r i t y . The o n e - p h o t o n s p e c t r u m s h o u l d h a v e two bands c o r r e s p o n d i n g t o t h e A -> B ^ t r a n s i t i o n s , w h i l e t h e t w o - p h o t o n s p e c t r u m s h o u l d have one b a n d c o r r e s ponding t o the A •> Β transition. M i k a m i a n d I t o (30) h a v e s t u d i e d t h i s s y s t i m and found such a s i m p l e a n a l y s i s t o be p a r t i ally correct. T h a t i s , t h e A-^ B^ t r a n s i t i o n a p p e a r s v e r y w e a k l y , i f a t a l l , i n the, o n e - f h o t o n s p e c t r u m ; w h i l e t h e A •> Bp l g
u
g
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
wnvra
AND LYTLE
Two-Photon Excited Molecular Fluorescence
(235 r
lg
D
2u
J
(315 nm)
3u
\
—
1
•
—
Figure 5. Electronic energy level diagram for naphthalene
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
38
NEW
APPLICATIONS
O F LASERS T O C H E M I S T R Y
t r a n s i t i o n appears v e r y weakly i n t h e two-photon spectrum. This a p r i o r i g e n e r a t e s l a r g e q u a l i t a t i v e d i f f e r e n c e s i n t h e shape o f t h e two s p e c t r a i n t h e s e w a v e l e n g t h r e g i o n s . On t h e o t h e r h a n d , t h e A ·> Β t r a n s i t i o n represents a f a i l ure o f such a simple a n a l y s i s . With one-photon e x c i t a t i o n t h i s t r a n s i t i o n i s symmetry f o r b i d d e n due t o o t h e r s e l e c t i o n r u l e s . As a result the absorption t o the Β s t a t e a p p e a r s a s weak s t r u c t u r e on t h e f a r s t r o n g e r B~ b a c k g r o u n d . ( S e e F i g u r e 6.) With twop h o t o n e x c i t a t i o n t h e " t r a n s i t i o n i s weak a n d a p p e a r s o n l y b e c a u s e of coupling t o b v i b r a t i o n s . (See F i g u r e 7·) Note t h e l a r g e q u a l i t a t i v e d i f f e r e n c e s b e t w e e n t h e s p e c t r a e v e n when t h e b a n d s a r e d e r i v e d f r o m t h e same e l e c t r o n i c t r a n s i t i o n . T h e r e a r e two reasons f o r t h i s dramatic change. F i r s t , t h e two-photon spectrum does n o t have a s t r o n g t r a n s i t i o n o f c o m p a r a b l e e n e r g y , w h i l e t h e one-photon s p e c t r u m does. T h i s f a c t i s r e s p o n s i b l e f o r t h e q u i t e d i f f e r e n t b a s e l i n e s . Second b r o n i c progression correspond two-photon e x c i t a t i o n they correspond t o b vibrations. The n a p h t h a l e n e example i l l u s t r a t e s t h a t t h e f e a t u r e s o f o n e and t w o - p h o t o n e x c i t a t i o n s p e c t r a d i f f e r b e c a u s e o f t h e change i n s e l e c t i o n r u l e s and u n p r e d i c t a b l e f e a t u r e s a r i s i n g from t h e p o s s i b i l i t y o f unique c o n f i g u r a t i o n i n t e r a c t i o n s . This supports t h e d r i v e t o u t i l i z e t w o - p h o t o n e x c i t a t i o n s p e c t r a a s a t o o l i n chem i c a l a n a l y s i s because a d d i t i o n a l i n f o r m a t i o n c a n be o b t a i n e d from t h e s p e c t r a and added degrees o f s e l e c t i v i t y a r e g r a n t e d . Q u a n t i t a t i v e A n a l y s i s I n Complex Samples V i a Two-Photon copy
Spectros
The a p p l i c a t i o n o f f l u o r i m e t r y as an a n a l y t i c a l t e c h n i q u e i s l i m i t e d i n many c a s e s b y t h e s p e c t r o s c o p i c p r o p e r t i e s o f t h e s a m p l e e n v i r o n m e n t . When a s i g n i f i c a n t f r a c t i o n o f t h e i n c i d e n t r a d i a t i o n i s absorbed by t h e m a t r i x , t h e measured f l u o r e s c e n c e i n t e n s i t y c e a s e s t o be a s i m p l e f u n c t i o n o f t h e f l u o r o p h o r c o n centration. This problem has been addressed by H o l l a n d , e t a l . ( 3 1 ) , who h a v e c o r r e c t e d t h e e m i s s i o n w i t h a s i m u l t a n e o u s l y m e a s u r e d v a l u e o f s o l u t i o n a b s o r b a n c e . S u c h a scheme was shown t o be v a l i d f o r o p t i c a l d e n s i t i e s £ 2. O f t e n , h o w e v e r , t h e a n a l y t e i s f o u n d i n a more h i g h l y a b s o r b i n g medium where c o n v e n t i o n a l t e c h n i q u e s a r e v e r y u n r e l i a b l e a n d s u c h c o r r e c t i o n p r o c e d u r e s c a n be d i f f i cult. The t w o - p h o t o n method i s q u a n t i t a t i v e i n a b s o r b i n g m e d i a b e c a u s e t h e r e i s n e g l i g i b l e a t t e n u a t i o n o f t h e beam i n t h i s p r o cess. F i r s t o f a l l , t h e i n c i d e n t beam i s t w i c e t h e w a v e l e n g t h o f the t r a n s i t i o n , thus t h e r a d i a t i o n i s not absorbed by t h e onephoton process. Second, t h e two-photon process i t s e l f i s o n l y weakly absorbing. F l u o r i m e t r i c A n a l y s i s i n Complex S a m p l e s . The r a t e o f f l u o r escence emission i s , by d e f i n i t i o n , equal t o t h e r a t e o f l i g h t a b s o r p t i o n t i m e s t h e f l u o r e s c e n c e quantum y i e l d .
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3. wiRTH
AND LYTLE
Two-Photon Excited Molecular Fluorescence
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
39
40
NEW
F=
APPLICATIONS O F
LASERS TO
CHEMISTRY
I 0 A
or F = I (1-T)0
Eq.
9
where F i s t h e e m i s s i o n i n t e n s i t y , I . i s t h e a b s o r b e d e x c i t a t i o n i n t e n s i t y , I i s the i n c i d e n t e x c i t a t i o n i n t e n s i t y , Τ i s the sample t r a n s m i s s i o n and 0 i s t h e quantum y i e l d . A linear relation b e t w e e n e m i s s i o n i n t e n s i t y and c o n c e n t r a t i o n h o l d s o n l y when t h e sample a b s o r b a n c e , A, i s a p p r o x i m a t e l y e q u a l t o 1-T, b e c a u s e A, r a t h e r t h a n 1-T, i s p r o p o r t i o n a l t o c o n c e n t r a t i o n . The a p p r o x i m a t i o n A - 1-T b r e a k s down a t h i g h a b s o r b a n c e s , r e s u l t i n g i n r o l l o f f o f the c a l i b r a t i o n curve. T h i s s i t u a t i o n i s r e f e r r e d t o as the i n n e r f i l t e r e f f e c t . Under t h e s e c o n d i t i o n s , t h e i n t e n s i t y o f the i n c i d e n t r a d i a t i o n i s attenuated before reaching the center of t h e sample c e l l . When t h e r e i s mor d i m i n i s h e d a c c o r d i n g t o the t o t a l absorbance o f t h e sample. U s i n g Eq. 9, and t h e f a c t t h a t t h e r a t i o s o f t h e a b s o r b a n c e s e q u a l s t h e r a t i o o f t h e e m i s s i o n i n t e n s i t i e s f o r a b s o r b i n g samples (32), the r e l a t i o n s h i p between the absorbance o f the f l u o r o p h o r e , A , t h e t o t a l sample a b s o r b a n c e , A^, and t h e e m i s s i o n i n t e n s i t y can be d e r i v e d as F
Eq.
10
where Τ i s now t h e t o t a l sample t r a n s m i s s i o n . A c c o r d i n g t o Eq. 10, i f the t o t a l s o l u t i o n absorbance i s s m a l l , then Α - 1-T, and t h e s p e c i e s i n s o l u t i o n have independent responses. The e m i s s i o n i n t e n s i t y i n t h i s case i s p r o p o r t i o n a l t o c o n c e n t r a t i o n . At h i g h o p t i c a l d e n s i t i e s , t h e amount o f r a d i a t i o n e x c i t i n g t h e f l u o r o p h o r i s r e d u c e d by t h e r a t i o o f i t s a b s o r b a n c e t o t h e t o t a l s o l u t i o n value. The r e s u l t i n g i n n e r f i l t e r e f f e c t a l t e r s t h e i n c i d e n t i n t e n s i t y a t d i f f e r e n t d i s t a n c e s a l o n g t h e c e l l p a t h l e n g t h . Under t h e s e c i r c u m s t a n c e s , t h e f l u o r e s c e n c e i s no longer simply pro p o r t i o n a l t o c o n c e n t r a t i o n and, a t v e r y h i g h a b s o r b a n c e s , w i l l n o t e v e n be v i s i b l e a t t h e c e n t e r o f t h e s a m p l e . U n l i k e p u r e s u b s t a n c e s , d i l u t i n g the s o l u t i o n i s not g e n e r a l l y a p r a c t i c a l approach t o r e d u c i n g the problem. Often the s i g n a l would f a l l below the d e t e c t i o n l i m i t or the nature o f the f l u o r o p h o r c o u l d change b e c a u s e o f a s h i f t e d e q u i l i b r i u m . The i n n e r f i l t e r e f f e c t can be r e d u c e d by o b s e r v i n g t h e f l u o r e s c e n c e e m i s s i o n i n t h e f r o n t s u r f a c e c o n f i g u r a t i o n . T h i s method u l t i m a t e l y f a i l s a t h i g h c o n c e n t r a t i o n s because the p e n e t r a t i o n depth of the i n c i d e n t r a d i a t i o n i s dependent upon t h e sample a b s o r b a n c e . φ
A b s o r p t i o n I n t e r f e r e n c e s at E x c i t a t i o n Wavelengths. The e f f e c t o f a b s o r b i n g i n t e r f e r e n c e s was s t u d i e d by u s i n g a m o d e l s y s t e m c o n s i s t i n g o f p - t e r p h e n y l as t h e f l u o r o p h o r and b i p y r i d i n e
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
wmTH
AND LYTLE
580
Two-Photon Excited Molecular Fluorescence
41
59 WAVELENGTH (nm)
Figure 7. Two-photon excitation spectrum of naphthalene
Figure 8. Spectral data for p-terphenyl and bipyridine. (a) Excitation and (b) emission spectrum of ip-terphenyl; (c) absorption spectrum of bipyridine.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
42
NEW
APPLICATIONS OF
LASERS TO
CHEMISTRY
as t h e i n t e r f e r i n g c h r o m o p h o r e , i n t h e s o l v e n t c y c l o h e x a n e . The s p e c t r a l d a t a f o r t h i s s y s t e m a r e shown i n F i g u r e 8. The a b s o r p t i o n band o f b i p y r i d i n e o v e r l a p s t h a t o f p - t e r p h e n y l almost u n i f o r m i l y , while the emission i s i n a transparent s p e c t r a l region. A f i x e d amount o f p - t e r p h e n y l was e x c i t e d i n t h e p r e s e n c e o f a v a r y ing concentration of b i p y r i d i n e while the fluorescence of p - t e r p h e n y l was m o n i t o r e d . The r e s u l t s , w h i c h a r e p l o t t e d i n F i g u r e 9, show t h a t t h e o n e - p h o t o n e x c i t e d f l u o r e s c e n c e v a r i e s w i t h t h e s a m p l e a b s o r b a n c e r a t i o , as p r e d i c t e d . The f l u o r o p h o r c o n c e n t r a t i o n i n f o r m a t i o n i s l o s t upon o n e - p h o t o n e x c i t a t i o n when t h e c o n c e n t r a t i o n o f t h e chromophore i s unknown. I n t w o - p h o t o n e x c i t e d fluorescence, the inner f i l t e r e f f e c t i s nonexistent because the w a v e l e n g t h o f t h e i n c i d e n t r a d i a t i o n i s f a r removed from t h e s o l u t i o n a b s o r b a n c e and a n e g l i g i b l e amount o f power i s e x t r a c t e d by the n o n l i n e a r process. Therefore t h e i n t e n s i t y o f t h e beam i s n o t a f f e c t e d by the v a r i a t i o h i g h s o l u t i o n absorbance is retained. A b s o r p t i o n I n t e r f e r e n c e s a t E m i s s i o n W a v e l e n g t h s . A common problem e n c o u n t e r e d i n t h e f l u o r e s c e n c e a n a l y s i s o f complex samples i s the reabsorption of emission. T h i s o c c u r s when t h e e m i s s i o n band o f t h e a n a l y t e i s o v e r l a p p e d by t h e absorbance band o f a n o t h e r species i n solution. In r i g h t angle d e t e c t i o n t h e e m i s s i o n must p a s s t h r o u g h a c e l l p a t h l e n g t h o f 0.5 cm b e f o r e r e a c h i n g t h e de t e c t o r , t h u s t h e a b s o r b a n c e o f t h e s o l u t i o n c a n n o t e x c e e d 0.025 u n i t s a t the e m i s s i o n w a v e l e n g t h i n o r d e r t o keep the q u a n t i t a t i o n e r r o r b e l o w 5?. F o r a chromophore w i t h a m o l a r a b s o r p t i v i t y o f 1 0 ^ , i t s c o n c e n t r a t i o n must be b e l o w 2.5 yM t o k e e p t h e e m i s s i o n i n t e n s i t y r e p r e s e n t a t i v e o f t h e f l u o r o p h o r c o n c e n t r a t i o n . As i n t h e p r e v i o u s l y d i s c u s s e d i n t e r f e r e n c e c a s e , d i l u t i o n o f t h e sample w o u l d n o t n e c e s s a r i l y be a p r a c t i c a l a p p r o a c h . The r e a b s o r p t i o n p r o b l e m i s c i r c u m v e n t e d w i t h t w o - p h o t o n s p e c troscopy because i t i s p o s s i b l e t o u t i l i z e the f a c t t h a t the e x c i t a t i o n e f f i c i e n c y 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 the transverse area o f t h e beam. By f o c u s i n g t h e i n c i d e n t beam c l o s e t o t h e f r o n t surface of the c e l l , the path length of the emission i s decreased t h u s t o l e r a t i n g a h i g h e r chromophore c o n c e n t r a t i o n . The p a t h l e n g t h o b s e r v e d i n t h i s w o r k was e s t i m a t e d t o be 100 ym, a l l o w i n g t h e s o l u t i o n a b s o r b a n c e a t t h e e m i s s i o n w a v e l e n g t h t o be as h i g h as 3. The m o d e l s y s t e m u s e d i n t h i s s t u d y c o n s i s t e d o f t h e f l u o r o p h o r s b i s - m e t h y l s t y r y l b e n z e n e ( b i s M S B ) and 2,5-diphenyloxazole (PP0). The f l u o r e s c e n c e s p e c t r a o f F i g u r e 10 show t h a t t h e ab s o r b a n c e b a n d o f b i s M S B s t r o n g l y o v e r l a p s t h e e m i s s i o n b a n d o f ΡΡ0. F o r t h e e x p e r i m e n t , t h e c o n c e n t r a t i o n o f ΡΡ0 was h e l d c o n s t a n t , t h e c o n c e n t r a t i o n o f b i s M S B was v a r i e d , and t h e f l u o r e s c e n c e i n t e n s i t y o f ΡΡ0 was m o n i t o r e d f o l l o w i n g one- and t w o - p h o t o n e x c i t a t i o n . The e x p e r i m e n t a l r e s u l t s are p r e s e n t e d i n F i g u r e 11. I n the one-photon a n a l y s i s , f r o n t s u r f a c e d e t e c t i o n was i m p l e m e n t e d i n o r d e r t o e f f e c t
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
wiRTH
AND LYTLE
Two-Photon Excited Molecular Fluorescence
Bipyridine
43
Absorbance
Figure 9. Fluorimetry data for the p-terphenyl determination, (a) Two-photon and (b) one-photon excitation.
250
300
350 Wave leng t h
400
450
500
( nm )
Figure 10. Spectral data for 2,5-diphenyloxazole (PPO) and bis-methylstyrylbenzene (bisMSB). (a) Excitation and (b) emission spectra of PPO; (c) excitation and (d) emission spectra of bisMSB.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
44
N E W APPLICATIONS
0
1 bisMSB
O F LASERS T O C H E M I S T R Y
2 Absorption
3 (350nm)
Figure 11. Fluorimetry data for the mixture analysis, (a) Onephoton and (b) two-photon excitation.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
wiRTH
AND LYTLE
Two-Photon Excited Molecular Fluorescence
45
a s h o r t p a t h l e n g t h , h o w e v e r , t h i s e m i s s i o n was s t i l l d i m i n i s h e d a t h i g h s a m p l e a b s o r b a n c e s due t o t h e r e a b s o r p t i o n p r o c e s s . The d i s t a n c e t h r o u g h w h i c h t h e f l u o r e s c e n c e t r a v e l e d was s u f f i c i e n t l y l a r g e t o r e s u l t i n q u a n t i t a t i o n e r r o r s . I n t h e two-photon case, the i n t e n s i t y remains constant, independent o f t h e i n t e r f e r e n c e c o n c e n t r a t i o n up t o s o l u t i o n a b s o r b a n c e s o f 3. I t s h o u l d be n o t e d t h a t t h e e f f e c t s o f e m i s s i o n r e a b s o r p t i o n can b e a l l e v i a t e d t o some e x t e n t i n o n e - p h o t o n s p e c t r o s c o p y b y c o n s t r u c t i n g a spectrometer w i t h very p r e c i s e f o c a l parameters. I t i s p o s s i b l e t o c o l l e c t fluorescence i n t e n s i t y from only t h e f i r s t 1Û0 ym o f t h e s a m p l e , b y u s i n g h i g h q u a l i t y o p t i c s . There a r e t w o l i m i t a t i o n s t o t h i s a p p r o a c h . F i r s t , 100 ym i s a b o u t t h e s m a l l e s t p r a c t i c a l d i s t a n c e t h a t c a n be a c h i e v e d , whereas t h e twop h o t o n method c a n g e t down t o < 10 ym. S e c o n d , t h e u s u a l f r o n t s u r f a c e i n t e r f e r e n c e s a r e s t i l l a b l e t o hamper t h e a n a l y s i s : a) t h e b a c k g r o u n d s c a t t e c e l l i s subject t o adsorptio s e l f may f l u o r e s c e . These p r o b l e m s a r e a v o i d e d w i t h t w o - p h o t o n e x c i t a t i o n because t h e i n c i d e n t l i g h t i s n o t focused through t h e s a m p l e c e l l i n t h e s p a t i a l r e g i o n where t h e f l u o r e s c e n c e i s o b served. F l u o r i m e t r i c A n a l y s e s i n A b s o r b i n g S o l v e n t s . An i m p o r t a n t s p e c i a l case o f an i n t e r f e r i n g chromophore i s t h e o p t i c a l l y dense s o l v e n t . W i t h one-photon e x c i t a t i o n , i t i s i m p e r a t i v e t h a t t h e s o l v e n t b e t r a n s p a r e n t where t h e s p e c i e s o f i n t e r e s t a b s o r b . When t h i s r e s t r i c t i o n precedes chemical c o n s i d e r a t i o n o f t h e solvent c h o i c e , t h e a n a l y s i s i s a p r i o r i l e s s t h a n t h e optimum. A s a w o r s t c a s e , t h e f l u o r o p h o r i s o f t e n i n , a n d c a n n o t b e removed f r o m , a n o p t i c a l l y dense m a t r i x . W i t h two-photon e x c i t a t i o n , t h e i n c i d e n t r a d i a t i o n i s twice t h e wavelength o f t h e solvent absorbance, thus i t e a s i l y penetrates into t h e matrix t o e x c i t e t h e species o f interest. The s p e c t r a l d a t a f o r a m o d e l s y s t e m , PPO i n a c e t o n e , a r e given i n F i g u r e 12. V i s u a l i n s p e c t i o n o f t h e s o l u t i o n s i n d i c a t e s t h a t v i r t u a l l y none o f t h e e x c i t i n g r a d i a t i o n r e a c h e s t h e c e n t e r o f t h e sample c e l l . As a r e s u l t , a f r o n t s u r f a c e c o n f i g u r a t i o n i s mandatory f o r one-photon e x c i t a t i o n . Even w i t h such an arrangement, s e n s i t i v i t y i s decreased by t h e numerical value o f t h e s o l v e n t , a c c o r d i n g t o Eq. 10. S i n c e two-photon e x c i t a t i o n i s n o t a t t e n u a t e d b y t h e s o l v e n t , a r i g h t a n g l e c o n f i g u r a t i o n may b e u s e d . F i g u r e 1 3 shows t h e f l u o r e s c e n c e c a l i b r a t i o n c u r v e s o b t a i n e d f o r t h e model systems d i s c u s s e d above. W i t h t h e l a s e r s o u r c e u s e d i n t h i s s t u d y , t h e d e t e c t i o n l i m i t (S/N = l ) f o r two-photon e x c i t a t i o n o f PPO i n a c e t o n e i s a p p r o x i m a t e l y 0.5 yM. T h i s r e s u l t i s p r i m a r i l y d e t e r m i n e d b y t h e p e a k power o f t h e s o u r c e , w h i c h i t s e l f c a n c e r t a i n l y b e i m p r o v e d b y a t l e a s t a n o r d e r o f m a g n i t u d e . The o n e - p h o t o n d e t e c t i o n l i m i t i s a p p r o x i m a t e l y t h e same a s t h a t f o r the two-photon case. Although t h e one-photon v a l u e can c o n c e i v a b l y be l o w e r e d , i t i s u l t i m a t e l y l i m i t e d b y t h e amount o f s c a t t e r e d
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
46
NEW
250
300
APPLICATIONS
350 W a v e l e n g th
O F LASERS T O C H E M I S T R Y
400
450
(nm)
Figure 12. Spectral data for PPO in acetone, (a) Excitation and (b) emission spectra of PPO. The dashed curve represents the acetone cut off.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
wiRTH
AND LYTLE
Two-Photon Excited Molecular Fluorescence
Figure 13. Fluorimetry data for the PPO in acetone determination, (a) Two-photon and (b) one-photon excitation.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
48
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
e x c i t a t i o n e n t e r i n g t h e e m i s s i o n monochromâtor. T h i s i s a n i n h e r e n t problem i n t h a t the e x c i t a t i o n and e m i s s i o n f r e q u e n c i e s are u s u a l l y c l o s e t o each other. N a t u r a l l y , t h ef r o n t surface arrangement maximizes t h e d i f f i c u l t y . T h e r e a r e no a p r i o r i r e a s o n s t o assume t h a t a n i n c r e a s e i n s o u r c e power w i l l i m p r o v e t h e d e t e c t i o n l i m i t , because the background noise i s i n c r e a s e d pro portionally. A g a i n , two-photon e x c i t a t i o n minimizes t h i s problem s i n c e the f r e q u e n c i e s are u s u a l l y f a r from each o t h e r and a r i g h t a n g l e o b s e r v a t i o n can be used. I t i s e v i d e n t f r o m F i g u r e 13 t h a t t h e o n e - p h o t o n c u r v e s e x h i b i t the well-known i n n e r f i l t e r r o l l - o f f a t h i g h concentrations. The l i n e a r i t y i n a n y f l u o r e s c e n c e c a l i b r a t i o n f a i l s a s s o o n a s t h e e m i t t e r a b s o r b a n c e becomes s u f f i c i e n t l y l a r g e a s t o b r e a k down t h e approximation that A - ( l - T ) . I n o p t i c a l l y dense s o l v e n t s t h i s r o l l o f f occurs a t c o n c e n t r a t i o n s s e v e r a l hundred times h i g h e r than i n t r a n s p a r e n t s o l v e n t s du s h o u l d be noted t h a t th i n c r e a s e d r o l l - o f f c o n c e n t r a t i o n because the lower l i m i t o f d e t e c t i o n i s s i m u l t a n e o u s l y r a i s e d b y t h e same f a c t o r . For two-photon e x c i t a t i o n t h e t o t a l a b s o r p t i o n i s s o s m a l l t h a t t h e r e i s no i n n e r f i l t e r e f f e c t o p e r a t i n g a n d , t h e r e f o r e , no c u r v a t u r e i n t h e c a l i b r a t i o n g r a p h . T h u s , f o r f l u o r o p h o r s i n o p t i c a l l y dense m a t r i c e s , t h i s method p r o d u c e s t h e l a r g e s t l i n e a r r a n g e . A c k n o w l e dgement s T h i s r e s e a r c h was s u p p o r t e d i n p a r t t h r o u g h f u n d s p r o v i d e d b y The N a t i o n a l S c i e n c e F o u n d a t i o n u n d e r G r a n t CHE77-2^312. M.J.W. g r a t e f u l l y acknowledges the American A s s o c i a t i o n o f U n i v e r s i t y Women f o r f e l l o w s h i p s u p p o r t . Literature Cited 1.
2. 3. 4. 5.
F o r a brief, b u t c o m p r e h e n s i b l e discussion of tensors, s e e F. Z e r n i k e a n d J. E. Midwinter, "Applied Nonlinear Optics", Wiley-Interscience, New Y o r k , 1973. B l o e m b e r g e n , N. in "Quantum Electronics: A Treatise", H. R a b i n and C. L. Tang, E d s . , A c a d e m i c Press, New Y o r k 1975. Maiman, Τ. Η., N a t u r e , (1960), 187, 493. F r a n k e n , P. A. et al., P h y s . Rev. Lett., (1961), 7, 118. K a i s e r , W. a n d Garrett, C. G., P h y s . Rev. Lett., (1961), 9,
455. 6. 7. 8. 9. 10. 11.
E c k h a r d t , G., et al., P h y s . Rev. Lett., (1962), 7, 229. B a s s , M., et al., P h y s . Rev. Lett., (1962), 8, 18. B a s s , M., et al., P h y s . Rev. Lett., (1962), 9 , 446. T e r h u n e , R. W., M a k e r , P. D., a n d S a v a g e , C. M. P h y s . R e v . Lett., (1962), 8, 404. C h a i o , R. Y., G a r m i r e , Ε . , a n d Townes, C. Η., P h y s . R e v . Lett., (1964), 12, 279. M a k e r , P. D. a n d T e r h u n e , R. W., P h y s . Rev., (1965), 137, 801.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. ron., 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
WIRTH
AND
LYTLE
Two-Photon Excited Molecular Fluorescence
49
M c C l a i n , W. Μ., A c c . Chem. R e s . , (1974), 7, 129. W i r t h , M. J. a n d Lytle, F. Ε . , Anal. Chem., (1977), 4 9 , 2054. Harris, J. M., C h r i s m a n , R. W., a n d Lytle, F. Ε . , Appl. P h y s . Lett., (1975), 2 6 , 16. Chan, C. K. a n d Sari, S. O., Appl. P h y s . Lett., (1974), 25, 403. Mahr, H. a n d Hirsch, M. D., Opt. Commun., (1975), 13, 96. Harris, J. Μ., G r a y , L. M., Pelletier, M. J., a n d Lytle, F. Ε . , M o l . Photochem., (1977), 8, 161. S t e i n m e t z , L. L . , R i c h a r d s o n , J. H., Wallin, B. W., a n d Book less, W. Α., presented at the Topical M e e t i n g for Picosecond Phenomena, Hilton Head, S o u t h Carolina, May 1978. K u h l , J., L a m b r i c h , R., a n d v o n d e r Linde, D., Appl. P h y s . Lett., (1977), 31, 657. J a i n , R. K. a n d Heritage, J. P. Appl. P h y s Lett., (1978) 32, 41. Ausschnitt, C. P. Jain, , Appl. P h y s Lett., ( 1 9 7 8 ) , 32, 727. Frigo, M. J., D a l e y , T., a n d Mahr, Η., I E E E J. Quant. Elect (1977), 1 3 , 101. G e o p p a r t - M a y e r , Μ., Ann. P h y s . (Leipzig), (1931), 9, 273. M c C l a i n , W. M. a n d Harris, R. Α., in "Excited States", Vol. 3 , (1977), E. C. L i m , E d . , A c a d e m i c Press, New Y o r k 1977. G e l b w a c h s , J. Α., Klein, C. F., a n d W e s s e l , J. Ε . , Appl. P h y s . Lett., (1977), 9 , 489. L e t o k h o v , V. S., Ann. Rev. P h y s . Chem., (1977), 2 8 , 1 3 3 . C o t t o n , F. Α., " C h e m i c a l Applications of Group T h e o r y " , WileyInterscience, New Y o r k 1971. Monson, P. R. a n d McClain, W. M., J. Chem. P h y s . , ( 1 9 7 0 ) , 5 3 , 29. M c C l a i n , W. M., J. Chem. P h y s . , (1971), 55, 2789. M i k a m i , N. a n d Ito, Μ., Chem. P h y s . Lett., (1975), 3 1 , 472. H o l l a n d , J. F., Teets, R. Ε . , Kelley, P. Μ., a n d T i m n i c k , Α., Anal. Chem., (1977), 4 9 , 706. M i c h a e l s o n , R. C. a n d L o u c k s , L. F., J. Chem. E d . , (1975), 5 2 , 652.
RECEIVED
August 25, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4 Laser-Excited Luminescence Spectrometry J. D. WINEFORDNER Department of Chemistry, University of Florida, Gainesville, F L 32611
Laser e x c i t e d atomi r e c e n t l y i n the d e t e c t i o n of u l t r a low concentrations of s p e c i e s . For example, atomic a b s o r p t i o n flame spectrometry (AAS) is l i m i t e d to ~10 atoms/cm . Mass spectrometry is g e n e r a l l y a lit tle l e s s s e n s i t i v e than AAS. Conventional atomic f l u o r e s c e n c e flame spectrometry (FAFS) i s capable of ~10 atoms/cm . Dop p l e r - f r e e , two photon atomic f l u o r e s c e n c e spectrometry (DAFS) i s capable of d e t e c t i n g ~10 species/cm but the absorption cross - s e c t i o n s must be l a r g e , n a t u r a l and collisional l i n e widths must be s m a l l , and r a d i a t i o n l i f e t i m e s must be s h o r t . Resonantly en hanced two-photon a b s o r p t i o n spectrometry (TPAAS) allows detec t i o n of ~10 atoms/cm , but the absorber must have an i n t e r m e d i ate s t a t e of the a p p r o p r i a t e energy to allow utilization of two counter-propagating or co-propagating l a s e r beams. Single-photon l a s e r e x c i t e d f l u o r e s c e n c e spectrometry (SAFS) has a l s o been used to detect ~ 1 0 Na atoms/cm . SAFS however, r e q u i r e s f r e quency-doubled dye l a s e r s f o r many t r a n s i t i o n s and i n some cases difficult frequency-mixing systems to o b t a i n good d e t e c t i o n lim its. L a s e r - e x c i t e d non-resonance AFS (LNRAFS) has been used to detect 10 atoms/cm , although d e t e c t i o n l i m i t s of ~10 atoms/cm have been p r e d i c t e d . In the LNRAFS case, the atom must have 3 l e v e l s with appropriate o p t i c a l and mixing p r o p e r t i e s . Finally, p h o t o i o n i z a t i o n d e t e c t i o n of a l a s e r - e x c i t e d gaseous system (PLE)L2>Ii of Cs has been found to y i e l d a d e t e c t i o n l i m i t of the order of 1 atom/cm^. The PLE system i s e x t r a o r d i n a r i l y s e n s i t i v e but i s plagued with problems d i f f i c u l t to circumvent i n a n a l y t i c a l spectrometry, such as the sampling method, i n c l u d i n g the means of producing atoms (or molecules), and the l a s e r source which must cover a wide-wavelength range. Photon c a t a l y s i s l i t which i n v o l v e s the use of a c t i v e n i t r o g e n produced by passing N2 through a microwave discharge, has been used to detect ~10^ atoms/cm^ of B i i n an Ar atmosphere. Photon c a t a l y s i s and mass spectrometry do not i n v o l v e the use of l a s e r e x c i t a t i o n but do 1,2
8
3
2,3
4
3
4
5
3
5
2
3
6
3
2
7
4
3
0-8412-0459-4/78/47-085-0 5 0$07.25/0 © 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3
4.
51
Laser-Excited Luminescence Spectrometry
wiNEFORDNER
8
r e s u l t i n good d e t e c t i o n l i m i t s , i . e . , order of or below 1 0 atoms/ cm . I t i s apparent that a c t i v e work i s c u r r e n t l y on-going i n the area of u l t r a - t r a c e d e t e c t i o n of atoms and molecules. The detec t i o n l i m i t s i n d i c a t e d above are t r u l y e x c i t i n g when one considers that a species with a molecular weight of 100 amu represents 1.67x10 g/cm assuming a d e t e c t i o n l i m i t of 1 atom/cm or 1.67 x l O ^ g/cm assuming a d e t e c t i o n l i m i t of 10° atoms/cm . I f the atoms are produced i n a t y p i c a l a n a l y t i c a l flame (the detec t i o n l i m i t of 1 cm" i s v a l i d only f o r a quartz v e s s e l ) , then the conversion f a c t o r of sample c o n c e n t r a t i o n ( i n pg/ml) a s p i r a t e d i n t o a chamber type laminar flame system to gas c o n c e n t r a t i o n (atoms/cm ) assuming a sample i n t r o d u c t i o n ( t r a n s p o r t ) r a t e of 3 cnr/min, an unburnt gas flow r a t e (room temperature) of 20 &/min, a flame temperature of 2500°K and a 10% n e b u l i z a t i o n e f f i c i e n c y i s ~10~9, i . e . atoms/cm corresponds t 3
2
2
3
- 1
3
3
Fundamental Considerations Pseudo-Continuum Laser E x c i t e d Atomic Fluorescence Spectrome t r y , AFS. B o u t i l i e r , et a l j X and others?> h» ^ ^ h a v e considered the atomic fluorescence radiance expressions f o r the case of "broad-band" (pseudo-continuum, i . e . , <5Vg>6v^, where 6vg = spec t r a l h a l f - w i d t h of source of e x c i t a t i o n and 6v^ = absorption l i n e half-width) e x c i t a t i o n of atoms present i n a medium at low o p t i c a l d e n s i t i e s . The general expression f o r Bp, the fluorescence r a d i ance i s given by: ην
τ»
Y F Ε Ρ a
r ι
So36
η
m-i
ττ
-ζ ο — ι±
(1)
v
τη
χ.
or AU F
IT A
n
j'
m
"2
"Ι
(2)
s r
where: f l u o r e s c e n c e path length i n d i r e c t i o n of observation, m, fluorescence power e f f i c i e n c y ,
pF
absorbed W
=
s p e c t r a l i r r a d i a n c e from source of e x c i t a t i o n ,
2
m"",
hv^ = absorbed BA
dimensionless,
photon energy, J , 1
J
E i n s t e i n induced absorption c o e f f i c i e n t , s (Jm Hz
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
)
,
NEW
APPLICATIONS
d e n s i t y o f a n a l y t e s p e c i e s i n lower
O F LASERS T O C H E M I S T R Y
l e v e l of absorption
-3 transition, m
,
d e n s i t y of a n a l y t e s p e c i e s i n upper l e v e l of a b s o r p t i o n 3
transition, m δ
g
£'
,
s t a t i s t i c a l w e i g h t s o f l e v e l s £ and m, r e s p e c t i v e l y , d i -
m
mensionless, A.p = E i n s t e i n c o e f f i c i e n t o f s p o n t a n e o u s e m i s s i o n f o r f l u o r escence t r a n s i t i o n
s
\
n. = d e n s i t y o f a n a l y t
3
-3 transition, m
F o r a t h r e e - l e v e l atom o f t h e N a - t y p e , B_ i s g e n e r a l l y g i v e n Γ
by
A
4π
u£
h V
(3)
n
u£ T
A
T
£->u
+k u£
1+
,+k • uu u£ u u
v, So 1+-*- [1+ p ^ ' l u' V £u g
E
f
f
w h e r e a l l t e r m s a r e as d e f i n e d above e x c e p t t h a t t h e k s a r e p s e u d o - f i r s t order r a d i a t i o n l e s s d e a c t i v a t i o n rate constants ( s " " ) , Ε* i s a m o d i f i e d s a t u r a t i o n s p e c t r a l i r r a d i a n c e and i s g i v e n by 1
Z
u
c A , u' '£u
Y
f
f
B
(4)
, W m
f
u £ u £
w h e r e Y i £ i s t h e f l u o r e s c e n c e quantum e f f i c i e n c y and u , i s t h e u p p e r l e v e l i n v o l v e d i n t h e e x c i t a t i o n p r o c e s s , and £ i s t h e l o w e r l e v e l i n v o l v e d i n t h e a b s o r p t i o n p r o c e s s ( n o t e f o r some t r a n s i t i o n s u = u ) , and η i s t h e t o t a l d e n s i t y o f a n a l y t e atoms i n a l l e l e c t r o n i c s t a t e s . The s a t u r a t i o n s p e c t r a l i r r a d i a n c e , Ef ,i s '£u' g i v e n by u
f
t
+
NiU Vu
+ A
f
k
f
C
u £~ u u
k +k +A u£ u u 0
f
T
'£u
f
I V £
e
ι
k .
n
+
u
u£
'
u£
u
uu'
uX.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
(5)
4.
wiNEFORDNER
53
Laser-Excited Luminescence Spectrometry
For a t h r e e - l e v e l atom of the T£-type, Β
i s generally
given
Γ
by A u
r
h
v
u * ' \
E*
.
,
(6)
£-m v
£u
f
£ £
where a l l terms are as d e f i n e d above except f o r c A u£ -2 , W m , Υ Β u£ u£ T T
'£u
(7)
and the s u b s c r i p t s u, £ uppe (metastable) l e v e l , and the lower l e v e l ( f o r some t r a n s i t i o n s £ = £ ) , respectively. F i n a l l y , the s a t u r a t i o n s p e c t r a l i r r a d i a n c e , i s given by £u A +A + k +k u£ u£' u u£ (8) £ £ ' u £ ' u£' £u £u— + 8» ££ £ £ T
ol
f
, t 0
k
J
+ A
k
l + k
+ k
T
I t should be noted that e i t h e r the Na-type case or the T£type case r e v e r t s to the 2 - l e v e l case i f i n the former u -Hi and i n the l a t e r £'+£, i . e . , f
B_
Α
ir>£
£-m
«ΛΛ
(9) 1+
1+
£u £a
From the above expressions, p a r t i c u l a r l y ^n the l i m i t i n g c a ses of high source s p e c t r a l i r r a d i a n c e ( E >>E ) and i n the case of low source s p e c t r a l i r r a d i a n c e ( E << Ε * ) , s e v e r a l i n t e r e s t i n g conclusions r e s u l t . (1) F o r low o p t i c a l d e n s i t i e s , B i s l i n e a r l y r e l a t e d to r ^ , the t o t a l atom density; (2) For low source s p e c t r a l i r r a d i a n c e , Bp i s l i n e a r l y r e l a ted to source s p e c t r a l i r r a d i a n c e and to the quantum e f f i c i e n c y of the f l u o r e s c e n c e t r a n s i t i o n ; (3) For high source s p e c t r a l i r r a d i a n c e , Bp i s independent of source s p e c t r a l i r r a d i a n c e ; f o r the 2 l e v e l atom case, a s i m p l i s t i c r e l a t i o n between Bp and n-p r e s u l t s , but f o r a v
v
p
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
54
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
3 - l e v e l atom, n o n - r a d i a t i o n a l r a t e constants must be known, as w e l l as the flame temperature, T, f o r the case of e x c i t a t i o n of an e x c i t e d lower l e v e l , i n order to ev a l u a t e absolute values of n^ from absolute B-p measure ments; f o r the case of s a t u r a t i o n fluorescence measure ments, B must be measured under steady s t a t e c o n d i t i o n s , i . e . , the r a d i a t i v e l i f e t i m e of the e x c i t e d s t a t e must be much l e s s than the temporal pulse width of the e x c i t i n g source. In the case of a Na-type atom, the measurement of B™ and the r a t i o n / n , i . e . , B-π* /B-p enables *Vl *2+l 3+1 1+3 1+3 1+3 F
0
3
0
2
absolute η·ρ values to be measured without the need f o r r a d i a t i o n l e s s r a t e constants. For atoms of a T£-type atom, i t i s not p o s s i b l e to determine absolute η,ρ-values unless s e v e r a unless A
>
+ k
i 32 32 k
8
. 3
+
f2 -E e
g
21
1 2
/kf
3
which i s simply the 2 - l e v e l approximation f o r a 3 - l e v e l atom. (4) The s a t u r a t i o n s p e c t r a l i r r a d i a n c e , E , i . e . , the source s p e c t r a l i r r a d i a n c e where B-p becomes 50% the maximum pos s i b l e value, depends upon the frequency, v, of the t r a n s i t i o n ( A £ / B £ = δπην-Vc ) and upon the r a d i a t i o n a l and r a d i a t i o n l e s s r a t e constants ( r e f e r to equations 5 and 8). Therefore, t h e o r e t i c a l e s t i m a t i o n of E^ values from fundamental aspects i s d i f f i c u l t even though experimental measurement i s q u i t e easy. For a 2 - l e v e l atom, measure ment of Ef) values allows e s t i m a t i o n of quantum e f f i c i e n c i e s , Y. However, f o r 3 - l e v e l atoms, e s t i m a t i o n of quantum e f f i c i e n c i e s r e q u i r e s not only measurement of E^ but a l s o e x p l i c i t knowledge of c e r t a i n r a d i a t i o n a l and r a d i a t i o n l e s s r a t e constants. v
3
U
U
Narrow L i n e Laser E x c i t a t i o n Molecular Luminescence Spectro metry, MLS, B o u t i l i e r and W i n e f o r d n e r — have considered the molecular luminescence radiance expressions f o r the case of "narrow-line e x c i t a t i o n of molecules (6v <6v ) i n a medium at low o p t i c a l d e n s i t i e s . The general expressions (Eqns. 1 and 2) f o r B L , the molecular luminescence radiance are i d e n t i c a l to those f o r atomic fluorescence (see Eqns. 1 and 2). However, with molecules, s e v e r a l a d d i t i o n a l f a c t o r s must be considered i n e v a l u a t i n g the E i n s t e i n expressions and the d e n s i t i e s of the v a r i o u s s t a t e s i n volved i n the molecule ( i t i s p o s s i b l e to s i m p l i f y the molecular energy s t a t e diagram to a 3 - l e v e l , Na-type system, where 1 i s the lower, 3 i s the upper s i n g l e t , and 2 i s the corresponding e x c i t e d 11
s
A
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
Laser-Excited Luminescence Spectrometry
wiNEFORDNER
55
triplet). These a d d i t i o n a l f a c t o r s i n c l u d e the v i b r a t i o n a l over lap i n t e g r a l between the v i b r a t i o n a l l e v e l s involved i n the two e l e c t r o n i c s t a t e s involved i n the a b s o r p t i o n and luminescence pro cesses and luminescence s p e c t r a l p r o f i l e and average luminescence frequency as compared to the e x c i t a t i o n frequency. For a 2 - l e v e l molecule, the general expression f o r Β i s given by
.f
A 0
h V
20,li
(10)
Ε* /ε \ ^2j,10 1+ Ε /δν 12
n
20,li T
1 2
1+ —
Α
V
where the summation i s over a l l i t h v i b r a t i o n a l l e v e l s i n the lower e l e c t r o n i c s t a t e 20,li correspondin e l e c t r o n i c s t a t e p o p u l a t i o n of the molecules, the g's are s t a t i s t i c a l weights of e l e c t r o n i c s t a t e s l^and 2, E ^ i s spectral - Γ ^ - Λ - Ϊ — ~ ~ *- ~ -·*-_·-™ i s the modified s a t u r a 6 v i s the h a l f - w i d t h of ^ s o r p t i o n band and ξ^. i s the v i b r a t i o n a l overlap-luminescence p r o f i l e term f o r tnè absorption t r a n s i t i o n from the 0 t h v i b r a t i o n a l l e v e l of the ground e l e c t r o n i c s t a t e and i s defined by n v
a
r
e
t
n
e
t
n
e
v
u
A
x
f
ξ
2α,10
=
V
3 2j,10
< V
L
-3 AV
V
2j,10
(11)
where
-
i=°° ^Σ '2 0 , l i 2 0 , l i i=0 i=°° Σ V,20, l i i=0
'2j,10
l<e (Q)|Q
'20,li
N
V
V
n
3
L
AV
and
2 j
2 0
(Q)|Q
1 0
U
(Q)>|
(12)
J
(Q)>l'
(13a)
(13b)
where || i s the v i b r a t i o n a l overlap i n t e g r a l (FranckCondon f a c t o r ) between v i b r a t i o n a l l e v e l s i n the two e l e c t r o n i c s t a t e s involved i n the a b s o r p t i o n and luminescence processes (Q i s the v i b r a t i o n a l c o o r d i n a t e ) ; the Born-Oppenheimer approximator i s assumed to apply here (the overlap term i s d i m e n s i o n l e s s ) . For a three l e v e l molecule, B o u t i l i e r , et al.Jâ showed that
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
56
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
the fluorescence radiance 3+1 f o r 1+3 e x c i t a t i o n or the forbidden process of 2+1 f o r 1+2 e x c i t a t i o n i s given by i=a, Σ A
.νυο,ιΛο,ιΛ *
1=0
1 +
—
(14)
/ε
E* 1 +
8„
V10, uj
/δν
Α
i=
Q
u'O.li
u'l
u'u
where a l l symbols have sents the r a d i a t i o n a l l other upper l e v e l ( i n case 1+3 e x c i t a t i o n , u = 3 and u = 2). For a three l e v e l molecule, B o u t i l i e r , et al.IS- showed that the luminescence radiance f o r 1+3 e x c i t a t i o n and 2+1 emission or f o r 1+2 e x c i t a t i o n and 3+1 emission i s given by i=oo
Σ A u 0,li i=0 tf
T
u 0,li Τ
(15) E
1+ 1 +
1 +
v
/ ? l u
uj,10
Ε
/6v
f
f
. u 0,li 1=0
u l
Λ
T
u u
A
10, uj where a l l terms have been defined above (note that u i s the r a d i a t i o n a l l y e x c i t e d upper s t a t e and u' i s the other upper s t a t e ; f o r 1+3 e x c i t a t i o n and 2+1 luminescence, u = 3 and u = 2. F i n a l l y , f o r the case of delayed fluorescence (1+3 e x c i t a t i o n , 3—>2 intersystem c r o s s i n g , 2—K3 intersystem c r o s s i n g , 3+1 emission) ?
DF
A
h V
n
.^ 30,li 30,li T 0
1+
+
^32 i=' Σ f 20,li 21 i=0 A
+ k
v - '3j,10 13
/δν + k
23jJ
Α
"l0,3j
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
• (eqn. cont.)
4.
wiNEFORDNER
Laser-Excited Luminescence Spectrometry
57
(Eqn. continued from previous page) K
i=°° 4-k Σ A, i=0 3 0 , l i
K
23 32
+k 3132
(16)
.f
A 0
20,li
The s a t u r a t i o n s p e c t r a l i r r a d i a n c e Ε molecule i s given by
rE
+ k
21
+ k
23
for a 2-level 10,2j v
1 2
δν. A (17)
10,2 j
g
l
+
g
6v
A
•2j,10 J
2
and f o r a three l e v e l molecul Ε V
18J
13 J
3,i 10
j
(18)
?
'10,3 j
32
1 + — + go i=°° .f 20,li A
+ k
0
and f o r a three l e v e l molecule with 1+2 Ε 8n 18 J 3
10,2j
v
21
+ k
23
excitation
δν · A Α
l 2
2,1,10
}
(19) x
1 + — + go i=°° E
A
23
. 30,li i=0 n
+ k
31
+ k
32
The major conclusions r e s u l t i n g from the molecular lumines cence radiance expressions are l i s t e d below. (1) The radiance expressions f o r molecular luminescence are s i m i l a r to those f o r atomic f l u o r e s c e n c e and reduce to the atomic case i f ξ = 1. (2) For low source ( s p e c t r a l ) i r r a d i a n c e s , the f l u o r e s c e n c e radiance depends d i r e c t l y upon the source i r r a d i a n c e , the f l u o r e s c e n c e quantum e f f i c i e n c y , the emission t r a n s i t i o n p r o b a b i l i t y , and the t o t a l d e n s i t y of a n a l y t e . (3) For high source ( s p e c t r a l ) i r r a d i a n c e s , the fluorescence radiance depends d i r e c t l y upon the emission t r a n s i t i o n p r o b a b i l i t y , and the t o t a l analyte d e n s i t y but i s inde pendent of the source i r r a d i a n c e and the f l u o r e s c e n c e
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
58
NEW APPLICATIONS OF LASERS TO CHEMISTRY quantum e f f i c i e n c y . (4) For the 2 - l e v e l case under s a t u r a t i o n c o n d i t i o n s , abso l u t e n-p values can be measured by measuring under steady s t a t e c o n d i t i o n s and by knowing A g-, g , and £. (5) The s a t u r a t i o n i r r a d i a n c e E f o r a 3 - l e v e l molecule at room temperature i s 1CH to ΙΟ* x l e s s than f o r a 2 - l e v e l atom or molecule at any temperature or f o r a 3 - l e v e l atom or molecule at h i g h temperature, as i n flames. Because molecules have broad bandwidths compared to atoms, s a t u r a t i o n can be achieved e i t h e r by a high source s p e c t r a l i r r a d i a n c e over a narrow s p e c t r a l r e g i o n or a lower source s p e c t r a l i r r a d i a n c e over a much wider bandwidth, <5ν , i . e . , E of the source must exceed E /6vg f o r narrow line excitation. (6) Assuming s a t u r a t i o t r i p l e t state i f i r s t s i n g l e t e x c i t a t i o n followed by r a d i a t i o n l e s s c r o s s over to the corresponding t r i p l e t s t a t e . Thus Ar ion-dye l a s e r s can be used to e x c i t e t r i p l e t s assuming they can be focussed down to ^10 ym to achieve ^MW cm"" (assuming Yp S 0.1). ? n
9
S
s
Α
v
2
Narrow-Line Laser E x c i t e d Luminescence Spectrometry. In t h i s case, the s p e c i f i c nature of the broadening processes, namely homo geneous vs heterogeneous broadened a b s o r p t i o n l i n e s , and the spe c i f i c source c h a r a c t e r i s t i c s , namely, s i n g l e narrow l i n e _vs source with m u l t i p l e d i s c r e t e s p e c t r a l components such as modes, must be considered. K i l l i n g e r , Wang, and Hanabusa,— and Ε. H. P i e p meier— *=2£?have considered these cases i n c o n s i d e r a b l e d e t a i l and have given f l u o r e s c e n c e radiance expressions. No attempt w i l l be made here to d u p l i c a t e these expressions because: (1) the reader can go to the c i t e d l i t e r a t u r e i f i t i s d e s i r e d ; (2) the expres sions are considerably more complex; and (3) the major a n a l y t i c a l use of l a s e r s has been w i t h the ones given above and not with the narrow l i n e systems. Nevertheless, s e v e r a l i n t e r e s t i n g c o n c l u sions of these s t u d i e s a r e : ( i ) with homogeneous broadened absorp t i o n ( L o r e n t z i a n broadening) t r a n s i t i o n s induced by a s i n g l e f r e quency t r a n s i t i o n , the f l u o r e s c e n c e radiance expression i s s i m i l a r to the ones given i n the s e c t i o n concerning atoms and i d e n t i c a l to the ones given i n the s e c t i o n concerning molecules except that f o r atoms the a b s o r p t i o n l i n e shape f a c t o r (Hz~l) must be considered e x p l i c i t l y ; ( i i ) with inhomogeneous (heterogeneous) broadened ab s o r p t i o n t r a n s i t i o n induced by a s i n g l e frequency t r a n s i t i o n , the f l u o r e s c e n c e radiance expression d i f f e r s c o n s i d e r a b l y from those i n both of the previous s e c t i o n s because of the need to consider both the a b s o r p t i o n l i n e shape f a c t o r (Hz"~l) and the f r a c t i o n a l number of absorbers with the proper frequencies ( v e l o c i t i e s ) to i n t e r a c t with the e x c i t a t i o n l i n e must be considered; ( i i i ) with homogeneous and/or inhomogeneous broadening induced by a continuum
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
wiNEFORDNER
Laser-Excited Luminescence Spectrometry
59
source with constant s p e c t r a l d i s t r i b u t i o n , the i d e n t i c a l f l u o r e s cence radiance expressions as i n the s e c t i o n f o r atoms r e s u l t ; and (iv) with inhomogeneous broadened a b s o r p t i o n t r a n s i t i o n s induced by r a d i a t i o n w i t h d i s c r e t e narrow l i n e s p e c t r a l components, as l a s e r modes, the expressions can vary from those i n the s e c t i o n for atoms and i n the s e c t i o n f o r molecules to those i n item ( i i ) above with the necessary change to i n c l u d e the e f f e c t of the source f l u x i n each s p e c t r a l component w i t h i n the a b s o r p t i o n l i n e width. I t should be s t r e s s e d that i n the case of narrow l i n e e x c i t a t i o n and homogeneous broadening, i n the case of continuum e x c i t a t i o n and homogeneous and/or inhomogeneous broadening, and i n the case of m u l t i p l e narrow l i n e e x c i t a t i o n and homogeneous broadening, the f l u o r e s c e n c e radiance expressions are i d e n t i c a l to or s i m i l a r to those i n the s e c t i o n s f o r atoms and f o r molecules However for the cases of inhomogeneou or m u l t i p l e narrow l i n p r o p o r t i o n a l to the square root (rather than the f i r s t power) of the source i r r a d i a n c e , the i n t e g r a t e d a b s o r p t i o n c o e f f i c i e n t , and the E i n s t e i n spontaneous emission t r a n s i t i o n p r o b a b i l i t y assuming high source i n t e n s i t y . 23—26 M u l t i p l e Photon Laser E x c i t e d Luminescence Spectrometry.— — No attempt w i l l be made here to give e x p l i c i t expressions f o r mul t i p l e photon e x c i t a t i o n luminescence spectrometry. The lumines cence radiances f o r atoms or molecules, however, are p r o p o r t i o n a l to the f o l l o w i n g parameters: Bp
α n
£
Ασ(ν)
Ε(ν ) χ
Ε(ν ) 2
. . .
Ε(ν ) ν
where η^ i s the d e n s i t y of a n a l y t e s p e c i e s i n the lower l e v e l i n volved i n the a b s o r p t i o n process, I i s the f l u o r e s c e n c e path length, σ(ν) i s the a b s o r p t i o n cross s e c t i o n f o r the m u l t i p l e (v) photon process, and E ( v ^ ) , Eiv^O, . . . ( ν ) source ex c i t a t i o n i r r a d i a n c e s of m u l t i p l e f r e q u e n c i e s , ν^,ν^, · · ·ν . I t should be s t r e s s e d that f o r the case of strong resonance i n t e r a c t i o n , the luminescence s i g n a l s w i l l approach those of the onephoton a b s o r p t i o n process. However, an a d d i t i o n a l and very impor tant advantage occurs with m u l t i p l e photon e x c i t a t i o n (2 and 3photons are the most common) namely a very s i g n i f i c a n t improvement i n s e l e c t i v i t y over the one-photon process, p a r t i c u l a r l y f o r mol ecules where s e l e c t i v i t y with one photon e x c i t a t i o n i s poor. Var ious e x c i t a t i o n modes are p o s s i b l e v i a m u l t i p l e photon e x c i t a t i o n , namely: ( i ) the f i r s t photon e x c i t e s a resonant l e v e l , the second e x c i t e s an intermediate energy l e v e l , and the t h i r d , i f used, e x c i t e s a higher l e v e l or even i o n i z e s the s p e c i e s ; ( i i ) the f i r s t photon e x c i t e s a resonant l e v e l which r e l a x e s to some metastable l e v e l as a t r i p l e t and the second and t h i r d photons e x c i t e higher intermediate l e v e l s and/or cause i o n i z a t i o n ; ( i i i ) the f i r s t photon e x c i t e s a metastable l e v e l and the second and Ε
a
r
e
t
n
e
γ
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
60
NEW
APPLICATIONS OF
LASERS TO
CHEMISTRY
t h i r d photons e x c i t e h i g h e r i n t e r m e d i a t e l e v e l s and/or cause zation.
ioni
Experimentation The p u l s e d l a s e r e x c i t e d a t o m i c f l u o r e s c e n c e f l a m e s p e c t r o m e t r i c s y s t e m u s e d by Weeks, H a r a g u c h i , and Winefordner^â i s shown i n F i g u r e 1. T h i s s y s t e m i s s i m i l a r t o t h e one u s e d by o t h e r wor kers. E x c e p t f o r t h e r e p l a c e m e n t o f t h e f l a m e c e l l by a c o n v e n t i o n a l quartz c e l l , the e x p e r i m e n t a l system f o r p u l s e d l a s e r e x c i t e d m o l e c u l a r l u m i n e s c e n c e of condensed p h a s e s p e c i e s i s s i m i l a r t o t h e one f o r a t o m i c f l u o r e s c e n c e f l a m e s p e c t r o m e t r y i n F i g u r e 1, and so a b l o c k d i a g r a m s e t u p w i l l n o t be g i v e n h e r e . Analytical
Results
Atomic Fluorescenc l a s e r s have b e e n u s e d i n c r e a s i n g l y a s e x c i t a t i o n s o u r c e s f o r atom i c f l u o r e s c e n c e f l a m e s p e c t r o m e t r y . Z 2 ~ h l The most r e c e n t and comp r e h e n s i v e s t u d y o f s u c h a s y s t e m has b e e n c o n d u c t e d by Weeks, H a r a g u c h i , and W i n e f o r d n e r . 2 2 T h e s e w o r k e r s u t i l i z e d a p u l s e d ^ - l a s e r - d y e l a s e r s o u r c e , f l a m e a t o m i z e r , and g a t e d d e t e c t i o n and ob t a i n e d d e t e c t i o n l i m i t s s u p e r i o r to or s i m i l a r to the best r e s u l t s p r e v i o u s l y o b t a i n e d by a l l a n a l y t i c a l methods ( r e f e r t o T a b l e s 1 and 2 ) . O t h e r a d v a n t a g e s d i s c u s s e d by t h e s e w o r k e r s w e r e : ( i ) t h e l a r g e l i n e a r dynamic r a n g e s ( r e f e r t o F i g u r e s 2-5 f o r t y p i c a l r e s u l t s ) f o r most e l e m e n t s s t u d i e d c o v e r e d 5- t o 7 - o r d e r s o f mag n i t u d e i n c o n c e n t r a t i o n o f a n a l y t e w h i c h compared f a v o r a b l y w i t h t h e r a n g e s l i s t e d by w o r k e r s u s i n g I C P s f o r a t o m i c e m i s s i o n s p e c t r o m e t r y and c o n s i d e r a b l y g r e a t e r t h a n t h e r a n g e s l i s t e d f o r atom i c a b s o r p t i o n flame or furnace spectrometry; ( i i ) s p e c t r a l i n t e r f e r e n c e s w e r e c o n s i d e r a b l y l e s s t h a n t h o s e o b t a i n e d i n a t o m i c ab s o r p t i o n flame- or furnace-spectrometry, atomic emission flame- or I C P - s p e c t r o m e t r y , and a t o m i c f l u o r e s c e n c e e x c i t e d w i t h a c o n t i n u u m s o u r c e s p e c t r o m e t r y ( r e f e r t o F i g u r e 6 and T a b l e 3 f o r a t y p i c a l example o f t h e e a s e o f c i r c u m v e n t i n g t h e r a r e c a s e o f a s p e c t r a l i n t e r f e r e n c e i n atomic f l u o r e s c e n c e flame spectrometry w i t h a pulsed l a s e r source. The e f f e c t i v e r e s o l u t i o n i n l a s e r e x c i t e d AFS i s d e t e r m i n e d by t h e l a s e r l i n e w i d t h r a t h e r t h a n t h e s p e c t r o m e t e r b a n d p a s s ( s e e F i g u r e 7 ) . The o n l y p o t e n t i a l l y - s e r i o u s i n t e r f e r e n c e i n p u l s e d l a s e r AFS w i t h g a t e d d e t e c t i o n w o u l d a r i s e f r o m m o l e c u l a r f l u o r e s c e n c e o f s p e c i e s formed i n t h e f l a m e s by c o n c o m i t a n t s and f l a m e g a s e s and by R a y l e i g h and M i e s c a t t e r . The f o r m e r has b e e n shown by Weeks, e t al.ZI t o be m i n i m a l i n most c a s e s a s l o n g as PH3 has b e e n removed f r o m t h e a c e t y l e n e f u e l (PH3 r e s u l t s i n f o r m a t i o n o f ΡΟΗ w h i c h f l u o r e s c e s i n t e n s e l y i n t h e f l a m e . ) The l a t t e r has b e e n shown by Weeks, e t al.ftfi. t o be a v o i d a b l e i n most c a s e s ; M i e s c a t t e r can be a v o i d e d by use o f e f f i c i e n t chamber t y p e n e b u l i z e r s , n e b u l i z a t i o n o f s o l u t i o n s c o n t a i n i n g l e s s t h a n 1% s o l i d s and use o f a c e t y l e n e f l a m e s and R a y l e i g h and M i e f
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
wiNEFORDNER
Laser-Excited Luminescence Spectrometry
Figure 1. Diagram of User-excited atomic fluorescence flame spectrometry system
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Na
Mo
Li Mg Mn
In
Cr Cu Fe Ga
Bi Ca Cd Co
Ba
Ag Al
d
Element
a
1
A
F
+
TA-SLF
RF
RFJ
RF^
R F |
RF;
R F
RF*J*
S-DLF
R F
S-DLF
RF+
S-DLF
RF
RF;
R F |
AS-DLF'
1
b
E-RF S-DLF RF * RF RF ' RF RF t
Type of A F 4 2 0.6 100 8 3 0.08 8 1000 1000 1 1 30 7 0.9 0.8 0.2 0.5 0.2 1 0.4 12 12 <.l 1
0.06 0.6 0.3 10 >1 0.5 0.006 0.03 >2 >2 0.3 0.1 >1 0.3 0.2 0.4 0.3 >0.001 0.02 0.08 0.1 >1 >1 0.05 0.06
4.2 5.4 5.7 5 >5 5.2 4.9 3.5 >3.3 >3.3 5.5 5 >4.5 4.6 5.4 5.7 6.2 >4.3 5 4.9 5.4 >4.9 >4.9 >5.7 4.8
27 Weeks et a l . log(LDR) LOD UL (mg/ml) (dimens) (ng/ml)
1000 300 0.1
0.3 10
100 20 10 2
20
200
5
300 5 8 40
LOD (ng/ml)
—
—
0.1 0.1
0.02
0.5 0.02 0.5 0.5
2 2.5
3.3
3.7 3 3.7 4.4
3
3
0.2 0.02
4
4 4.6 >5
0.05
0.1 0.2 >1
0
Previous Works UL log(LDR) (mg/ml) (dimens)
DETECTION LIMITS BY LASER-EXCITED ATOMIC FLUORESCENCE FLAME SPECTROMETRY
328.1 396.1 394.4/396.1 553.7 553.7 306.8 422.7 228.8 357.5/347.4 230.9 359.3 324.7 296.7/373.5 403.3 403.3/417.2 410.4 410.4/451.1 670.8 285.2 403.1 279.5 379.8 390.3 589.0 589.6/589.0
Ex/Fl (nm)
TABLE I.
28 28 4_0
36 28
28 28 28 28
28
28
28
28 28 38 40
f
Ref n.
C/)
g
w
s
>
ο
δ
η >
H-1
r
>
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
a
^N/A or n i t r o u s oxide/acetylene
A
1
RF S-DLF " E-RF E-S-SLF
f
RF E-AS-DLF E-RFÏ AS-DLF RF S-DLF RFj RF~T
A/A or a i r / a c e t y l e n e .
232.0 361.0/352.4 405.8 405.8/283.3 283.3 283.3/405.8 460.7 399.9 365.4 377.6 377.6/535.0 411.2 370.4/411.2
flame.
12 2 180 30 20 13 0.3 5 2 4 7 50 30
0.1 1 >1 >1 1 >1 0.03 1 0.4 0.3 0.6 >1 >1 3.9 5.7 >3.7 >4.5 4.7 >4.9 5 5.3 5.2 4.9 4.9 >4.3 >4.5 500
1000 200
30 10 100
50 6
0.05 0.1
0.02 0.2
0.2
1.7 3.7
3.3 3.3
3.6
32
28 28
36 28 28
28 37
^Experimental d i f f i c u l t y was encountered i n o b t a i n i n g good l a s e r output at the Co l i n e s attempted.
°L0D = L i m i t of D e t e c t i o n , UL = Upper L i m i t of l i n e a r i t y , and LDR = l i n e a r dynamic range=UL/LOD.
^RF = resonance fluorescence; E-RF = e x c i t e d resonance f l u o r e s c e n c e ; S-DLF = Stokes d i r e c t l i n e fluorescence; AS-DLF = anti-Stokes d i r e c t l i n e f l u o r e s c e n c e ; TA-SLF = thermally a s s i s t e d stepwise l i n e f l u o r e s c e n c e ; E-AS-DLF = e x c i t e d anti-Stokes d i r e c t l i n e f l u o r e s c e n c e ; and E-S-SLF = e x c i t e d Stokes stepwise l i n e f l u o r e s c e n c e .
E x / F l = e x c i t a t i o n wavelength/fluorescence wavelength ( i f d i f f e r e n t than e x c i t a t i o n wavel e n g t h ) . The energy l e v e l s i n v o l v e d i n the t r a n s i t i o n and t h e i r gf and gA values can be found i n C. H. C o r l i s s and W. R. Bozman, "Experimental T r a n s i t i o n P r o b a b i l i t i e s f o r S p e c t r a l L i n e of Seventy Elements," NBS Monograph 53, 1962.
V
Tl
Sr Ti
Pb
Ni
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Ag Al Ba Bi Ca Cd CoS Cr Cu Fe Ga In Li Mg Mn Mo Na Ni Pb Sr Ti Tl V
Element
0.43 123 <0.1 23 133 0.3 23 43 30
0.23
4 0.63 8 33 0.083 8 1000 13 1 30 0.93 0.23 0.5
AF (laser)
a
33(5) 103(3) 30 4000 83(20) 70
0.13(1) 1(5) 500
5 0.5(1) 8(10) 10(50) 100
53(D
53(10) 20(0.1) 0.0013(0.01)
0.13 (0.4) 100
e
AF conventional source
b
b
800 30 23 0.13 5 10* 0.43 0.023 5 1 100 0.13* 20 100 0.2 30 20 7
0.13
2 3 1 20,000
AE
b
1(0.01) 30*(0.1) 20(0.6) 50(0.4) 1*(0.04) 1(0.008) 23(0.2) 23(0.2) 1*(0.06) 4(1) 50(0.1) 30(0.04) 1(0.3) 0.13(0.004) 0.8(0.02) 30(0.3) 0.8 53(0.9) 103(0.2) 5(0.1) 90*(4) 20(1) 20(0.3)
AA
D e t e c t i o n l i m i t , ng/ml
43 0.2+3 43 103 0.02+3 3+3 200+ 13
0.13
0.7t
14t 30t
0.23 0.23
0.93
23
50+ 0.07t3 1.0
0.1+3
4t 2
pneumatic nebulization
TABLE I I . COMPARISON OF DETECTION LIMITS IN FLAME AND FURNACE SPECTROMETRY AND INDUCTIVELY COUPLED PLASMA (ICP)
ICP
0.06
0.003 0.01* 0.2 0.02 0.2 1* 0.003 0.03
0.0001 0.07* 0.1* 0.08* 0.04* 0.09 0.6
0.5** 0.01
ultrasonic , Q n e b u l i z af t i o n
4.
wiNEFORDNER
Footnotes
Laser-Excited Luminescence Spectrometry
65
to Table I I :
a L i m i t s of d e t e c t i o n represent concentrations r e q u i r e d t o produce a l i n e s i g n a l three times as great as the standard d e v i a t i o n of the background n o i s e . A l l other values l i s t e d i n the t a b l e represent concentrations r e q u i r e d t o produce a l i n e s i g n a l twice as great as the standard d e v i a t i o n of the background n o i s e , except where noted. Values taken from S. J . Weeks, H. Haraguchi, and J . D. Winefordner, A n a l . Chem., i n p r e s s . ^Values taken from J . D. Winefordner, J . J . F i t z g e r a l d , and N. Omenetto, Appl. Spectrosc., 29, 369 (1975) except f o r those designated *, which were taken from V. A. F a s s e l and R. N. K n i s e l e y , Anal. Chem., 46, 1110A (1974). Values i n parantheses are f o r furnace AF and AA A l l values taken A. F a s s e l , Anal. Chem., 49, 632 (1977) except those with t , which are from V. A. F a s s e l and R. N. K n i s e l e y , A n a l . Chem., 4(6, 1110A (1974). ^ A l l values taken from P. W. J . M. Boumans and F. J . deBoer, Spectrochim. Acta, 30B, 309 (1975) except those designated *, which were taken from K. W. Olson, W. J . Haas, J r . , and V. A. F a s s e l , A n a l . Chem., 49, 632 (1977) and those designated **, which were taken from M. H. Abdallah, R. Diemiaszonek, J . J a r o s z , J . M. Mermet, J . Robin, and C. Trassy, Anal. Chim. Acta, 84, 271 (1976). L i m i t of d e t e c t i o n designated ** represent concentrations required to produce a l i n e s i g n a l s i x times as great as the standard d e v i a t i o n of the background n o i s e . 3 designates best value with pneumatic n e b u l i z a t i o n . A l l values w i t h i n a f a c t o r of 3 a r e considered t o be equal. ^
i n d i c a t e s no v a l u e reported.
e x p e r i m e n t a l d i f f i c u l t i e s were encountered l a s e r output at the Co l i n e s .
i n o b t a i n i n g good
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
66
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
/CO
10* H
I
-Μη
ιο · 3
8
5
to H
Ca^
Να
10 '
10 Analyte
10"
10
10
10°
Concentration (ng ml )
Figure 2. Analytical calibration curves for elements having only strong reso nance transitions above 355 nm in air-acetylene flame. \ resonancefluorescenceof Ba o - resonancefluorescenceof Ca resonancefluorescenceof Cr Θ - resonancefluorescenceof Li resonancefluorescenceof Mn 9 i -- resonancefluorescenceof Na o - resonancefluorescenceof Sr (O - indicates the limit of detection)
e x
: \fi(nm) 554 422 359 670 403 589 460
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Laser-Excited Luminescence Spectrometry
WINEFORDNER
10
I
ΙΟ
ΙΟ
Κ)
ΙΟ
ΙΟ
ΙΟ
Analyte Concentration (ngml ) 1
Figure 3. Analytical calibration curves for elements having both strong reso nance and nonresonance transitions excited above 355 nm in an air—acetylene flame.
• - resonancefluorescenceof Ga ο - nonresonancefluorescenceof Ga • - resonancefluorescenceof In ο - nonresonancefluorescenceof In nonresonancefluorescenceof Ni • - resonance fluorescenceof Pb
οχ - nonresonancefluorescenceof Pb - ο - resonancefluorescenceof Tl Δ - nonresonancefluorescenceof Tl (Ο - indicates the limit of detection)
\ex(nm) 403 403 410 410 361 405 405 377 377
\fi(nm) 403 417 410 450 352 405 283 377 535
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
68
NEW
Andyte
APPLICATIONS
O F LASERS T O C H E M I S T R Y
Concentration (ng ml" ) 1
Figure 4. Analytical calibration curves for elements having transitions excited above 355 nm in a nitrous oxide-acetylene flame. resonancefluorescenceof Al ο - nonresonancefluorescenceof Al resonancefluorescenceof Ba fluorescenceof Mo + - resonance fluorescenceof Mo • - resonance resonance fluorescenceof Ti • - resonancefluorescence of Ti χV - nonresonancefluorescenceof V resonance fluorescence of V 6«5 -indicates the limit of detection)
• -
Xex(nm) 396 394 553 380 390 365 400 370 411
Xfi(nm) 396 396 553 380 390 365 400 411 411
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WINEFORDNER
Laser-Excited Luminescence Spectrometry
e
io -l
Analyte Concentration (ng ml" ) 1
Figure 5. Analytical calibration curves for elements having transitions ex cited below 855 nm in an air—acetylene flame. X-o-
resonancefluorescenceof Ag resonancefluorescenceof Bi resonancefluorescenceof Cd resonance fluorescenceof Cu τnonresonancefluorescenceof Fe jro - resonancefluorescenceof Mg resonancefluorescenceof Mn V - resonancefluorescenceof Ni resonancefluorescenceof Pb nonresonancefluorescenceof Pb Φ - indicates the limit of detection) (O -
+ ••-
\ex(nm) 328 306 228 325 296 285 280 232 283 283
Xfi(nm) 328 306 228 325 373 285 280 232 283 405
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
N E W APPLICATIONS
I ppm Mn
O F LASERS T O C H E M I S T R Y
I ppm Mn + 5ppm Ga
00
3 9
J I I 4CB.0
Iι
I
J
I 4036
4030
W τ—τ
4036
Wavelength (nm)
Figure 6. Fluorescence excitation spectrum for (A) a 1 ppm Μ η solution and (B) a 1 ppm Mn + 5 ppm Ga solu tion
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WINEFORDNER
TABLE I I I .
(i)
Laser-Excited Luminescence Spectrometry
INVESTIGATION OF SPECTRAL INTERFERENCES BETWEEN MANGANESE AND GALLIUM
E x c i t e d at 403.08 ran/Observed a t 403.08 ran [Observe manganese atomic fluorescence]
Composition
R e l a t i v e atomic fluorescence i n t e n s i t y
Mn 1 ppm
69
Mn 1 ppm + Ga 5 ppm
70
Mn 1 ppm + Ga 100 pp
68
Mn 0.5 ppm + Ga 100 pp Mn 0.1 ppm + Ga 100 ppm (ii)
E x c i t e d at 403.30 nm/Observed a t 403.30 nm [Observe g a l l i u m atomic fluorescence]
Composition Ga 5 ppm
R e l a t i v e atomic fluorescence i n t e n s i t y 67
Ga 5 ppm + Mn 1 ppm (iii)
7
108
E x c i t e d at 403.30 nm/Observed a t 417.01 nm [Observe g a l l i u m atomic fluorescence]
Composition
R e l a t i v e atomic fluorescence i n t e n s i t y
Ga 5 ppm
151
Ga 5 ppm + Mn 1 ppm
151
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
Loser Scon
Monochromotor Soon
I,
I
589.0
589.6
ι
ι
592
ι 590
ι
ι 588
Wavelength (nm)
Figure 7. Fluorescence excitation and fluorescence emis sion profiles of sodium Ό lines (at 589.0 and 589.6 nm) in the air-acetylene flame (slit width was 800 \xm in both cases)
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
WINEFORDNER
73
Laser-Excited Luminescence Spectrometry
s c a t t e r can be avoided by use of non-resonance fluorescence (however, even f o r resonance fluorescence, e l a s t i c s c a t t e r does not r e s u l t i n s i g n a l s more than s e v e r a l times the dark current l e v e l ) . Detection l i m i t s i n l a s e r - e x c i t e d AFS could most l i k e l y be improved by up to s e v e r a l orders of magnitude by:^Z ( i ) o p t i m i z a t i o n of t h e i r o p t i c a l system; ( i i ) by o p t i m i z a t i o n of the l a s e r beam diameter; ( i i i ) by use of a b e t t e r q u a l i t y l a s e r beam shape over the e n t i r e s p e c t r a l range; ( i v ) by use of higher output peak powers, e s p e c i a l l y i n the frequency-doubled region; (v) by use of u l t r a s o n i c n e b u l i z a t i o n ; ( v i ) by r e d u c t i o n of flame i n s t a b i l i t y v i a an improved flow system and mixing chamber design; ( v i i ) by use of an improved burner shape; ( v i i i ) by o p t i m i z a t i o n of the e l e c t r o n i c measurement system; and ( i x ) by r e d u c t i o n of r a d i o frequency i n t e r f e r e n c e noise due to the N£-laser. Atomic FLuorescenc l a s e r e x c i t e d atomic fluorescenc conducted where the atoms have been produced i n a furnace designed to atomize r e a l samples. s. Neumann and Kriesei±-2 using a f r e quency-doubled flashlamp pumped dye l a s e r to e x c i t e lead atoms produced with a graphite rod atomizer were able to detect 0.2 pg BP ( d i r e c t l i n e f l u o r e s c e n c e , 283.3 nm e x c i t a t i o n and 405.8 nn fluorescence) and to achieve a l i n e a r dynamic range of >10 x. Bolshov^ et a l . i t i t w i t h a frequency-doubled dye l a s e r pumped by a YAG:Nd l a s e r and a graphite tube atomizer have detected 25 pg/ml r e l a t i v e and 0.75 pg absolute Fe ( d i r e c t l i n e f l u o r e s c e n c e with e x c i t a t i o n at 296.7 nm and fluorescence of 373.5 nm) and 2.5 pg/ml r e l a t i v e and 0.075 pg absolute Pb ( d i r e c t l i n e f l u o r e s c e n c e , with e x c i t a t i o n at 283.3 nm and fluorescence at 405.8 nm); the l i n e a r dynamic ranges were >10^x and >10^x, r e s p e c t i v e l y , f o r Fe and Pb. D e t e c t i o n l i m i t data v i a l a s e r e x c i t e d atomic furnace f l u o r e s c e n c e spectrometry are not given i n t a b u l a r form due to t h e i r s c a r c i t y . Several groups of workers have u t i l i z e d l a s e r e x c i t a t i o n of Na-atoms produced i n quartz c e l l s maintained at elevated temperatures (quartz tube w i t h i n a furnace). These s t u d i e s have included the c l a s s i c work i n 1975 of Fairbanks, Hansen, and Schawlowii-5 who detected 1 0 atoms of Na/cm (upper l i m i t was v L 0 atoms/cm- ) and h o p r e d i c t e d that under optimal c o n d i t i o n s 0.1 Cs atom/cm should be d e t e c t a b l e ; these same workers a l s o p r e d i c t e d that many other atoms and molecules should be d e t e c t a b l e to 10 -10^ atoms/cm^ depending upon the o s c i l l a t o r strength of the a b s o r p t i o n t r a n s i t i o n . Kuhl and Marowskyi+2 s e v e r a l years e a r l i e r i n 1971 had detected Na i n a resonance c e l l at concentrations of 3 pg/cm . Mayo, et a l . ^ i n 1976 detected Na atoms i n an open contaminated quartz tube heated to 1000°C and obtained a d e t e c t i o n l i m i t of 5x10 atoms/ cm**. Gelbwachs, Klein,^and W e s s e l 3 9 i 1977 obtained a d e t e c t i o n l i m i t of 10 Na atoms/cm f o r a flashlamp pumped dye l a s e r e x c i t a t i o n of the atomic fluorescence of Na produced i n an enclosed environment. F i n a l l y , Brod and Y e i n g ^ determined a d e t e c t i o n l i m i t of 7 fg/cm f o r Na produced i n a quartz container and e x c i z
5
w
n
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3
5
lxlO"
514 415
480 360
3.3 1.4
Fluorescein
Acridine
52 5Q 57 55
5
5
5
6
7
lxlO" 3xl0" 2xl0~ lxlO" 4.7xl0"
460 400 390 660 540
337 337 337 442 375
150 150 150 75 mW
Fluoranthene
Phenanthrene
Pyrene
Riboflavin
1-15
50
5
3xl0"
388
337
150
Chrysene
(cw)
50
5
lxlO"
397
337
150
Anthracene
(a or b)
5Q
5
3xl0"
415
337
150
Acridine
Chlorophyll
50
-4 1x10
514
337
150
Fluorescein
50
lxKf
456
337
50
42
49
150
-4
49
?
Ref n,
Quinine S u l f a t e
1x10
5
454
365
2.5
Quinine S u l f a t e
lxlO"
Emission Wavelength (nm)
Molecule
Excitation Wavelength (nm)
Detection Limit (ug/ml)
DETECTION LIMITS BY LASER EXCITED FLUORIMETRY
Laser Peak Power (kW)
TABLE IV.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 100 100
Rhodamine 6G
Aflatoxin ( B , G 1:1
•k 1.5-5
(cw)
3 4 0 , 360 404 450 395 Visible Visible
273 254 287 273 337 337
545
300-400
350-450
302, 273
260
600
Visible
325
phot
4.6x10*
1.1x10"
2.5x10'
3.9x10'
5x10'
1x10"
4.4x10"
1.3x10"
1.9x10*
1.8x10
*Two p h o t o n e x c i t e d m o l e c u l a r f l u o r e s c e n c e — t h e r e m a i n d e r a r e a l l s i n g l e molecular fluorescence.
p-Terphenyl
2,5 D i p h e n y l o x a z o l e *
1.5-5
2-5
Pyrene
Mixture)
2-5
Fluoranthene
1
2-5
Anthracene
1
2-5
Naphthalene
8 mW 2-5
(B^B^G^G^
Benzene
Aflatoxin
76
NEW APPLICATIONS OF LASERS TO CHEMISTRY
ted w i t h a flashlamp pumped dye l a s e r . Despite the rather e x o t i c and e x c i t i n g d e t e c t i o n l i m i t s ob tained f o r Na (and p r e d i c t e d f o r other atoms and molecules) i n quartz containers, the a n a l y t i c a l s p e c t r c s c o p i s t must temper the e x c e l l e n t r e s u l t s with the d i f f i c u l t i e s encountered i n converting r e a l samples to a form to allow use of a quartz sample c e l l with the concomitant i d e a l environment, e.g., an i n e r t gas. Therefore, the world record d e t e c t i o n l i m i t s i n quartz c e l l s are only t r a n s f e r a b l e with sampling d i f f i c u l t i e s to the world of the analyte. Molecular Luminescence Spectrometry. Smith, et a l . — were the f i r s t to demonstrate the p o t e n t i a l of pulsed tunable dye l a s e r molecular f l u o r i m e t r y of condensed phase molecules. Van Geel and W i n e f o r d n e r ^ f u r t h e r evaluated pulsed tunable dye l a s e r s f o r an a l y t i c a l condensed phase molecular f l u o r i m e t r y Berman and Z a r e — obtained an absolute d e t e c t i o B^, G^, and G2) by t h i by time r e s o l v e d l a s e r e x c i t e d f l u o r i m e t r y . Bradley and Z a r e — used pulsed l a s e r (N^-laser) e x c i t e d f l u o r i m e t r y of molecules i n the condensed phase f o r s u b - p a r t - p e r - t r i l l i o n d e t e c t i o n l i m i t s . Diebold and Z a r e — have used modulated l a s e r e x c i t a t i o n (He-Cd l a ser) of a suspended drop (^4 μ il) of solvent e l u t i n g from a column and obtained an absolute d e t e c t i o n l i m i t of 7 f g f o r a f l a t o x i n . Richardson, et a l . , — and Richardson and A n d o — h a v e u t i l i z e d a system s i m i l a r to the one of Smith et a l . ^ - except f o r a more elaborate e x c i t a t i o n o p t i c s and sample c e l l and a more powerful ^ - l a s e r pump to detect r i b o f l a v i n at the s u b - p a r t - p e r - t r i l l i o n level. F i n a l l y , Wirth and Lytle-^- have u t i l i z e d two photon e x c i tation-molecular f l u o r i m e t r y to detect s e l e c t i v e l y , low concentra t i o n s of s e v e r a l molecules (a cavity-dumped, synchronously-pumped cw dye l a s e r was used). In Table 4, the s t a t e of the a r t d e t e c t i o n l i m i t s f o r s e v e r a l organic molecules e x c i t e d by l a s e r sources are given. The a n a l y t i c a l advantages of using pulsed l a s e r sources f o r molecular f l u o r i m e t r y of condensed phase species are l e s s obvious than f o r atomic fluorescence spectrometry. For example, the d i f f i c u l t i e s with removal of u l t r a - t r a c e luminescent i m p u r i t i e s from solvents and reagents can lead to d e t e c t i o n l i m i t s determined by luminescent background f l i c k e r noise and so conventional and l a s er sources should g i v e s i m i l a r d e t e c t i o n l i m i t s . However, m u l t i photon e x c i t e d (with and without resonance enhancement) f l u o r e s cence should allow an extension of the upper concentration l i m i t p r i o r to d e v i a t i o n from n o n - l i n e a r i t y and freedom from many, i f not most, s c a t t e r and p o t e n t i a l s p e c t r a l i n t e r f e r e n c e s as compared to conventional (single-photon) l a s e r e x c i t e d f l u o r i m e t r y . In a d d i t i o n , o p t i c a l l y - d e n s e s o l u t i o n s ( o p t i c a l density due to some interfèrent) which may be impossible to do v i a conventional l a s e r e x c i t e d molecular f l u o r i m e t r y may be r e a d i l y p o s s i b l e by m u l t i photon l a s e r e x c i t e d molecular f l u o r i m e t r y . F i n a l l y , multiphoton l a s e r e x c i t e d f l u o r i m e t r y can be r e a d i l y used i n the r i g h t - a n g l e
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
WINEFORDNER
Laser-Excited Luminescence Spectrometry
77
c o n f i g u r a t i o n which i s l e s s prone t o w a l l a d s o r p t i o n e f f e c t s and a l s o should be f r e e from the inner (pre) f i l t e r e f f e c t (however, i t i s j u s t as s u s c e p t i b l e t o the post f i l t e r e f f e c t j u s t as i n s i n g l e photon e x c i t a t i o n ) . M u l t i p h o t o n - e x c i t e d resonance enhanced molecular luminescence of low pressure gas p h a s e — and even con densed p h a s e — s p e c i e s has tremendous a n a l y t i c a l p o t e n t i a l d e s p i t e i t s l a c k of use i n published a n a l y t i c a l s t u d i e s ; the great analy t i c a l p o t e n t i a l should encompass great s e n s i t i v i t y and low detec t i o n l i m i t s due t o resonance enhancement, great s e l e c t i v i t y , e s p e c i a l l y at low pressures, but, to some extent, a l s o f o r con densed phase species due t o s e l e c t i v e e x c i t a t i o n of v i b r a t i o n a l l e v e l s ; and freedom from most s p e c t r a l i n t e r f e r e n c e s and c a l i b r a t i o n curvature problems of c o n v e n t i o n a l s i n g l e photon lumines cence. Literature Cited 1.
2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17.
L'vov, Β. V., "Atomic Absorption Spectroscopy," T r a n s l a t e d from Russian, I s r a e l Program f o r Scientific Translation, Jerusalem, 1969. Winefordner, J. D., Svoboda, V., and C l i n e , L. J., CRC Crit. Rev. A n a l . Chem. (1970), 1, 233. Sychra, V., Svoboda, V., and Rubeska, I . , "Atomic F l u o r e s cence Spectrometry," T r a n s l a t e d by M. Cresser, Van NostrandReinhold Co., London, 1975. Gelbwachs, J. Α., Appl. O p t i c s (1976), 15, 2654. Bjorkholm, J . E., and L i a o , P. F., O p t i c s Commun. (1976), 18, 4. Fairbank, W. Μ., Hänsen, T. W., and Schawlow, A. L.,J.Opt. Soc. Amer. (1975), 65, 199. Omenetto, N., and Winefordner, J . D., Appl. Spectrosc. Rev. (1973), 7, 147. Omenetto, Ν., Omenetto, P., Hart, L. P., Winefordner, J. D., and Alkemade, C. Th. J., Spectrochim. Acta (1973), 28B, 289. Gelbwachs, J. Α., K l e i n , C. F., and Wessel, J. E., Appl. Phys. L e t t . (1977), 30, 489. Hurst, G. S., Nayfeh, Μ. Η., and Young, J. P., Phys. Rev. (1977), 15A, 2283. Nayfeh, Μ. Η., Phys. Rev. (1977), 16A, 927. C a p e l l e , G. Α., and Sutton, D. G., A p p l . Phys. L e t t . (1977), 30, 407. B o u t i l i e r , G. D., Blackburn, Μ. Β., Mermet, J. M., Weeks, S. J . , Haraguchi, Η., and Winefordner, J. D., Appl. O p t i c s , in press. Measures, R. Μ., J. Appl. Phys. (1968), 39, 5232. O ' l i v a r e s , D. R., Ph.D. T h e s i s , Indiana U n i v e r s i t y , Bloomington, IN, 1976. D a i l y , J.W., A p p l . Optics (1976), 15, 955. Omenetto, N., and Winefordner, J . D., Chapter on Atomic Fluorescence Spectroscopy w i t h Laser E x c i t a t i o n in " A n a l y t i —
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
78
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.
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
c a l Laser Spectrometry," N. Omenetto, ed., John Wiley, New York, in p r e s s . B o u t i l i e r , G. D., Winefordner, J. D., and Omenetto, N., Appl. O p t i c s , in p r e s s . K i l l i n g e r , P. Κ., Wang, C. C., and Hanabusa, M., Phys. Rev. (1976), 13A, 13. P a n t e l l , P. Η., and Puthoff, Η. Ε., "Fundamentals of Quantum E l e c t r o n i c s , " John Wiley, New York, 1969. Piepmeier, Ε. Η., Spectrochim. Acta (1972) 27B, 431. I b i d . (1972), 445. Piepmeier, Ε. Η., Chapter on Atomic Absorption Spectroscopy w i t h Laser Primary Sources, in " A n a l y t i c a l Laser Spectrome t r y , " N. Omenetto, ed., John Wiley, in p r e s s . Gelbwachs, J. Α., Jones, P. F., and Wessel, J . E., Appl. Phys. L e t t . (1975) 27, 40 Gelbwachs, J . Α. 27, 551. McClain, W. M., Acc. Chem. Res. (1974), 7, 129. Weeks, S. J., Haraguchi, Η., and Winefordner, J. D., A n a l . Chem., in p r e s s . F r a s e r , L. M., and Winefordner, J. D., A n a l . Chem. (1971), 43, 1693. Denton, Μ. Β., and Malmstadt, H. V., Appl. Phys. L e t t . (1971), 18, 485. F r a s e r , L. Μ., and Winefordner, J. D., A n a l . Chem. (1972), 44, 1444. Omenetto, Ν., Hatch, Ν. Ν., F r a s e r , L. Μ., and Winefordner, J . D., A n a l . Chem. (1973), 45, 195. Omenetto, Ν., Hatch, Ν. Ν., F r a s e r , L. Μ., and Winefordner, J. D., Spectrochim. Acta (1973), 28B, 65. Omenetto, N., Hart, L. P., B e n e t t i , P., and Winefordner, J . D., Spectrochim. Acta (1973), 28B, 301. Omenetto, Ν., B e n e t t i , P., Hart, L. P., Winefordner, J . D., and Alkemade, C. Th. J., Spectrochim. A c t a (1973), 28B, 289. Kuhl, J . , Neumann, S., and K r i e s e , Μ., Z. N a t u r f o r s c h (1973), 28a, 273. Kuhl, J . , and Spitschan, H., Opt. Commun. (1973), 7, 256. de O l i v a r e s , D. R., Ph.D. T h e s i s , Indiana U n i v e r s i t y , 1976. Green, R. B., T r a v i s , J . C., and K e l l e r , R. Α., A n a l . Chem. (1976), 48, 1954. Gelbwachs, J. Α., K l e i n , C. F., and Wessel, J . E., Appl. Phys. L e t t . (1977), 30, 489. Smith, B. W., Blackburn, Μ. Β., and Winefordner, J . D., Can. J. Spectrosc. (1977), 22, 57. D a i l y , J. W., and Chan, C., U n i v e r s i t y of C a l i f o r n i a , Berke l e y , CA, unpublished work. Kuhl, J . , and Marowsky, G., Opt. Commun. (1971), 4, 125. Neumann, S., and K r i e s e , Μ., Spectrochim. Acta (1974), 29B, 127. Bolshov, Μ . Α., Zybin, Α. V., Zybina, L. Α., Koloshnikov, V.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
WINEFORDNER
45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
Laser-Excited Luminescence Spectrometry
79
G., and Majorov, I . Α., Spectrochim. Acta (1976), 31B, 493. Fairbanks, Jr., W. M., Hänsen, T. W., and Schawlow, A. L., J . Opt. Soc. Am. (1975), 65, 199. Mayo, S., K e l l e r , R. Α., T r a v i s , J. C., and Green, R. Β., J . Appl. Phys. (1976), 47, 4012. Brod, H. L., and Yeung, E. S., A n a l . Chem. (1976), 48, 344. Sharp, B. L., and Goldwasser, Α., Spectrochim. Acta (1976), 31B, 431. Smith, B. W., Plankey, F. W., Omenetto, Ν., Hart, L. P., and Winefordner, J. D., Spectrochim. Acta (1974), 30A, 1459. Van Geel, T. F., and Winefordner, J . D., A n a l . Chem. (1976), 48, 335. Berman, M. R., and Zare, R. N., A n a l . Chem. (1975), 47, 1200. Bradley, A. B., and Zare, R. N., J. Amer. Chem. Soc. (1976), 98, 620. D i e b ó l d , G. J . , an Richardson, J . H. besh, L. W., A n a l . Chim. Acta (1976), 86, 263. Richardson, J. Η., and Ando, M. E., A n a l . Chem. (1977), 49, 955. Wirth, M. J . , and L y t l e , F. E., A n a l . Chem. (1977), 49, 2054. LeBlanc, R. M., G a l i n i e r , G., T e s s i e r , Α., and Lemieux, L., Can. J . Chem. (1974), 52, 3723. Bjorkholm, J. E., and L i a o , P. F., Phys. Rev. L e t t . (1974), 33, 128. Adams, G. Ε., F i e l d e n , Ε. Μ., and M i c h a e l , B. D., "Fast Pro cesses in R a d i a t i o n Chemistry and B i o l o g y , " John Wiley, New York, 1975.
RECEIVED August 7, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5 Laser Fluorimetry: Detection of Aflatoxin B in 1
Contaminated Corn 1
G. J. DIEBOLD and R. N. ZARE Department of Chemistry, Stanford University, Stanford, CA 94305
A f l a t o x i n s , metabolite the most potent n a t u r a l l presence of these toxins i n v a r y i n g amounts i n a wide v a r i e t y of g r a i n and food products i n virtually every country of the world has given r i s e to concern over the p o t e n t i a l p u b l i c h e a l t h hazard caused by a f l a t o x i n s in the human food supply.(2) The carcinogenic a c t i v i t y of these compounds at the low ppb level, documented i n feeding experiments with l a b o r a t o r y animals, (1) ipso f a c t o demands a n a l y t i c a l d e t e c t i o n methods f o r a f l a t o x i n s at t h i s l e v e l or b e t t e r . From the feeding e x p e r i ments, the most pronounced e f f e c t s of a f l a t o x i n , both accute and c h r o n i c , are known to occur i n the liver. In t r o u t fry, a f l a t o x i n has been shown to induce hepatoma in statistically s i g n i f i c a n t percentages at a l e v e l of only 100 ppt (1)—a d i s q u i e t i n g f a c t given that conventional a n a l y t i c a l techniques are g e n e r a l l y capable of d e t e c t i n g a f l a t o x i n at ten to a hundred times t h i s concentration. By f a r , the most widely used method f o r a f l a t o x i n d e t e c t i o n r e l i e s upon separation of the a f l a t o x i n s on a TLC p l a t e followed by v i s u a l observation of t h e i r n a t u r a l fluorescence when e x c i t e d by a UV lamp. Depending upon the s k i l l of the i n d i v i d u a l experimenter, t h i s method has a d e t e c t i o n l i m i t from 1 to 10 ppb i n corn. Attempts to improve upon t h i s procedure by forming an e x t r a c t from a l a r g e r g r a i n sample, or e q u i v a l e n t l y by s p o t t i n g a l a r g e r amount of g r a i n e x t r a c t on the TLC p l a t e are of l i t t l e value s i n c e overloading of the TLC p l a t e r e s u l t s , i . e . band spreading of various components i n the e x t r a c t occurs i n the region of a f l a t o x i n fluorescence, p r e c l u d i n g more s e n s i t i v e detection. Taking advantage of the p a r t i a l cleanup of the e x t r a c t a f f o r d e d by an overloaded TLC p l a t e , we report here a two-step chromatography procedure, p r e p a r a t i v e TLC followed by reverse phase HPLC, f o r q u a n t i t a t i o n of a f l a t o x i n B^ i n corn. Detection "Current address: Department of Chemistry, Brown University, Providence, RI 02912 0-8412-0459-4/78/47-085-080$05.00/0 © 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
DiEBOLD
AND ZARE
Aflatoxin Bi in Corn
81
of a f l a t o x i n (formed from a f l a t o x i n B.^) i s c a r r i e d out with a high s e n s i t i v i t y l a s e r f l u o r i m e t e r that can detect as l i t t l e as 750 f g of a f l a t o x i n . The procedure o u t l i n e d i s capable of l i n e a r l y q u a n t i t a t i n g a f l a t o x i n B^ from 0.1 to 10 ppb i n white and yellow corn. Apparatus The l a s e r f l u o r i m e t e r (3) i s based on phase s e n s i t i v e d e t e c t i o n of fluorescence e x c i t e d by an amplitude modulated, 8 mW, He-Cd i o n l a s e r . The normal d r i f t i n output power of the l a s e r i s v i r t u a l l y eliminated by a feedback loop c o n s i s t i n g of an acousto-optic l i g h t modulator, d i f f e r e n c e a m p l i f i e r , and photodiode employed i n the L i c o n i x Model 405 UV l a s e r . As shown i n F i g . 1, an o s c i l l a t o r d r i v e s the d i f f e r e n c e a m p l i f i e r at 50 kHz, which by v i r t u amplitude modulated bea A f t e r passing through a UV pass f i l t e r (Corning 7-60) to remove background r a d i a t i o n from the plasma tube, the beam i s focused by a quartz lens i n t o a flowing l i q u i d d r o p l e t of eluent from the HPLC column. By p o s i t i o n i n g the c o l l i m a t o r so that the surface of the d r o p l e t where the l a s e r enters i s excluded from view, s c a t t e r i n g of the l a s e r r a d i a t i o n i n t o the d e t e c t i o n o p t i c s i s minimized. Fluorescence e x c i t e d i n the d r o p l e t passes through an i n t e r f e r e n c e f i l t e r , a quartz l e n s , and a v i s i b l e pass f i l t e r l o c a t e d i n a separate p o l i s h e d brass chamber. The f i r s t f i l t e r a f t e r the c o l l i m a t o r i s a f r o n t s u r f a c e , long wavelength pass i n t e r f e r e n c e f i l t e r on a s p e c t r o s i l quartz s u b s t r a t e . The 1.25 i n . f o c a l length quartz lens focuses l i g h t from the d r o p l e t onto the photocathode of a low n o i s e p h o t o m u l t i p l i e r (Centronix 4249BA). The p h o t o m u l t i p l i e r s i g n a l i s fed i n t o a l o c k - i n a m p l i f i e r and detected i n phase with the o s c i l l a t o r s i g n a l . The output of the l o c k - i n a m p l i f i e r , which i s proport i o n a l to the fluorescence i n t e n s i t y from the d r o p l e t , i s d i s p l a y e d on a s t r i p c h a r t recorder. The fluorescence c e l l i s a l i q u i d d r o p l e t of eluent i n the shape of Plateau's unduloid (4) which i s supported by surface tension i n a gap between the 1/16 i n . O.D. tubing from the chromatography column and a s o l i d s t a i n l e s s s t e e l rod of the same diameter. The d r o p l e t forms a d e t e c t i o n c e l l with a volume of only 4 y l and yet does not s u f f e r from problems of c e l l w a l l fluorescence. Bubbles are prevented from entering the d r o p l e t by notching the tubing near the end, and plugging the t i p so that the eluent i s forced to flow down the sides of the tubing allowing bubbles to r i s e to the surface. A f t e r passing down the outside of the s t a i n l e s s s t e e l rod, the eluent i s removed by an aspirator. Some s c a t t e r i n g of the beam by the d r o p l e t cannot be avoided. To minimize the r e s u l t i n g f l u o r e s c e n c e from the l i g h t s h i e l d , a c o a t i n g of f i n e carbon ( F i s h e r N o r i t A i n chloroform) was a p p l i e d to the i n t e r i o r surfaces of the l i g h t s h i e l d .
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
82
N E W APPLICATIONS
O F LASERS T O C H E M I S T R Y
Figure 1. Diagram of the feedback stabilized laser, flowing droplet fluorescence cell, and detection electronics (3)
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
DIEBOLD AND ZARE
AfldtOXin
83
Βι ΙΠ Com
The s e n s i t i v i t y of t h i s device f o r a f l a t o x i n d e t e c t i o n i s demonstrated i n the chromatograms shown i n F i g . 2. Here, the aflatoxins and G i have been d e r i v a t i z e d to the more f l u o r e s cent a f l a t o x i n s B2A d &2k As a r e s u l t of the p a r t i t i o n i n g processes i n the HPLC column, the a f l a t o x i n s e l u t e from the column considerably more d i l u t e than the s o l u t i o n i n j e c t e d onto the column; thus, the detector must respond to a correspondingly l e s s f l u o r e s c e n t s o l u t i o n . The peak e l u t i n g from the column has a Gaussian p r o f i l e given by f(χ) = (2ττ)~^ σ~1 e x p ( - x / 2 c ) where 3.56 σ i s the f u l l width at h a l f maximum i n t e n s i t y of the peak. A t y p i c a l peak has a f u l l width at h a l f maximum of 0.4 min which c o r r e sponds to σ = 0.17 ml at a flow r a t e of 1.5 ml/min. Since f ( x ) i s normalized to u n i t y , the concentration of a f l a t o x i n at the d e t e c t i o n l i m i t (Fig.2) becomes c(x) = 750 f ( x ) i n fg/ml At the maximum i n the peak 6 χ 10"~12^ Since th of a f l a t o x i n i n the d r o p l e t i s only 7 f g corresponding to 1 χ 10? molecules. a
n
9
2
2
e
A f l a t o x i n B^ Detection i n Contaminated Corn E x t r a c t i o n o f a f l a t o x i n from corn i s c a r r i e d out using the method of S e i t z and Mohr.(5) The f i n a l e x t r a c t represents 10 g of g r a i n and i s d i s s o l v e d i n 0.5 ml of a b e n z e n e - a c e t o n i t r i l e s o l u t i o n . The f i r s t cleanup step c o n s i s t s of s p o t t i n g 50 y l of t h i s e x t r a c t onto a Brinkman SIL-G-25-HR TLC p l a t e which i s developed i n an 88:12 v/v chlororform-acetone s o l u t i o n . Now, even at 1 ppb, the 50 y l of e x t r a c t corresponds to only 1 ng of a f l a t o x i n — a n amount d i f f i c u l t to i d e n t i f y on a TLC p l a t e . To provide c e r t a i n i d e n t i f i c a t i o n of the p o s i t i o n of the a f l a t o x i n s i n the unknown samples, 10 ng standards of a f l a t o x i n Βχ are spotted adjacent to the unknowns. Following development of the p l a t e , the p o r t i o n of the s i l i c a g e l c o n t a i n i n g the unknown sample can be e a s i l y found by n o t i n g the p o s i t i o n of the 10 ng standards under a UV lamp. The s i l i c a g e l c o n t a i n i n g the a f l a t o x i n i s then removed from the TLC p l a t e , a g i t a t e d s e v e r a l minutes i n a v i a l with 2 ml of chloroform and the supernatant c o l l e c t e d . The sample i s evaporated to dryness under a stream of n i t r o g e n on a steam bath and the a f l a t o x i n B^ converted to a f l a t o x i n B2A> (1) known to have a high f l u o r e s c e n t quantum e f f i c i e n c y i n hydrogen bonded solvents.(6) Although t r i f l u o r o acetic a c i d has been used, (7) we p r e f e r to use 100 y l of IN HC1 (8) which i s allowed to r e a c t f o r 10 min. The a c i d i s then evaporated under n i t r o g e n and the e x t r a c t d i s s o l v e d i n 100 y l of the HPLC e l u t i o n s o l v e n t . The HPLC column used i n these experiments i s a Waters Inc. C18 \^onàa.pàk reverse phase column operated at 1.5 ml/min with a 75:25 water-ethanol s o l u t i o n . Solvents are of extremely high p u r i t y . Ethanol i s prepared (9) by d i s t i l l i n g 95% "gold s h i e l d " y
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW APPLICATIONS OF LASERS TO CHEMISTRY
15
'
'
'
'
10
'
'
'
'
5
'
'
'
'
0
Minutes Science
Figure 2. (a) Chromatogram of aflatoxins B G B and G eluting from a C18 nBondapak HPLC column at aflowrate of 1.5 mL/min; 30 pg each. The aflatoxins B and G have been converted to aflatoxins B and G , respectively, with HCl. (b) The fluorimeter response at the detection limit of 750 fg. A three-second time constant was used in (b); at higher levels a one-second or shorter time constant on the lock-in amplifier can be used (3). u
t
t
u
2y
2
2A
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2A
5.
DIEBOLD AND ZARE
AflatOXlTl Bi
ÏYI
Com
85
e t h a n o l t o w h i c h 1 g KOH p e l l e t s p e r l i t e r has b e e n added. A f t e r d i s c a r d i n g t h e f i r s t few m l o f d i s t i l l a t e , a p p r o x i m a t e l y 2/3 o f t h e o r i g i n a l v o l u m e i s c o l l e c t e d . Water i s f i l t e r e d , d e i o n i z e d , p a s s e d o v e r a c t i v a t e d c h a r c o a l , and d i s t i l l e d . After m i x i n g , s o l v e n t s a r e d e g a s s e d i n an u l t r a s o n i c c l e a n e r f o r 1/2 h o u r . G r e a t c a r e must be t a k e n t o i n s u r e t h a t a l l s u r f a c e s coming i n t o c o n t a c t w i t h t h e s o l v e n t s a r e f r e e o f c o n t a m i n a t i o n . T h o r o u g h c l e a n i n g w i t h a 10% HF s o l u t i o n i s recommended. The p u r i t y o f t h e s o l v e n t s can be g i v e n a f i n a l t e s t by v i s u a l l y o b s e r v i n g f l u o r e s c e n c e e x c i t e d by t h e l a s e r . In a completely d a r k e n e d room, f l u o r e s c e n c e o v e r a few cm p a t h l e n g t h s h o u l d be f a i n t l y v i s i b l e , o r n o t v i s i b l e a t a l l . The c o l u m n a l s o must be f r e e o f c o n t a m i n a t i o n . Passing h i g h p u r i t y ethanol through t h e c o l u m n , as recommended by t h e m a n u f a c t u r e r , i s e f f e c t i v e i n removing v a r i o u s f l u o r e s c e n t contaminants from the column. After a stable baselin of the e x t r a c t are i n j e c t e s e n s i t i v i t y , and c o l u m n r e t e n t i o n t i m e f o r a f l a t o x i n B2A d e t e r m i n e d by p r e p a r a t i o n o f known q u a n t i t i e s o f a f l a t o x i n B i w h i c h a r e s p o t t e d on TLC p l a t e s , d e r i v a t i z e d , e t c . i n t h e same manner as t h e unknown s a m p l e . The a f l a t o x i n c o n t e n t o f t h e c o n t a m i n a t e d g r a i n i s d e t e r m i n e d by c o m p a r i n g t h e peak i n t h e unknown s a m p l e t o t h o s e f r o m s t a n d a r d s . I f t h e above p r o c e d u r e i s f o l l o w e d e x a c t l y , 1 ppb o f a f l a t o x i n c o n t a m i n a t i o n corresponds to 100 pg o f a s t a n d a r d i n j e c t e d d i r e c t l y o n t o t h e HPLC c o l u m n . a r e
Results I n a p r e v i o u s p a p e r , (3_) t h e l i n e a r r e s p o n s e o f t h e d e t e c t o r was d e m o n s t r a t e d o v e r t h e r a n g e f r o m 750 f g t o 30 ng f o r a f l a t o x i n s B 2 , 2A> 2 * 2* l i n e a r i t y o f t h i s method f o r a f l a t o x i n q u a n t i t a t i o n r e s t s on a c o n s t a n t c o n v e r s i o n o f a f l a t o x i n Bj_ t o B 2 , and t h e r e f o r e t h e c o n v e r s i o n e f f i c i e n c y , a l t h o u g h r e p o r t e d t o be h i g h , (8) must be s c r u t i n i z e d o v e r t h e e n t i r e range of i n t e r e s t . T h u s , a f l a t o x i n B^ s t a n d a r d s r a n g i n g f r o m 10 pg t o 5 ng w e r e i n d i v i d u a l l y d e r i v a t i z e d , r e d i s s o l v e d i n e l u t i o n s o l v e n t , and i n j e c t e d o n t o t h e HPLC c o l u m n . The m a g n i t u d e s o f t h e f l u o r e s c e n c e s i g n a l s , as shown i n F i g . 3, e x h i b i t the expected l i n e a r i t y over roughly three orders of m a g n i t u d e . The s t r a i g h t l i n e s a r e l e a s t s q u a r e s f i t s t o t h e d a t a d e t e r m i n e d by m i n i m i z i n g t h e f r a c t i o n a l e r r o r b e t w e e n t h e d a t a p o i n t s and a l i n e t h r o u g h t h e o r i g i n . (This procedure weights each p o i n t e q u a l l y , whereas a c o n v e n t i o n a l l e a s t squares f i t strongly favors data points w i t h large values.) The r o o t mean s q u a r e (rms) e r r o r was t h e n c a l c u l a t e d f o r e a c h r u n g i v i n g an a v e r a g e o f 28% f o r t h e two r u n s . G
B
a n c
G
T n e
A
A
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW APPLICATIONS OF LASERS TO CHEMISTRY
86
The o v e r a l l p r e c i s i o n o f t h i s method i s d e t e r m i n e d b y e r r o r s i n r e c o v e r y f r o m t h e TLC p l a t e , dérivâtization, and quantitation i n the fluorimeter. Further errors introduced i n the e x t r a c t i o n p r o c e d u r e have been p r e v i o u s l y investigated.(5) S t a n d a r d s o f a f l a t o x i n B]_ w e r e s p o t t e d on TLC p l a t e s , r e c o v e r e d , d e r i v a t i z e d , and i n j e c t e d o n t o t h e HPLC c o l u m n . The r e s u l t s shown i n F i g . 4 s u r p r i s i n g l y show a marked d e p a r t u r e f r o m l i n e a r i t y above 1 n g i n d i c a t i n g an i n c r e a s e d r e c o v e r y f r o m t h e TLC p l a t e s . S i n c e t h i s change i n r e c o v e r y i s n o t p a r t i c u l a r l y p r o n o u n c e d , s t a n d a r d s c o u l d be u s e d t o c a l i b r a t e t h e p r o c e d u r e p e r m i t t i n g a c c u r a t e q u a n t i t a t i o n a t any l e v e l o f a f l a t o x i n c o n t a m i n a t i o n f r o m 0.1 t o 50 ppb (10 pg t o 50 n g ) . However, s i n c e t h e s i m p l e r method o f f l u o r i m e t r i c q u a n t i t a t i o n d i r e c t l y on TLC p l a t e s c a n be u s e d above t h e 10 ppb l e v e l i t a p p e a r s prudent t o l i m i t the range of the HPLC-laser f l u o r i m e t r i c method t o l e v e l s b e l o analysis. With t h i s c o n s t r a i n o f b o t h r u n s i n F i g . 4) o b t a i n s f o r t h e r a n g e b e t w e e n 0.1 and 10 ppb. The e f f i c a c y o f t h e t w o - s t e p c h r o m a t o g r a p h y p r o c e d u r e f o r e l i m i n a t i n g i n t e r f e r i n g components i n t h e c o r n e x t r a c t i s shown i n F i g . 5 a . The s m o o t h n e s s o f t h e b a s e l i n e c a n be compared t o t h e s i g n a l i n F i g . 5 b , r e p r e s e n t i n g a c o n t a m i n a t i o n o f 250 p p t , t h u s i n d i c a t i n g a l i m i t o f d e t e c t i o n f o r t h i s method o f a p p r o x i m a t e l y 100 p p t . Note t h a t s i n c e t h e d e t e c t i o n l i m i t i s d e t e r m i n e d by t h e c o n c e n t r a t i o n o f c o n t a m i n a n t s s t i l l r e m a i n i n g i n t h e f i n a l e x t r a c t , h i g h e r f l u o r i m e t e r s e n s i t i v i t y ( b e l o w 10 pg) w o u l d n o t f u r t h e r i m p r o v e d e t e c t i o n l i m i t s . Thus a r e d u c t i o n i n t h e above f i g u r e f o r d e t e c t i o n o f a f l a t o x i n i n c o r n i s c o n t i n g e n t upon t h e d e v e l o p m e n t o f e i t h e r more s p e c i f i c d e t e c t o r s , o r f u r t h e r improvements i n c l e a n u p t e c h n i q u e s . Discussion An i n h e r e n t a d v a n t a g e o f t h i s t e c h n i q u e l i e s i n t h e a b i l i t y t o use t h e d e r i v a t i z a t i o n s t e p as a c o n f i r m a t o r y procedure f o r t h e p r e s e n c e o f a f l a t o x i n B^. S i n c e a f l a t o x i n B^ h a s a l o w f l u o r e s c e n t quantum e f f i c i e n c y i n s o l u t i o n , c o m p a r i s o n o f c h r o m a t o g r a m s where t h e d e r i v a t i z a t i o n s t e p h a s b e e n d e l e t e d provides a d d i t i o n a l evidence of i t s presence i n the g r a i n sample. The u l t i m a t e s e n s i t i v i t y o f t h e f l u o r i m e t e r d e s c r i b e d h e r e i s c o n t i n g e n t upon a f o r t u i t o u s c o i n c i d e n c e b e t w e e n t h e l a s e r l i n e a t 325 nm and a n a b s o r p t i o n maximum o f t h e s p e c i e s o f interest. However, i n p r a c t i c e r e m a r k a b l e s e n s i t i v i t y i s a t t a i n a b l e even when t h i s c o n d i t i o n i s n o t f u l f i l l e d . F l u o r e s c e i n , f o r i n s t a n c e , h a s a n a b s o r p t i o n maximum a t 500 nm, a c o n s i d e r a b l e s h i f t from t h e l a s e r l i n e . While the o p t i c a l d e n s i t y a t 325 nm d i f f e r s f r o m t h a t a t t h e p e a k b y f o u r , (10) corresponding to a factor of 1 0 i n absorption, the fluorimeter 4
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
DIEBOLD AND ZARE
87
Aflatoxin Bi IYI Com
of aflatoxin plotted as a function of the quantity of aflatoxin B derivatized to determine the linearity of the HCl-derivative formation procedure. The displacement of the two lines on the log-log plot corresponds to slightly differentfluorimetergains for the two runs. (- · -) Run 1. (- A ~) Bun 2. t
lOOpg QUANTITY
lOng
Figure 4. Aflatoxin B standards were spotted on TLC plates, removed, deriva tized, and quantitated with the fluorime ter. Thefluorimeterresponse, plotted on the ordinate, shows a slight nonlinearity above 1 ng. (- · -) Run 1. (-- A ~) Run 2. t
10pg
lOOpg Ing QUANTITY OF AFLATOXIN
lOng
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
N E W APPLICATIONS
O F LASERS T O C H E M I S T R Y
(a)
(b)
10
5 Minutes
0
Figure 5. {a) Chromatogram of an aflatoxin-free sample of yellow corn using the cleanup procedure described in the text. The flatness of the baseline compared with the signal from the 25 pg aflatoxin B (Bi) standard in (b) indicates a sensitivity of approximately 100 ppt. The tailing seen in these chromât ο grams is attributable to deterioration in the two-year-old column. The sharper peaks expected from a new column would suggest a slightly improved sensitivity. 2A
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5. DIEBOLD AND ZARE
AflatoxinB1in Corn
89
is still capable nevertheless of detecting 4 pg of this dye injected onto the HPLC column. In the case of the aflatoxins, absorption maxima lie at 360 nm, yet the detection limit of this device is 750 fg of each of the four aflatoxins injected onto the HPLC column. We believe that laser fluorimetry is applicable to a number of trace analysis problems, and has special advantage in those cases where sensitivity is a limiting factor. Acknowledgments We would like to thank Dr. Larry Seitz for supplying us with grain extracts. The assistance of N. Karny, J . Kinney and S. Lidofsky in various phases of this work is gratefully acknowledged. This work was supported by National Cancer Institute grant 1-R01-CA-18271-01 Abstract A high sensitivity laser fluorimeter designed specifically for use with high pressure liquid chromatography (HPLC) is described. An amplitude modulated He-Cd ion laser at 325 nm irradiates a flowing droplet of eluent from an HPLC column, and fluorescence is detected in phase with the modulation by a lock-in amplifier. The fluorimeter is capable of detecting 750 fg of each of the four commonly occurring aflatoxins, and has a linear range of over three orders of magnitude. Using a two-step chromatography procedure, this device is capable of quantitating aflatoxin B to 100 ppt in corn. Following extraction of the aflatoxin from corn, the extract is given a preliminary cleanup on a normal phase thin layer chromatography (TLC) plate. The aflatoxin B is recovered, and injected onto a reverse phase HPLC column. Experiments with aflatoxin B1 standards show a constant ratio for conversion of aflatoxin B1 to aflatoxin B over roughly three orders of magnitude. The recovery of aflatoxin B from the TLC plates, although slightly nonlinear above 10 ppb permits linear quantitation of aflatoxin B in white and yellow corn in the range from 0.1 to 10 ppb with an average root mean square (rms) error of 26%. 1
1
2A
1
1
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
90
NEW APPLICATIONS OF LASERS TO CHEMISTRY
Literature Cited 1.
G o l d b l a t t , L. Α., " A f l a t o x i n : S c i e n t i f i c Background, C o n t r o l , and Implications," Academic Press, New York, 1969.
2.
Shank, R. C., in "Mycotoxins and Other Fungal Related Food Problems," Rodricks, J . , Ed. American Chemical Society, Washington, D.C., 1976.
3.
Diebold, G. J . , and Zare, R. Ν., Science (1977) 196, 1439.
4.
Bickerman, J . J., " P h y s i c a l Surfaces," Academic Press, New York, 1970.
5.
S e i t z , L. M. and Mohr 179.
6.
Maggon, Κ. K., Gopal, S., Viswanathan, L., Venkitasubramanian, Τ. Α., R a t h i , S., Ind. J . Biochem. Biophys., (1972) 9, 195.
7.
Thorpe, W., and S t o l o f f , L., in "89th Annual Meeting of the A s s o c i a t i o n of Official A n a l y t i c a l Chemists," A s s o c i a t i o n of Official A n a l y t i c a l Chemists, Washington, D.C., 1975, a b s t r . 58.
8.
Pohland, A. E., Cushmac, M. E., A n d r e l l o s , P. J . , J. Assoc. O f f . Anal. Chem. (1968) 51, 907.
9.
Parker, C. Α., "Photoluminescence of S o l u t i o n s , " p. 421, E l s e v i e r , Amsterdam, 1968.
10.
Η
Ε.
Cereal Chemistry (1977) 54
Hansen, P. Α., "Fluorescent Compounds used in P r o t e i n T r a c i n g , " U n i v e r s i t y o f Maryland, MD, 1964.
RECEIVED August 7, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6 Laser-Enhanced Ionization for Trace Metal Analysis in Flames J. C. TRAVIS and G. C. TURK Center for Analytical Chemistry, U.S. National Bureau of Standards, Washington, D.C. 20234 R. B. GREEN Department of Chemistry, West Virginia University, Morgantown, WV
26506
I o n i z a t i o n of atom e x c i t e d s t a t e than a groun c r e t e atomic t r a n s i t i o n will sufficiently b i a s the e x c i t e d s t a t e p o p u l a t i o n of the atom to produce a change in i o n i z a t i o n r a t e which is e a s i l y measured w i t h conventional e l e c t r o n i c s . The excess i o n i z a t i o n i n the flame due to l a s e r e x c i t a t i o n has been g e n e r a l l y c h a r a c t e r i z e d as an optogalvanic e f f e c t but l a s e r enhanced i o n i z a t i o n (LEI) is more d e s c r i p t i v e , p a r t i c u l a r l y i n terms of the mechanism . Generalized p l o t s of the Saha equation3 (Figure 1) show that most elements ( i o n i z a t i o n p o t e n t i a l ≥ 5eV) are predominantly n e u t r a l at t y p i c a l flame temperatures. The a b s o r p t i o n of o p t i c a l energy, moving an atom c l o s e r to its i o n i z a t i o n l i m i t , will significantly increase the i o n p o p u l a t i o n in the flame. According to F i g u r e 1, an e l e c t r o n v o l t of e x c i t a t i o n energy will provide approximately one order of magnitude increase i n i o n i z a t i o n at 2500 K. LEI spectrometry is a h y b r i d technique which depends on both l a s e r e x c i t a t i o n and thermal i o n i z a t i o n . The process may proceed by p h o t o e x c i t a t i o n and thermal i o n i z a t i o n or a combination of thermal e x c i t a t i o n , p h o t o e x c i t a t i o n and thermal i o n i z a t i o n (Figure 2). The experimental system used i s illustrated i n F i g u r e 3. The sample i s a s p i r a t e d i n t o a f u e l lean a i r - a c e t y l e n e flame of a standard premix burner w i t h a 5 cm s i n g l e s l o t burner head. The atomized species are e x c i t e d w i t h a flashlamp-pumped tunable dye l a s e r wi£h c a p a b i l i t y f o r frequency-doubled o p e r a t i o n . The data reviewed- and presented here were obtained at l a s e r bandwidths of 0.05-0.1 nm, although f u r t h e r narrowing i s r e a d i l y p o s s i b l e . The l a s e r dyes used included Fluoral-7GA, Rhodamine 575, Rhodamine 6G, and Rhodamine 640, w i t h frequency doubling i n most cases. The s i g n a l i s detected with a p a i r of 1 mm diameter tungsten welding rods 1 cm apart and ^ 1 cm above the burner head. These dual cathodes run p a r a l l e l to the burner s l o t and are maintained at -500V to -1000V with respect to the burner head, which i s used I
2
This chapter not subject to U.S. copyright. Published 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
Number
Density= 1 0
APPLICATIONS O F LASERS T O C H E M I S T R Y
1
°/cm
3
10
3000K
0.1 ^10-2
Lu Q Q_
ζ Ç2ioUJ >
4
_i
Lu 10"
6
10-
4
5
6
7
8
E N E R G Y TO IONIZE ( e V )
Figure 1. Relative degree of ionization of a generalized element (3) as a function of its ionization potential for flames of (a) 2000, (b) 2500, and (c) 3000 Κ
IONIZATION POTENTIAL
ΔΕ· I ΔΕ;
hi/ hi/
GROUND STATE USING A RESONANCE LINE
USING A NON-RESONANCE LINE
Figure 2. Typical avenues of User-enhanced ionization: (a) photo excitation from the ground state followed by collisional ioniza tion; (b) photo excitation from a thermally populated excited state followed by collisional ionization
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
TRAVIS
Trace Metal Analysis in Flames
93
as the anode. The e l e c t r o d e s remain v i s u a l l y outside the flame, although s t i l l i n e l e c t r i c a l contact with the flame. This c o n f i g u r a t i o n i s s l i g h t l y l e s s s e n s i t i v e than when the e l e c t r o d e s are immersed i n the flame, but i t avoids f o u l i n g and e r o s i o n which lead to long-term s i g n a l d e t e r i o r a t i o n . The burner head i s e l e c t r i c a l l y i n s u l a t e d from the burner body by a s t r i p of e l e c t r i c a l tape, so that the current may be monitored on the low v o l t a g e s i d e of the flame. The s i g n a l pulse i s separated from the dc background current with a high-pass f i l t e r , a m p l i f i e d , and pro cessed w i t h sample-and-hold c i r c u i t s and a minicomputer (or a boxcar s i g n a l averager w i t h d i g i t a l s t o r a g e ) . F i g u r e 4 shows LEI s i g n a l s from a s o l u t i o n of 25 ng/mL Mg and 7 ng/mL Na. The LEI i n t e n s i t i e s f o r these t r a n s i t i o n s are not i n d i c a t i v e of the r e l a t i v e concentrations and absorption c o e f f i c i e n t s alone. Indeed, (285.2 nm) t o Na (285. O s c i l l a t o r s t r e n g t h , i o n i z a t i o n p o t e n t i a l , and f r a c t i o n a l popula t i o n a l l p l a y important r o l e s i n determining the s i g n a l s t r e n g t h . The most s e n s i t i v e l i n e f o r atomic absorption, atomic f l u o r e s cence, and atomic emission, may not n e c e s s a r i l y be the best l i n e f o r LEI spectrometry. Table I shows the d e t e c t i o n l i m i t s f o r pure, aqueous s o l u t i o n s of s e v e r a l elements. These d e t e c t i o n l i m i t s were deter mined by using the average and standard d e v i a t i o n of the mean of 150 l a s e r pulses (^ 0.8 ys pulsewidth) a t a pulse r e p e t i t i o n r a t e of 5 pulses per second. For comparison, Table I a l s o contains d e t e c t i o n l i m i t s reported f o r other flame spectrometric t e c h n i ques. With one exception, LEI d e t e c t i o n l i m i t s a r e comparable to or b e t t e r than those reported f o r other flame spectrometric methods. A c l o s e r i n s p e c t i o n of the LEI d e t e c t i o n l i m i t s i s i n s t r u c t i v e . Table I I shows p e r t i n e n t parameters f o r the t r a n s i t i o n s used. One f a c t o r which i s p a r t i c u l a r l y important i s the energy d i f f e r e n c e between the l a s e r populated e x c i t e d s t a t e and the i o n i z a t i o n p o t e n t i a l . T h i s energy d i f f e r e n c e i s r e f e r r e d to as ΔΕ.. Sodium i s a s t r i k i n g example of the combined e f f e c t of t r a n s i t i o n p r o b a b i l i t y and proximity to the i o n i z a t i o n l i m i t . The d e t e c t i o n l i m i t s are comparable f o r both the strong l i n e a t 589.0 nm (which i s commonly used f o r s p e c t r o s c o p i c a n a l y s i s ) and the weak l i n e a t 285.3 nm, although the l a s e r output i s 100 times more powerful a t the strong l i n e . The e f f e c t of ΔΕ^ on s e n s i t i v i t y i s a l s o i l l u s t r a t e d by the low d e t e c t i o n l i m i t s obtained using e x c i t e d - e x c i t e d t r a n s i t i o n s of Cr, Cu, Mn, Pb, Sn, and T l . In these cases, the l o s s of s e n s i t i v i t y r e s u l t i n g from the small f r a c t i o n a l p o p u l a t i o n of the lower l e v e l i s s i g n i f i c a n t l y counter balanced by the decrease i n ΔΕ.. L E I a n a l y s i s of copper a t 324.8 nm i s r e l a t i v e l y i n s e n s i t i v e , s i n c e i t s ΔΕ^ i s l a r g e r than any of the other t r a n s i t i o n s attempted. Indium enjoys the dual advantages of a s m a l l ΔΕ. and a r e l a t i v e l y l a r g e a b s o r p t i o n coefficient, B . 0 t
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
94
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
PULSED LASER
PHOTODIODE
AMPLIFIER
AMPLIFIER SAMPLE
SAMPLE
&
&
OSCILLOSCOPE
COMPUTER
TTY
Figure 3. Block diagram of the instrument
Na V
j 0.1 nm
Mg t
\
285.3 285.2 WAVELENGTH (nm)
Figure 4. LEI spectrum of a solution of 25 ng/ mL Mg and 7 ng/mL Na in a C H /air flame 2
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
TRAVIS
95
Trace Metal Analysis in Flames
TABLE I Comparative D e t e c t i o n L i m i t s by LEI and Other Flame Techniques
Element Cr
a
2
FAA°
FAE
2
2
ioo
Fe
2
Ga
0.07
50
In
0.008
30
Mg Mn Na
C
FAF
b
Cu
Κ
a
LEl
0.3
0.4 .05
3
1 o.i
60
3
b
b
0.05
b
1
0.8
0.5
1
0.
1
10
0.9
100
0.2 —
—
0.1
0.2
1
0.4 0.1
—
5
20
3
10
100
10
6
50
100
50
0.09
20
20
Ni
8
Pb
0.6
Sn Tl
b
,d Laser FAF*
5
70
0.8
C
8
2 13
4
T h i s work, except as noted. A l l l i m i t s a r e i n ng/mL. o r i g i n a l l y i n r e f e r e n c e 2.
b
Reported
C
Taken from J . D. Winefordner, J . J . F i t z g e r a l d , and N. Omenetto, Appl. Spectrosc. 29, 369 (1975). FAA = Flame Atomic A b s o r p t i o n . FAE = Flame Atomic Emission. FAF = Flame Atomic Fluorescence.
d
S . J . Weeks, H. Haraguchi, and J . D. Winefordner, A n a l . Chem. _50, 360 (1978).
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
298.6 301.8 282.4 324.8 298.4 302.1 287.4 294.4 303.9 294.3 285.2 279.5 280.0 285.3 589.0 300.2 280.2 283.3 284.0 286.3 291.8
Wavelength (nm)
3
a
.065 .065 1.0 1.0 .4 .4 .2 .2 .6 .2 .6 .6 .6 .9 .9 1.0 .7 .7 .04 .04 .5
:
TT
2.53 4.35 .298 1.77 .223 .426 1.76 1.76 2.51 7.5x10 5.5 .76 1.11 „ 7.0x10 4.63 .65 4.76 1.03 2.42 3.13 2.35
Β χ 10 L -2„ -1,-1 ) S~ (W cm Hz
7
8308 8095 11203 0 0 0 0 826 0 0 0 0 17052 0 0 205 10650 0 3428 0 7793
\
1.3 1 3 1 1 1 1 2 1 1 1 1 1.7 1 1 .8 5 1 5 1 2
12788 13345 15719 31533 30193 30604 13606 13601 13778 1037 26620 24200 7212 6407 24476 28078 13491 24533 20603 24318 7218
the LEI Figure of M e r i t ΔΕ. 1 g (cm ) V o (cm )
2
4.2x10^ 2.6x10^ 1.6x10^ 5x10" 2x10 30 8x10^ 7x107 9.6x10 17 „ 9.5x10. 1.6xl0 29 32 z 6.2x10
2.6x10" 3.6x10 17 6.9 .78
Figure of Merit
Computed f r o m E q u a t i o n 1, u s i n g t h e f a c t o r s g i v e n and a n assumed s p e c t r a l i r r a d i a n c e o f -9 -2 -1 -7 -? -1 3 χ 10 W cm Hz f o r a l l uv t r a n s i t i o n s and 3 χ 10 W cm Hz f o r Na ( 5 8 9 . 0 ) .
Taken f r o m R e f e r e n c e 5.
Tl
Sn
Ni Pb
Na
In Κ Mg Mn
Ga
Fe
Cu
Cr
Element
1
Factors Determining
TABLE I I
2.0 2.0 100 100 4 2 0.07 0.1 0.008 1 0.1 0.3 5 0.05 0.1 8 0.6 3 6 10 0.09
Limit of Detection (ng/mL)
1
H O
W
t>
O
o
>
2 M
05
6.
TRAVIS
97
Trace Metal Analysis in Flames
A zero-order f i g u r e of merit i s u s e f u l f o r p r e d i c t i n g the r e l a t i v e s e n s i t i v i t y of atomic t r a n s i t i o n s : F i g u r e of M e r i t = 31 Β
Here 3 i s the atomization e f f i c i e n c y of the element i n the flame; 1^ the l a s e r s p e c t r a l i r r a d i a n c e ; the E i n s t e i n c o e f f i c i e n t f o r the p r o b a b i l i t y of a b s o r p t i o n from s t a t e I t o u; E^ the energy of the lower s t a t e of the t r a n s i t i o n ; g^ and g a r e lower s t a t e and ground s t a t e s t a t i s t i c a l weights; Τ the flame tempera ture; and k the Boltzmann constant. The f i r s t exponential term expresses the r e l a t i v e p r o b a b i l i t y that a c o l l i s i o n i n the flame w i l l provide the thermal energy r e q u i r e d (ΔΕ.) to complete the i o n i z a t i o n process. Th the s t a t i s t i c a l weight r a t i o of the lower l e v e l of the t r a n s i t i o n , r e l a t i v e t o the ground state. The uv L E I d e t e c t i o n l i m i t s are p l o t t e d on a l o g - l o g s c a l e against the corresponding f i g u r e s of merit (shown i n Table II) i n F i g u r e 5. The trend of the data towards a slope of -1 r e s u l t s from the nominal r e c i p r o c a l r e l a t i o n s h i p between d e t e c t i o n l i m i t and s e n s i t i v i t y ( f o r constant l i m i t i n g noise) and supports the v a l i d i t y of the f i g u r e of m e r i t . Current i n v e s t i g a t i o n s using three and four l e v e l model c a l c u l a t i o n s — c a l l f o r s e v e r a l r e f i n e ments to the f i g u r e of merit, e s p e c i a l l y f o r e x c i t e d s t a t e t r a n s i t i o n s . The simple expression i s nonetheless more v a l i d than cross s e c t i o n s alone f o r p r e d i c t i n g the s e n s i t i v i t y of a t r a n s i tion. Given the f a v o r a b l e d e t e c t i o n l i m i t s of Table I , and the s p e c t r a l s e l e c t i v i t y a v a i l a b l e with tunable l a s e r s , i t i s worth while to study observed and p r e d i c t e d matrix i n t e r f e r e n c e s f o r r e a l sample a n a l y s i s by LEI. Such i n t e r f e r e n c e s may g e n e r a l l y be c l a s s i f i e d as chemical, s p e c t r a l , or e l e c t r i c a l . Chemical i n t e r f e r e n c e s w i l l g e n e r a l l y be the same as experienced by other flame s p e c t r o s c o p i c methods*-, and w i l l not be f u r t h e r discussed here. S p e c t r a l i n t e r f e r e n c e s , on the other hand, have some unique f e a t u r e s f o r LEI. Although atomic s p e c t r a l overlaps wjiich have been documented f o r other flame spectrometric methods- are s t i l l present, the r e l a t i v e degree of i n t e r f e r e n c e may d i f f e r d r a s t i c a l l y from o p t i c a l spectrometry due to the LEI f i g u r e of merit of the i n t e r f e r i n g t r a n s i t i o n . Thus, some of the documented i n t e r f e r ences may be i n c o n s e q u e n t i a l , because of a low f i g u r e of merit f o r the i n t e r f e r i n g l i n e , and conversely, some undocumented coincidences of weak s p e c t r a l l i n e s may become important due to a high f i g u r e of m e r i t . An example of t h i s l a t t e r case i s the "weak" 285.3 nm Na t r a n s i t i o n which y i e l d s a strong L E I s i g n a l and i n t e r f e r s s i g n i f i c a n t l y with the 285.2 nm Mg t r a n s i t i o n , (Figure 4 ) . Q
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
! r
Cu Cu*
en "'In'
:Fe Te
Μη* Pb * » CrCr
Ξ~
Pb Μη Mg
Γ
Gaj|*Na Ga Na In
Γ
1
10
1 ου Figure
10 of
3
10
4
10
s
l u
6
Merit
Figure 5. LEI detection limits as a function of predictedfigureof merit
Figure 6. Percent recovery of 100 ppb lead LEI signal as a function of sodium concentration for applied potentials of —600, —800, and —1000 V
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
TRAVIS
Trace Metal Analysis in Flames
100
A P P L I E D POTENTIAL (Volts)
Figure 7. Percent recovery of 100-ppb lead LEI signal as a function of applied potential for so dium concentrations of 0,10, 20, and 30 ppm
TABLE I I I R e l a t i v e I n t e r f e r e n c e of D i f f e r e n t M a t r i c e s on 100 ppb Lead S i g n a l Matrix
I.P.
(10 ppm)
(eV)
Percent S i g n a l Recovery -50QV
-750V
-1000V
Κ
4.3
0
0
180
Na
5.1
45
90
110
Li
5.4
100
110
110
Ca
6.1
82
100
100
Cu
7.7
100
100
100
Analyte:
100 ppb Pb
λ = 280.2 nm
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
100
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
Molecular s p e c t r a l i n t e r f e r e n c e s seem to be l e s s of a problem with LEI than conventional flame spectrometry, s i n c e most of the t r a d i t i o n a l i n t e r f e r i n g molecules have high (a 10 eV) i o n i z a t i o n p o t e n t i a l s . R e l a t i v e l y few molecular species have been observed i n flames to date using LEI . E l e c t r i c a l i n t e r f e r e n c e s are unique to LEI ( i n l i e u of such o p t i c a l background i n t e r f e r e n c e s as flame background, ambient l i g h t , and s c a t t e r e d source l i g h t , to which LEI i s impervious). Two types of e l e c t r i c a l i n t e r f e r e n c e may be i d e n t i f i e d : 1) the e f f e c t of ambient e l e c t r o n d e n s i t y i n the flame on i o n i z a t i o n / recombination r a t e s ; and, 2) the e f f e c t of the ambient e l e c t r o n and i o n d e n s i t y on the s i g n a l c o l l e c t i o n process. As an example, F i g u r e 6 i l l u s t r a t e s the e f f e c t of sodium on the s i g n a l from 100 ppb lead f o r s e v e r a l values of a p p l i e d p o t e n t i a l . At each p o t e n t i a l , the a d d i t i o n of sodium i s f i r s t seen to enhance the s i g n a l p o t e n t i a l s — completel obtained from the same data as F i g u r e 6, and i s u s e f u l f o r e x p l a i n i n g the s i g n a l behavior. The threshold v o l t a g e — below which no s i g n a l i s observed f o r a given matrix — i s r e l a t e d to the sheath, or space-charge, of p o s i t i v e ions which surrounds the two cathode w i r e s . For a given flame temperature and ambient i o n d e n s i t y , a corresponding e l e c t r i c a l s h i e l d i n g i s provided by t h i s sheath, reducing the magnitude of the e l e c t r i c a l p o t e n t i a l at the measurement s i t e . The s h i e l d i n g e f f e c t , and hence the t h r e s h o l d , i n c r e a s e s with i n c r e a s i n g matrix i o n c o n c e n t r a t i o n . Above t h r e s h o l d , e l e c t r o n s generated by enhanced i o n i z a t i o n are d r i v e n by the e l e c t r i c f i e l d toward the burner head with a v e l o c i t y p r o p o r t i o n a l to the a c t u a l e l e c t r i c f i e l d . E l e c t r o n s which reach the burner head before recombining w i t h a p o s i t i v e ion provide the LEI s i g n a l . Table I I I compares the r e l a t i v e degree of i n t e r f e r e n c e f o r matrices of v a r i o u s i o n i z a t i o n p o t e n t i a l s . Only the most e a s i l y i o n i z e d elements are seen to provide a problem. The e l e c t r i c a l i n t e r f e r e n c e s are subject to m o d i f i c a t i o n by instrumentation design and parameter o p t i m i z a t i o n , and an a c t i v e program to minimize i n t e r f e r e n c e s i s underway. Laser enhanced i o n i z a t i o n may be seen to be a s e n s i t i v e and s e l e c t i v e method. Although p r e s e n t l y subject to unique matrix i n t e r f e r e n c e s , these are subject to f u r t h e r instrument development or sample pre-treatment. The method i s " b l i n d " to such common o p t i c a l i n t e r f e r e n c e sources as flame background emission, ambient l i g h t , and s c a t t e r e d e x c i t a t i o n l i g h t . Because of the i n s e n s i t i v i t y to s c a t t e r e d l a s e r l i g h t , the a b i l i t y of l a s e r s to s a t u r a t e o p t i c a l t r a n s i t i o n s may be u t i l i z e d to the f u l l e s t advantage. F i n a l l y , the v a s t l y modified c r i t e r i a f o r s p e c t r a l s e n s i t i v i t y gives the method multi-element p o t e n t i a l , and the c a p a b i l i t y of avoiding t r a d i t i o n a l s p e c t r a l i n t e r f e r e n c e s . Implementation of LEI spectrometry r e q u i r e s minimal m o d i f i c a t i o n of a l a s e r induced f l u o r e s c e n c e (LIF) spectrometer. Though
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
TRAVIS
Trace Metal Analysis in Flames
101
l e s s thoroughly developed than L I F a t t h i s time, LEI represents a complementary measurement which may be made simultaneously, i f d e s i r e d . Comparative LEI and LIF measurements w i l l o b v i o u s l y be r e q u i r e d f o r a wide v a r i e t y of samples and flames to a c c u r a t e l y e s t a b l i s h the dominant r o l e s o f the complementary methods of l a s e r e x c i t e d flame spectrometry.
Literature Cited 1. 2. 3.
4. 5.
6.
7. 8. 9.
Green, R. Β., R. A. K e l l e r , P. K. Schenck, J. C. T r a v i s , and G. G. Luther, J . Am. Chem. Soc. 98, 8517 (1976). Turk, G. C., J. C. T r a v i s , J . R. DeVoe, and T. C. O'Haver, A n a l . Chem. 50, 817 (1978). In order to render th p l o t element-independent th ratio of ion-to-atom p a r t i t i o u n i t y . The e r r o r in one order of magnitude f o r any given element. The n a t u r a l e l e c t r o n background p o p u l a t i o n o f the flame is assumed t o be negligible. See P. J. W. Boumans, Theory of Spectrochemical E x c i t a t i o n , H i l g e r and Watts, London (1966), p 161, for a more complete d i s c u s s i o n . T r a v i s , J . C., P. K. Schenck, and G. C. Turk, i n p r e p a r a t i o n . W i l l i s , J . B., in CRC Handbook of Spectroscopy, Volume I , J . W. Robinson, ed., CRC P r e s s , Cleveland (1974) p 799. Values from Table 12, p 814, w i t h c o r r e c t i o n s from Table 13 f o r Na and K. For a b r i e f d i s c u s s i o n , and p e r t i n e n t Tables, see M. L. Parsons, B. W. Smith, and G. E. Bentley, Handbook of Flame Spectroscopy, Plenum Press, NY (1975), p 61. See, for instance, R. J. L o v e t t , D. L. Welch, and M. L. Parsons, Appl. Spectrosc. 29, 470 (1975). Schenck, Peter K., W. Gary M a l l a r d , John C. T r a v i s , and Kermit C. Smyth, submitted f o r p u b l i c a t i o n . Weeks, S. J., H. Haraguchi, and J. D. Winefordner, A n a l . Chem. 50, 360 (1978).
RECEIVED August 7, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7 The Study of Biological Surfaces by Laser Electrophoretic Light Scattering B. R. WARE Department of Chemistry, Harvard University, 12 Oxford Street, Cambridge, MA 02138
One of the newest a p p l i c a t i o n s of l a s e r s is the measurement of very small v e l o c i t i e light which has been s c a t t e r e This technique, c a l l e d l a s e r Doppler velocimetry, has been a p p l i e d t o a number of i n t e r e s t i n g biological problems, and a general review of the progress i n t h i s area has r e c e n t l y been p u b l i s h e d (1). This l e c t u r e will focus on the a p p l i c a t i o n of l a s e r Doppler velocimetry t o the d e t e c t i o n of e l e c t r o p h o r e s i s , a technique which I s h a l l call e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g (ELS). The theory and the first s u c c e s s f u l experiments of t h i s type were reported by myself and Bill Flygare i n 1971 (2). Since then the technique has been developed and a p p l i e d by s e v e r a l groups t o a wide v a r i e t y of problems. For reviews see references (1/3/4/5). I shall s t a t e briefly the p r i n c i p l e s of e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g , describe the methodology of the experiments as we do them, and then summarize the r e s u l t s of a few of the p r o j e c t s which are being pursued in my l a b o r a t o r i e s at Harvard. A block diagram of an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g apparatus i s shown i n Figure 1. Laser l i g h t i l l u m i n a t e s the p a r t i c l e s or molecules t o be s t u d i e d , and l i g h t which has been s c a t t e r e d from these p a r t i c l e s at a s e l e c t e d angle θ i s c o l l e c t e d and d i r e c t e d t o a p h o t o m u l t i p l i e r tube. When an e l e c t r i c f i e l d i s a p p l i e d to the suspension, the p a r t i c l e s migrate toward the e l e c t r o d e of opposite p o l a r i t y . L i g h t s c a t t e r e d from them i s t h e r e f o r e s l i g h t l y s h i f t e d i n frequency by the Doppler e f f e c t . In order t o measure t h i s s h i f t , i t i s necessary t o combine with the s c a t t e r e d l i g h t a beam of u n s h i f t e d l i g h t which i s obtained by s p l i t t i n g a second beam from the i n c i d e n t l a s e r l i g h t and bypassing the chamber. These two beams, the s c a t t e r e d l i g h t and the s o - c a l l e d l o c a l o s c i l l a t o r beam, are mixed at the photocathode t o produce a low-frequency beat which i s e x a c t l y the Doppler s h i f t frequency. The low-frequency o s c i l l a t i o n s of the photocurrent are then a m p l i f i e d and processed by a real-time spectrum analyzer, the output of which i s the complete spectrum 0-8412-0459-4/78/47-085-102$05.00/0
© 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WARE
Laser Electrophoretic Light Scattering
VARIABLE SLIT VIEWING SCREEN VARIABLE ATTENUATOR
X MIRROR
MIRROR
LOCAL OSCILLATOR BEAM
BEAM"^ SPLITTER
TIMER
DATA RECORDING AND DISPLAY
CONSTANT CURRENT POWER SUPPLY
I
REAL TIME SPECTRUM ANALYZER
AMPLIFIER
Contemporary Topics in Analytical and Clinical Chemistry
Figure 1.
Diagram of an electrophoretic light-scattering apparatus.
The coherent, monochromatic beam from the laser is split into two beams. The forward beam is focused into the electrophoretic light-scattering chamber to illumhMe the moving particles. The chamber sits at an angle to the beam in order to obtain a higher scattering angle Θ, so the beam is refracted on entrance and exit. A constant electric field is applied to the scattering region by applying pulses of constant current to the electrodes. Duration of and interval between pulses are controlled by a specially constructed timing circuit. Light is scattered from the particles which are drifting in the electric field, and the scattered light is therefore shifted slightly in frequency by the Doppler effect. The scattered light is collected by an optical system and focused onto the surface of a photodetector. The split-off beam is recombined with the scattered light at the window of the chamber in order to form the so-called local oscillator. The alignment of the scattered light and the local oscilhtor is facilitated by the formation of real images of both, which are mewed on a screen when deflected by a reflex mirror. Once the two have been aligned at the proper point on the screen correspond ing to the center of the photocathode, the reflex mirror is moved out of the way, and the two beams pass through a calibrated slit to the photocathode. There they produce a beat or spectrum of beats equal to the Doppler shift magnitudes between the shifted scattered light and the unshifted local oscillator. The photocurrent or voltage is then amplified and analyzed in frequency by a real-time spectrum analyzer, which is triggered by the timing circuit to accept data only when the field is on. The diagram of the scattering chamber is shown greatly enlarged and is highly schematic. For actual designs and descriptions, see Ref. 5.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
104
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
o f a l l Doppler s i g n a l s corresponding t o the histogram of v e l o c i t i e s i n the sample. The equipment r e q u i r e d f o r an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g experiment i s r e l a t i v e l y simply and inexpensive. For many experiments on suspensions of large p a r t i c l e s such as blood c e l l s , a small He-Ne l a s e r c o s t i n g only a few hundred d o l l a r s i s s u f f i c i e n t . The e s s e n t i a l o p t i c a l components can be kept t o as l i t t l e as one lens and two p i n h o l e s , though i n p r a c t i c e we use a more complicated real-image viewing system t o f a c i l i t a t e alignment of the s c a t t e r e d l i g h t and the l o c a l o s c i l l a t o r (5). In some cases the l o c a l o s c i l l a t o r may be taken from one or both of the spots formed by entry and e x i t of the l a s e r beam through the chamber. We g e n e r a l l y p r e f e r t o have a separate o p t i c a l path f o r the l o c a l o s c i l l a t o r which bypasses the s c a t t e r i n g chamber and then i s recombined with the s c a t t e r e d l i g h t , u s u a l l y a f t e r being r e f l e c ted from the chamber window o s c i l l a t o r path allows adjustmen o s c i l l a t o r i n t e n s i t y t o the detected s c a t t e r e d l i g h t i n t e n s i t y . T h i s r a t i o i s u s u a l l y i n the range from 10:1 t o 40:1. An e x t e r n a l l o c a l o s c i l l a t o r path i s e s s e n t i a l f o r s c a t t e r i n g angles above about 20°. We g e n e r a l l y work at angles between 50° and 60° when analyzing l a r g e p a r t i c l e s such as blood c e l l s , f o r which s p e c t r a l broadening due t o d i f f u s i o n i s n e g l i g i b l e . Much lower angles must be u t i l i z e d f o r the study of s o l u t i o n s of s m a l l e r p a r t i c l e s such as p r o t e i n s , i n order t o optimize the r a t i o o f the Doppler s h i f t t o the d i f f u s i o n - c o n t r o l l e d h a l f - w i d t h (2). However, f o r blood c e l l s t u d i e s we employ the higher s c a t t e r i n g angles t o i n c r e a s e the Doppler s h i f t f o r a given e l e c t r o p h o r e t i c v e l o c i t y . This adaptation i s e s s e n t i a l f o r performing e x p e r i ments at p h y s i o l o g i c a l i o n i c s t r e n g t h , where the e l e c t r o p h o r e t i c m o b i l i t i e s are lower and the a t t a i n a b l e f i e l d strength f o r a given t o l e r a b l e amount o f Joule h e a t i n g i s a l s o lower. The b e a t i n g d e t e c t i o n system r e q u i r e d f o r an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g measurement i s simpler and cheaper than the high-speed photon counting apparata necessary f o r many other l a s e r l i g h t s c a t t e r i n g a p p l i c a t i o n s . The photodetector can be an inexpensive p h o t o m u l t i p l i e r tube o r an even cheaper p h o t o r e s i s t o r or p h o t o v o l t a i c c e l l . The s i g n a l s are g e n e r a l l y i n the region between 1 Hz and 200 Hz, so a m p l i f i c a t i o n and spectrum a n a l y s i s are t r i v i a l low-speed problems. I t i s e s s e n t i a l t h a t the spectrum a n a l y s i s , whether done by a spectrum analyzer, an autoc o r r e l a t o r , or a d i g i t a l computer o r c a l c u l a t o r , be done i n r e a l time; i . e . , i n the minimum time r e q u i r e d f o r measurement with a given frequency r e s o l u t i o n . For example, to measure a spectrum with a r e s o l u t i o n of 1 Hz r e q u i r e s 1 sec. We need t h e r e f o r e apply the e l e c t r i c f i e l d f o r one second only, i f the data p r o c e s s i n g device can make use of a l l the information a v a i l a b l e during t h a t one second t o produce a complete spectrum. The a b i l i t y t o use pulsed f i e l d s i s an i n t r i n s i c advantage o f e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g over c l a s s i c a l e l e c t r o p h o r e s i s
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
WARE
Laser Electrophoretic Light Scattering
105
techniques, s i n c e i t permits the use o f a higher f i e l d s t r e n g t h , which i n many cases leads t o g r e a t e r e l e c t r o p h o r e t i c r e s o l u t i o n . We g e n e r a l l y apply constant-current pulses with a duty c y c l e o f about 1:10; the i n t e r v a l between pulses allows the d i s s i p a t i o n o f Joule heat. The pulses are of a l t e r n a t i n g p o l a r i t y so that there i s no net t r a n s p o r t o f mass during the experiment. Timing and t r i g g e r i n g are c o n t r o l l e d by a s p e c i a l l y designed clock c i r c u i t . The use o f a constant-current power supply i s important f o r maintaining a constant e l e c t r i c f i e l d during each p u l s e , s i n c e i t a u t o m a t i c a l l y c o r r e c t s f o r e l e c t r o d e p o l a r i z a t i o n e f f e c t s and changes i n v i s c o s i t y which accompany Joule h e a t i n g . The design o f the chamber i s a c r i t i c a l feature of the experiment. A l l chambers have two e l e c t r o d e s f o r a p p l i c a t i o n of the f i e l d and an o p t i c a l path f o r entry of the l a s e r beam and e x i t of the s c a t t e r e d l i g h t However a number of chamber c o n f i g u r a t i o n s have been employed I n t e r e s t e d persons are r e f e r r e technique (1,3,4,5) f o r d i s c u s s i o n and r e f e r e n c e s . We c u r r e n t l y have s e v e r a l d i f f e r e n t chambers i n use f o r various a p p l i c a t i o n s . Common features o f these chambers which we have found t o be important are low volume, e f f i c i e n t heat d i s s i p a t i o n , and subs t a n t i a l s e p a r a t i o n between the e l e c t r o d e and the s c a t t e r i n g r e g i o n , so t h a t p a r t i c l e s , bubbles, and/or l o c a l pH gradients formed a t the e l e c t r o d e s w i l l not be able t o reach the p o r t i o n of the s o l u t i o n which i s being viewed during the measurement. Although the experiment i s not easy, e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g i n i t s current s t a t e of development i s capable of measuring complete e l e c t r o p h o r e t i c m o b i l i t y d i s t r i b u t i o n s i n a few seconds. Once the apparatus i s s e t up, operation i s f u l l y automatic, and adaptation t o o n - l i n e p r o c e s s i n g of m u l t i p l e samples would be a s t r a i g h t f o r w a r d extension of current capabilities. In the absence of an e l e c t r i c f i e l d , the l i g h t s c a t t e r e d from macromolecules i n s o l u t i o n i s frequency-broadened by the random thermal motions of d i f f u s i o n . The measured spectrum i s a Lorentz i a n l i n e centered at the i n c i d e n t frequency, or, when b e a t i n g d e t e c t i o n i s used, centered at zero. A p p l i c a t i o n of the e l e c t r i c f i e l d causes the p a r t i c l e s to migrate, and, i f they a l l have the same e l e c t r o p h o r e t i c m o b i l i t y , the r e s u l t i n g spectrum i s a s h i f t e d L o r e n t z i a n l i n e whose width i s s t i l l determined by d i f f u s i o n and the magnitude of whose s h i f t i s d i r e c t l y r e l a t e d t o the e l e c t r o p h o r e t i c d r i f t v e l o c i t y , which, when d i v i d e d by the f i e l d s t r e n g t h , gives the e l e c t r o p h o r e t i c m o b i l i t y . This pred i c t i o n has been v e r i f i e d experimentally, and an example i s shown i n F i g u r e 2. The p o i n t s i n the spectrum shown i n Figure 2 are data from an e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum on a d i l u t e s o l u t i o n of human carbon monoxyhemoglobin (1.6 mg/ml) at pH 9.5. The l i n e i s a L o r e n t z i a n f u n c t i o n f i t t o the s h i f t of the peak and with a h a l f - w i d t h corresponding t o the known d i f f u s i o n c o e f f i c i e n t of hemoglobin. We are c u r r e n t l y pursuing an
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
106
NEW
APPLICATIONS O F
LASERS T O
CHEMISTRY
i n t e r e s t i n the study of simultaneous d i f f u s i o n and e l e c t r o phoresis o f concentration f l u c t u a t i o n s under c o n d i t i o n s f o r which f l u c t u a t i o n s o f d i f f e r e n t species i n s o l u t i o n cannot be con s i d e r e d t o be uncoupled. This spectrum i s presented only t o show t h a t i n the simple case of uncoupled f l u c t u a t i o n s , the p r e d i c t i o n s o f the simple theory are observed. We have a l s o used ELS t o study the d i s s o c i a t i o n o f hemoglobin at high pH (8). I f there are more than one type o f macroions i n s o l u t i o n i n appreciable concentrations, and i f the d i f f e r e n t species have d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t i e s , then the e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum can be used t o detect and q u a n t i f y r e l a t i v e amounts of the s p e c i e s . A common example of a u s e f u l a n a l y t i c a l e l e c t r o p h o r e s i s determination i s the a n a l y s i s of human blood plasma. An e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum of human plasma i s shown i strong resemblance t o plasm niques, except t h a t the lower-mobility peaks, presumably due t o the various g l o b u l i n f r a c t i o n s , are enhanced with respect t o the l a r g e albumin peak because of t h e i r higher molecular weight. This p a r t i c u l a r measurement i s an extremely important c l i n i c a l t e s t , and some of our f r i e n d s i n i n d u s t r y t e l l us t h a t e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g may be cost-competitive with c l a s s i c a l methods f o r t h i s a p p l i c a t i o n . When b i o l o g i c a l p a r t i c l e s l a r g e r than p r o t e i n s , p a r t i c u l a r l y membranous p a r t i c l e s , are analyzed, i t i s observed t h a t there i s s u b s t a n t i a l e l e c t r o p h o r e t i c heterogeneity. Moreover, these l a r g e r p a r t i c l e s have correspondingly low d i f f u s i o n c o e f f i c i e n t s , and the d i f f u s i o n broadening i s t h e r e f o r e o f t e n i n s i g n i f i c a n t . For such p a r t i c l e s the e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum i s a determination of the e l e c t r o p h o r e t i c m o b i l i t y d i s t r i b u t i o n of the sample. As an i l l u s t r a t i o n I present the spectrum i n F i g u r e 4. The sample i n t h i s case was a mixture o f human and r a b b i t red blood c e l l s ; human c e l l s have the higher m o b i l i t y . The sharp r e s o l u t i o n i s a v i v i d demonstration t h a t ELS can be used t o detect s e v e r a l species simultaneously. The e l e c t r o p h o r e t i c m o b i l i t y o f red c e l l s i s q u i t e uniform, and i n a recent p u b l i c a t i o n with two other groups we have demonstrated t h a t pre vious r e p o r t s t h a t the e l e c t r o p h o r e t i c m o b i l i t i e s of red blood c e l l s decrease with age were erroneous (9). We have been using e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g t o study the surface p r o p e r t i e s of l i v i n g c e l l s and o r g a n e l l e s , and the remainder of t h i s l e c t u r e w i l l be a b r i e f summary o f some of our work i n t h i s area. One of the o r i g i n a l areas o f i n t e r e s t was the c h a r a c t e r i z a t i o n o f c e l l s i n v o l v e d i n the immune response, f o r which surface c h a r a c t e r i s t i c s are p a r t i c u l a r l y important. For example, lymphocytes are the white blood c e l l s i n v o l v e d with immunological r e c o g n i t i o n and response. Lymphocytes are u s u a l l y d i v i d e d i n t o two c a t e g o r i e s : Τ c e l l s , which are p r i m a r i l y i n volved with d i r e c t c e l l u l a r immunity, and Β c e l l s , which are r e s p o n s i b l e f o r the s y n t h e s i s of s p e c i f i c a n t i b o d i e s . We have
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WARE
107
Laser Electrophoretic Light Scattering
Analytical Biochemistry
Figure 2. Electrophoretic light-scattering spec trum (points) of carhoxyhemoglobin tetramers, 100 μΜ in heme. The experimental conditions are Ε = 88.8 V/cm, θ = 4.18°, bath temperature = 20.0°C, and a glycineNaOH-NaCl-EDTA buffer of ionic strength 0.01M and pH 9.5. The solid line is a theoretical curve for these conditions, assuming diffusion is the only source of spectral broadening with a diffusion coefficient D = 6.9 X 10' cm /sec and an electrophoretic mobility u = 2.74 X 10' cm /V-sec (7). 2 0
0.
40. 8 0 . 120. 160. 2 0 0 . FREQUENCY (HZJ
7
2
20
4
2
w
S(U)
Frequency
(Hz)
Contemporary Topics in Analytical and Clinical Chemistry
Figure 3. Electrophoretic light-scattering spectrum of human blood plasma. Fresh human plasma was dialyzed and then diluted severalfold to final solution conditions of pH 9.1 and ionic strength 0.004. This spectrum was taken with a high field strength (183 V'/cm) to maximize the Doppler shift and at a low scattering angle (3.2°) to minimize the diffusion broadening of each peak. The Urge peak at the highest frequency can be identified as albumin from its relative magnitude and its electrophoretic mobility (3.9 X 10~ cm /V-sec). Positive identification of the peaks at lower mobility cannot be made from the Doppler spectrum alone, but the form of the spectrum is similar to the known electrophoretic pattern of normal human plasma (5). 4
2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
108
NEW
APPLICATIONS O F LASERS T O
CHEMISTRY
been studying human lymphocytes and t h e i r surface r e a c t i o n s , and we have been comparing them w i t h p a t h o l o g i c a l c o n d i t i o n s such as acute lymphocytic leukemia, i n which the lymphocytes, or more p r o p e r l y lymphoblasts, produced are malignant, d i v i d i n g c e l l s which have g r e a t l y reduced immunological c a p a c i t y . An e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g spectrum o f normal human lymphocytes i s shown i n Figure 5. The spectrum i s l a b e l l e d i n u n i t s of e l e c t r o p h o r e t i c m o b i l i t y , because f o r these c e l l s d i f f u s i o n broadening i s i n s i g n i f i c a n t , and there i s a one-to-one c o r respondence between frequency and m o b i l i t y . The s o l i d l i n e i s the spectrum from a normal sample o f lymphocytes i n one-tenthp h y s i o l o g i c a l - s a l t medium at pH 7.4. Note the bimodal c h a r a c t e r . To determine whether these two peaks could be a t t r i b u t e d t o Τ and Β c e l l s we prepared samples from which Τ or Β c e l l s had been s e l e c t i v e l y removed. The dotted l i n e i s the spectrum f o r t h i s sample from which Τ c e l l r o s e t t i n g techniques. Not s e l e c t i v e l y diminished. By performing numerous experiments o f t h i s type we have e s t a b l i s h e d t h a t the h i g h e r - m o b i l i t y peak i s due p r i m a r i l y t o Τ c e l l s and the lower-mobility peak i s due p r i m a r i l y t o Β c e l l s (10) , which i s i n agreement with s e v e r a l groups who have obtained t h i s same r e s u l t and i n c o n f l i c t w i t h some groups who have claimed t h a t the two c e l l subtypes are e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e . Our method i s now by f a r the f a s t e s t means o f measuring the Τ c e l l / B c e l l r a t i o , which i s a parameter o f both research and c l i n i c a l i n t e r e s t . In a f u r t h e r attempt to c h a r a c t e r i z e these lymphocytes we have performed experiments t o determine the o r i g i n of the c e l l s u r f a c e charge and the d i s t i n c t i o n between c e l l subtypes on t h i s b a s i s . For example, s i a l i c a c i d (N-Acetylneuraminic acid) i s a ubiquitous source of charge i n c e l l membranes. S i a l i c a c i d can be removed by the a c t i o n o f the enzyme neuraminidase, and we have t r e a t e d lymphocytes with neuraminidase and analyzed separated subf r a c t i o n s . The r e s u l t s are i l l u s t r a t e d i n Figure 6. Again the s o l i d l i n e represents the whole f r a c t i o n o f the same sample seen i n the p r e v i o u s f i g u r e except a f t e r neuraminidase treatment. The m o b i l i t i e s are lower as expected. The dotted l i n e again r e p r e sents t h i s same sample from which Τ c e l l s had been s e l e c t i v e l y removed. Note t h a t the lower-mobility peak shows the only reduc t i o n i n i n t e n s i t y . By repeated experiments with both Τ and Β depleted samples we have demonstrated t h a t a f t e r neuraminidase treatment the Τ c e l l s , which were o r i g i n a l l y o f h i g h e r m o b i l i t y , become the lower-mobility f r a c t i o n , i n d i c a t i n g t h a t they have much more a v a i l a b l e s i a l i c a c i d on t h e i r s u r f a c e s . In f a c t the e l e c t r o p h o r e t i c d i s t i n c t i o n between the two types o f c e l l s i s even g r e a t e r a f t e r neuraminidase treatment. Comparison w i t h diseased s t a t e s i s i n t e r e s t i n g both p o s s i b l e development o f the e l e c t r o p h o r e t i c m o b i l i t y as i n d i c a t o r and f o r a c h a r a c t e r i z a t i o n o f the fundamental ences of the abnormal c e l l s . We have been p a r t i c u l a r l y
f o r the a clinical differ interested
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WARE
Laser Electrophoretic Light Scattering
109
Figure 4. Electrophoretic light scat tering spectrum for a mixture of rabbit and human erythrocytes in approxi mately equal concentrations. Rabbit erythrocytes have the lower mobility. The measurement was made in an electro phoresis buffer which had an ionic strength of 0.0097. The electric field was 44 V/cm, the frequency range 200 Hz, and the scat tering angle 58°. The chamber tempera ture was 20°C. The mobility distributions for the two cell types are completely re solved (6).
>
0
0.5
1.0
Electrophoretic
1.5 Mobility
2.0 ( cm /volt · sec ) 2
2.5 Χ
I0
3.0 4
Journal of Immunology
Figure 5. A comparison of the electrophoretic mobility distributions (at 0.015M ionic strength) of a fresh human mononuclear, white-blood-cell sample before (solid line) and after (dotted line) E rosette depletion (T-cell depletion). The whole sample (solid line) contained 44% cells which form E rosettes and 32% cells which form Ε AC rosettes (primarily Β cells). The horizontal axis indicates the magnitude of the electrophoretic mobility since the Doppler tech nique does not determine the sign of the mobility, which for these cells is nega tive. The vertical axis is approximately proportional to cell number (10). AET
AET
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
110
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
ι
0
1
0.5
1
1.0
Electrophoretic
1
1
1.5 Mobility
2.0 2
( cm /volt · s e c )
1
2.5 X 10
1
3.0
4
Journal of Immunology
Figure 6. A comparison of the electrophoretic mobility distributions (at 0.015M ionic strength) for the same pair of samples shown in Figure 5 after both had been treated with neuraminidase. Again, the solid line represents the whole sample and the dotted line represents the E -rosette-depleted (T-cell depleted) sample. Τ cells are therefore represented primarily in the low-mobility peak between 0.5 and 1.0 mobility units (10). AET
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
WARE
Laser Electrophoretic Light Scattering
111
i n comparisons with leukemic c e l l s (10,11). Electrophoretic comparison of normal lymphocytes and c e l l s obtained i n the same way from p a t i e n t s w i t h acute lymphocytic leukemia i s shown i n Figure 7. The s o l i d l i n e i s a normal sample w i t h i t s charac t e r i s t i c bimodal appearance. The dotted l i n e i s a sample of leukemic c e l l s . Note t h a t t h e i r mode m o b i l i t y i s lower and t h a t the d i s t r i b u t i o n i s narrower, w i t h no bimodal c h a r a c t e r , i n d i c a t i v e o f the l a c k o f d i f f e r e n t i a t i o n of these c e l l s . The m o b i l i t y of leukemic c e l l s i s q u i t e v a r i a b l e , ranging from 5% t o 25% lower than normal c e l l s at t h i s s a l t c o n c e n t r a t i o n (0.015 M). Whether the m o b i l i t y i s a meaningful c l i n i c a l i n d i c a t o r has not y e t been determined, though a group i n France u s i n g c l a s s i c a l micro e l e c t r o p h o r e s i s has r e c e n t l y r e p o r t e d t h a t i t may be (12). We have a l s o s t u d i e d the response of leukemic c e l l s t o neuraminidase. Those experiments are summarized i n Figure 8 The s o l i d l i n e i s the norma leukemic sample. Not narrow m o b i l i t y d i s t r i b u t i o n and t h a t i t f a l l s s l i g h t l y below the Τ c e l l mobility. This s l i g h t difference i s reproducible. Recall t h a t the leukemic c e l l s had a m o b i l i t y s i m i l a r t o the Β c e l l m o b i l i t y b e f o r e treatment; a f t e r neuraminidase treatment they show a m o b i l i t y f a r from Β c e l l s and c l o s e r t o Τ c e l l s . Clearly the surface o f the leukemic c e l l i s markedly d i f f e r e n t from either Τ or Β c e l l s . T h i s f a c t has a l s o been i l l u s t r a t e d by experiments we have performed on the i o n i c strength dependence o f the m o b i l i t i e s , which i s d i f f e r e n t f o r leukemic c e l l s than f o r Τ or Β c e l l s . In f a c t , leukemic c e l l s have a mode m o b i l i t y which i s about the same as normal samples at p h y s i o l o g i c a l i o n i c strength. These experiments on lymphocytes and leukemic lymphoblasts represent an almost s t r i c t l y a n a l y t i c a l approach t o the a p p l i c a t i o n o f the technique. Are there more fundamental questions which we can address? I wouldn't ask the question i f the answer were not yes, and i n p a r t i c u l a r I want t o describe some e x p e r i ments we have been performing on the general q u e s t i o n of the r o l e of e l e c t r o s t a t i c f o r c e s i n c e l l - c e l l i n t e r a c t i o n s . A l l b i o l o g i c a l membranes are negative and i t i s reasonable t o expect e l e c t r o s t a t i c r e p u l s i o n between them. However, we know t h a t many d i f f e r e n t kinds o f c e l l adhesion and aggregation r e a c t i o n s occur f r e q u e n t l y . This i s much e a s i e r t o accept when we a p p r e c i a t e t h a t the charges on c e l l s are screened e x p o n e n t i a l l y by the counterions i n s o l u t i o n , w i t h a space constant equal t o the r e c i p r o c a l o f the Debye-Huckel constant, which a t p h y s i o l o g i c a l i o n i c strength i s about 8 A. S t i l l one sees and hears many arguments i n c e l l adhesion problems on the r o l e o f e l e c t r o s t a t i c f o r c e s , so we have s e t about the task o f measuring c e l l charge and c o r r e l a t i n g with aggregation phenomena. The f i r s t system we s t u d i e d was the g r a n u l o c y t e s , o r p o l y morphonuclear white b l o o d c e l l s , which are the phagocytes respon s i b l e f o r e a t i n g and d i g e s t i n g f o r e i g n m a t e r i a l . These c e l l s
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
112
N E W APPLICATIONS O F LASERS T O C H E M I S T R Y
I
1
1
1
0
0.5
1.0
1.5
Electrophoretic
Mobility
1
1
2.0
2.5
(cm /volt · sec) Χ I 0 2
1 3.0 4
Journal of Immunology
Figure 7. A comparison of the electrophoretic mobility distributions for normal (solid line) and leukemic (dotted line) human mononuclear white blood cells at 0.015M ionic strength. The leukemic cells have a distinctly lower mode mobility than the normal cells. In this case, the leukemic cell distribution almost coincides with that portion of the normal distribution that has been identified as Β cells (10).
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
WARE
Laser Electrophoretic
Electrophoretic
113
Light Scattering
Mobility
( cm /volt · sec) 2
X
I 0
4
Journal of Immunology
Figure 8. A comparison of the electrophoretic mobility distributions of the same pair of samples shown in Figure 7, after neuraminidase treatment. Again, the solid line represents the normal sample and the dotted line represents the leukemic sample. The mobility of the leukemic cells is reduced by a much larger frac tion than that of the Β cells, so that the leukemic-cell mobility is now slightly lower than the Τ-cell mobility. Thus the leukemic cells more nearly resemble Τ cells in their response to neuraminidase treatment (10).
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
114
NEW APPLICATIONS OF LASERS TO CHEMISTRY
concentrate i n areas o f i n f e c t i o n i n response t o s o l u b l e p r o t e i n f a c t o r s c a l l e d lymphokines, which are s e c r e t e d by lymphocytes i n a f f e c t e d areas. One p o s s i b i l i t y advanced was t h a t the lymphok i n e s , c a l l e d LIF f o r leukocyte i n h i b i t i o n f a c t o r , b i n d t o the surface o f the g r a n u l o c y t e s , reduce t h e i r charge, and thereby cause them t o adhere t o each other and t o other s u r f a c e s i n the area. In experiments which are not y e t p u b l i s h e d , we have compared the e l e c t r o p h o r e t i c m o b i l i t i e s of granulocytes b e f o r e and a f t e r treatment with LIF and before and a f t e r treatment with cont r o l f r a c t i o n s i s o l a t e d i n the same way as the L I F . N e i t h e r the LIF nor the c o n t r o l s induced any measurable change i n the e l e c t r o p h o r e t i c m o b i l i t i e s o f the granulocytes at p h y s i o l o g i c a l i o n i c s t r e n g t h (6). T h e r e f o r e , the charge-reduction mechanism d e r i v e s no support from our data. But what i f the s u r f a c charg reduced? Would t h i e s s a r i l y cause an i n c r e a s t i o n , we have i n i t i a t e y (strai 3T3 MIT), which are d i v i d i n g connective t i s s u e c e l l s . These experiments were a l s o done at p h y s i o l o g i c a l i o n i c s t r e n g t h i n phosphate b u f f e r e d s a l i n e t o approximate as c l o s e l y as p o s s i b l e the r e l e v a n t p h y s i o l o g i c a l parameters. We considered three d i f f e r e n t types o f m o d i f i c a t i o n s o f the c e l l s u r f a c e s : neuraminidase treatment, v i r a l t r a n s f o r m a t i o n , and urea treatment. Electrop h o r e t i c m o b i l i t y histograms were measured by e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g , and the degree of change o f s u r f a c e charge was c o r r e l a t e d with the c e l l aggregation r a t e f o r the same c e l l s measured i n a hemacytometer by our c o l l a b o r a t o r s , Morris Karnovsky and Tom Wright o f the Harvard Medical School. The r e s u l t s have been d e s c r i b e d i n the t h e s i s of Barton Smith (6) and w i l l be p u b l i s h e d i n the near f u t u r e . To summarize, we found t h a t neuraminidase treatment o f these c e l l s lowers the s u r f a c e charge and i n c r e a s e s the aggregation rate by more than a f a c t o r o f two. V i r a l t r a n s f o r m a t i o n with SV-40 and polyoma v i r u s e s produces o n l y a few percent decrease i n the average s u r f a c e charge and y e t i n c r e a s e s the aggregation rate by more than a f a c t o r of t h r e e . Treatment o f the transformed c e l l s w i t h neuraminidase produced a l a r g e r e d u c t i o n i n t h e i r average e l e c t r o p h o r e t i c m o b i l i t y but no p e r c e p t i b l e change i n t h e i r r a t e o f aggregation. F i n a l l y t r e a t ment with 0.20 M urea produced no change i n the e l e c t r o p h o r e t i c histogram but i n c r e a s e d the rate o f aggregation by more than a f a c t o r o f t h r e e . We can only conclude that f o r t h i s system the c e l l s u r f a c e charge does not c o r r e l a t e w e l l with the p r o p e n s i t y o f the c e l l s t o adhere t o each other. C e l l adhesion must be understood on the b a s i s o f more d e t a i l e d molecular r e c o g n i t i o n and with an accounting f o r the balance between the a t t r a c t i v e van der Waals f o r c e s and the screened coulombic r e p u l s i o n s . The f i n a l type of study which I would l i k e t o d e s c r i b e i s an i n v e s t i g a t i o n i n t o the process o f v e s i c u l a r s e c r e t i o n . The sec r e t i o n of v a r i o u s hormones and neurotransmitters i n v o l v e s the r e l e a s e o f these molecules from s p h e r i c a l v e s i c l e s i n which they
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
WARE
Laser Electrophoretic Light Scattering
Figure 9. Electrophoretic mobilities of chromaffin granules vs. con centration of Ca , Mg" , and Mg-ATP; and electrophoretic mobility of plasma membrane vesicles vs. concentration of Ca . +2
2
+2
Mobilities are negative, viscosity corrected to pure water at 20°C, and are given in units of μm-cm/V-sec. The ionic strength of the suspension medium was 15 mM and the pH was 6.9. Note the Ca and Mg have identical effects on the mobilities, and hence on the surface charge, of chromaffin granules. +2
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
+2
116
NEW
APPLICATIONS O F LASERS T O C H E M I S T R Y
are s t o r e d . The s e c r e t o r y v e s i c l e s are thought t o r e l e a s e t h e i r contents by f u s i n g with the plasma membrane o f the c e l l i n which they r e s i d e . T h i s f u s i o n i s g e n e r a l l y t r i g g e r e d by an i n f l u x o f calcium i o n , which i s s p e c i f i c f o r t h i s process. We have been i n v e s t i g a t i n g the e f f e c t s o f calcium and magnesium ions on the surface charge and aggregation o f s e c r e t o r y v e s i c l e s . As an example I show the r e s u l t o f one such study on the chromaffin granules from the adrenal medulla, i n t h i s case from a cow. These granules are v e s i c l e s which c o n t a i n a d r e n a l i n and noradrenalin. They are e a s i l y i s o l a t e d and p u r i f i e d . We have t i t r a t e d them with C a and M g and have monitored the e f f e c t o f these ions on granule s u r f a c e charge by e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g . The r e s u l t s o f t h i s t i t r a t i o n are shown i n F i g u r e 9. Broken l i n e s represent the t i t r a t i o n o f granules and the s o l i d l i n e shows a t i t r a t i o n f vesicle formed fro th plasm branes o f the chromaffi r e s u l t s o f t h i s study wa g Mg same a f f i n i t y f o r the granule s u r f a c e , which means t h a t the s p e c i f i c r o l e o f calcium i n inducing e x o c y t o s i s cannot be d e t e r mined by i t s b i n d i n g constant. We have a l s o performed e x p e r i ments i n which mixtures o f the granules and the plasma membrane v e s i c l e s are observed f i r s t s e p a r a t e l y and then i n the same s o l u t i o n t o d e t e c t aggregation o f the two s p e c i e s . Our e x p e r i ments show no aggregation below 1 mM C a and considerable aggregation above t h a t c o n c e n t r a t i o n . We are c u r r e n t l y pursuing experiments o f t h i s type t o study the neurosecretory process. I t r u s t t h a t these examples serve adequately t o i l l u s t r a t e the various types o f a p p l i c a t i o n s f o r which e l e c t r o p h o r e t i c l i g h t s c a t t e r i n g can be u s e f u l . Probably the two most u s e f u l techniques i n b i o p h y s i c a l s t u d i e s have been spectroscopy and e l e c t r o p h o r e s i s . This new technique, which i s a spectroscop i c a l l y - d e t e c t e d e l e c t r o p h o r e s i s , combines the advantages o f speed, accuracy, r e s o l u t i o n , and o b j e c t i v i t y and w i l l , I b e l i e v e , be a p p l i e d t o an i n c r e a s i n g number o f important problems i n b i o l o g y and s u r f a c e chemistry i n the years t o come. + 2
+2
+ 2
LITERATURE CITED 1.
Ware, B. R., " A p p l i c a t i o n s o f Laser Velocimetry in Biology and Medicine," in Chemical and Biochemical Applications of Lasers, C. B. Moore, ed., Chapter 5, Academic Press, New York, 1977.
2.
Ware, B. R., and F l y g a r e , W. Η., "The Simultaneous Measure ment o f the E l e c t r o p h o r e t i c M o b i l i t y and D i f f u s i o n C o e f f i c i e n t in Bovine Serum Albumin S o l u t i o n s by L i g h t S c a t t e r i n g , " Chem. Phys. Lett. (1971) 12, 81.
3.
Ware, B. R., " E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Adv. Interface Sci. (1974) 4, 1.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Colloid
7.
WARE
Laser Electrophoretic Light Scattering
117
4.
F l y g a r e , W. Η., Ware, B. R., and H a r t f o r d , S. L., " E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " in Molecular Electro-Optics, C. T. O'Konski, ed., Chapter 9, Marcel Dekker, Inc., New York, 1976.
5.
Smith, Β. Α., and Ware, B. R., "Apparatus and Methods for Laser Doppler E l e c t r o p h o r e s i s , " in Contemporary Topics in Analytical and Clinical Chemistry, Hercules et al., eds., Plenum Press, New York, in press.
6.
Smith, Β. Α., "The Study o f Cell Surface Charge by E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Ph.D. Thesis (1977) Harvard U n i v e r s i t y , Cambridge, Massachusetts.
7.
Haas, D. D., and Ware, B. R., "Design and Construction o f a New E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g Chamber and A p p l i c a t i o n s t o S o l u t i o n s o f Hemoglobin," Anal. Biochem. (1976) 74, 175.
8.
Haas, D. D., and Ware D i f f u s i o n C o e f f i c i e n t s o f Hemoglobin for publication.
9.
a t High pH,
submitted
Luner, S. J . , Szklarek, D., Knox, R. J., Seaman, G. V. F., Josefowicz, J . Υ., and Ware, B. R., "Red Cell Charge is Not a Function o f Cell Age," Nature (London), (1977) 269, 719.
10. Smith, Β. Α., Ware, B. R., and Yankee, R. Α., " E l e c t r o p h o r e t i c M o b i l i t y D i s t r i b u t i o n s o f Normal Human Τ and Β Lymphocytes and o f P e r i p h e r a l Blood Lymphoblasts in Acute Lymphocytic Leukemia: E f f e c t s o f Neuraminidase and o f Solvent I o n i c Strength," J . Immunol. (1978) 120(3), 921. 11. Smith, Β. Α., Ware, B. R., and Weiner, R. S., " E l e c t r o p h o r e t i c D i s t r i b u t i o n s o f Human P e r i p h e r a l Blood Mononuclear White C e l l s from Normal Subjects and from P a t i e n t s with Acute Lymphocytic Leukemia," Proc. Natl. Acad. Sci. (USA) (1976) 73, 2388. 12. Sabolovic, D., Pompidou, Α., and Amiel, J . L., "Blood Lympho cytes in Acute Lymphoid Leukaemia in Remission and in Relapse. P r e d i c t i v e Value o f E l e c t r o p h o r e t i c M o b i l i t y and Refringence," Biomed. (1975) 23, 283. 13. S i e g e l , D. P., Ware, B. R., Green, D. J . , and Westhead, E. W., "The E f f e c t s o f Ca and Mg on the Surface Charge o f Chro maffin Granules Measured by E l e c t r o p h o r e t i c L i g h t S c a t t e r i n g , " Biophys. J. (1978) 22, 341. +2
+2
RECEIVED August 7, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8 New Laser-Based Methods for the Measurement of Transient Chemical Events GARY M. HIEFTJE and J. MICHAEL RAMSEY Department of Chemistry, Indiana University, Bloomington, IN 47401 GILBERT R. HAUGEN Department of Chemistry, Lawrence Livermore Laboratory, Livermore, CA 94550 One of the trends i moder chemical h is toward th observation and measuremen observations and measurement migh spe c i e s , of e x c i t e d s t a t e l i f e t i m e s , o f energy t r a n s f e r processes, or of chemical r e a c t i o n r a t e s themselves. Obviously, the ability to c h a r a c t e r i z e ever b r i e f e r chemical phenomena will lead to an improved understanding of many areas o f chemistry; consequently, the methodology o f high-speed observations has almost become an end i n itself. In keeping with these improvements in methodology, our d e f i n i t i o n s o f " b r i e f " and " t r a n s i e n t " have changed over the y e a r s . Whereas before 1950 it was common t o consider m i l l i s e c o n d events b r i e f , measurements on such a time s c a l e became commonplace a f t e r that time and we learned to consider microsecond events r e a d i l y measurable. In the 1960's, nanosecond processes were the subjects of study while the present decade has extended our t h i n k i n g to the picosecond and sub-picosecond time s c a l e . T h i s movement toward f a s t e r and f a s t e r time s c a l e s is not without i t s l i m i t . As f a r as s p e c t r o s c o p i c monitoring i s concerned, there i s l i t t l e reason to attempt examination o f events f a s t e r than one femtosecond ( 1 0 ~ s e c ) , since measurements on such a time s c a l e have a Heisenberg energy u n c e r t a i n t y approximately equal to that of the chemical bond. S i g n i f i c a n t l y , measurements on a femtosecond time s c a l e are already f e a s i b l e (1) and might be expected to become commonplace i n the next decade. In t h i s chapter, a new approach w i l l be presented f o r the measurement o f t r a n s i e n t chemical events. B a s i c a l l y , the new measurement techniques are an outgrowth o f i n f o r m a t i o n theory, p a r t i c u l a r l y that branch o f i n f o r m a t i o n theory d e a l i n g with l i n e a r response theory, c o r r e l a t i o n a n a l y s i s and s p e c t r a l power measurement. Although the methods have p o t e n t i a l l y broad a p p l i c a t i o n to areas as d i v e r s e as chemical k i n e t i c s and n u c l e a r magnetic resonance spectroscopy, t h e i r use i n f l u o r e s c e n c e l i f e t i m e determinat i o n w i l l serve here to i l l u s t r a t e t h e i r u t i l i t y . To begin, i t i s necessary to l a y some b a s i c groundwork so the 1 5
0-8412-0459-4/78/47-085-118$05.00/0 © 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8.
HiEFTjE
ET AL.
Measurement of Transient Chemical Events
nature and scope of the new approach can be a p p r e c i a t e d . f o l l o w i n g s e c t i o n e s t a b l i s h e s t h i s groundwork. F l u c t u a t i o n A n a l y s i s S p e c t r o s c o p i c Techniques
119 The
(FAST).
The b a s i s f o r the new family o f techniques f o r studying t r a n s i e n t phenomena can be found i n l i n e a r response theory. Accordi n g l y , l e t us adopt a linear-response-theory view of chemical measurements to understand the techniques. From such a view, any chemical measurement can be thought of as an attempt to a s c e r t a i n the response f u n c t i o n o f a chemical substance, instrument, energy l e v e l , or whatever. Conveniently, the time response f u n c t i o n i s one o f the most f r e q u e n t l y and e a s i l y determined kinds of r e s ponse f u n c t i o n s , and w e l l - e s t a b l i s h e d l i n e a r - r e s p o n s e - t h e o r y approaches f o r i t s determination have been e s t a b l i s h e d Perhaps the most e a s i l y understoo que i n v o l v e s the determinatio Measurement o f an impulse-response f u n c t i o n i s s t r a i g h t f o r ward. As shown i n F i g u r e 1, any system (substance, energy l e v e l , etc.) can be considered to have one or more inputs and one or more outputs, each o f which can be used to l e a r n something about the system. For convenience, l e t us assume that the system to be measured has only one input and one output. As w i l l be obvious l a t e r , t h i s simple two-terminal model can be used to represent many systems of chemical i n t e r e s t . The impulse response o f the system i s then j u s t the observed output which r e s u l t s from a p p l i c a t i o n o f a s u i t a b l e impulse to the input. In t h i s treatment, i t i s assumed that the t e s t e d system responds l i n e a r l y to the a p p l i e d stimulus. For example, i f the system to be measured were a simple r e s i s t o r - c a p a c i t o r (RC) e l e c t r o n i c network, i t s time response would be e x p o n e n t i a l . A p p l i c a t i o n o f an impulse (or pulse) to the input o f the RC network would then produce the expected s t r e t c h e d pulse at the output o f the network. From t h i s simple example, i t can be a p p r e c i a t e d that the time response f u n c t i o n i s merely a c o n v o l u t i o n (2, _3) o f the input impulse and the syst e m ^ impulse response f u n c t i o n and, indeed, i f we do apply a perf e c t impulse ( d e l t a f u n c t i o n ) , the observed output w i l l be the impulse response f u n c t i o n i t s e l f . S i g n i f i c a n t l y , the impulse response f u n c t i o n i s merely one of a f a m i l y of response f u n c t i o n s , each o f which r e s u l t s from a p p l i c a t i o n o f a d i f f e r e n t kind of input to the system under i n v e s t i g a t i o n . In general, i t can be shown that each p a r t i c u l a r response f u n c t i o n i s approximated by the c o n v o l u t i o n of a s p e c i f i e d input with the t r a n s f e r f u n c t i o n o f the t e s t e d system. More e x a c t l y , the t r a n s f e r f u n c t i o n o f the system (which i s the i n f o r m a t i o n a c t u a l l y being sought) i s the r a t i o o f the Laplace transform of the observed output to that o f the p e r t u r b i n g input. From a more q u a l i t a t i v e standpoint, the impulse response f u n c t i o n can be viewed as a time-domain r e p r e s e n t a t i o n o f the
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
120
APPLICATIONS O F LASERS T O C H E M I S T R Y
frequency response o f the system being t e s t e d . Because an i d e a l impulse ( e f f e c t i v e l y a d e l t a function) contains a l l frequencies, a p p l i c a t i o n o f the impulse t o a t e s t e d system involves sending a l l frequencies simultaneously i n t o the t e s t e d network. The r e s u l t i n g output then r e f l e c t s the frequency response o f the network, but r e v e a l s the network s phase response as w e l l . The importance o f the impulse-response f u n c t i o n to chemistry can be r e a d i l y i l l u s t r a t e d . For i n s t a n c e , temperature-jump and pressure-jump methods o f r e a c t i o n - r a t e measurement are e s s e n t i a l l y impulse-response approaches. In a d d i t i o n , the measurement o f luminescence decay times i s o r d i n a r i l y accomplished by a p p l i c a t i o n of an impulse o f o p t i c a l r a d i a t i o n and measurement o f the r e s u l t i n g response; o b v i o u s l y , t h i s procedure c o n s t i t u t e s another impulse-response measurement. From these simple examples, i t should be appreciated that the input impulse need not be an e l e c t r i c a l f u n c t i o n but coul produces a meaningful respons An a l t e r n a t i v e approach can be used to o b t a i n impulseresponse f u n c t i o n s . In t h i s second approach, a random or pseudorandom input p e r t u r b a t i o n i s employed and the r e s u l t i n g output observed. The output i s then e i t h e r a u t o c o r r e l a t e d or c r o s s c o r r e l a t e d with the s t o c h a s t i c p e r t u r b i n g f u n c t i o n to y i e l d , r e s p e c t i v e l y , the a u t o c o r r e l a t i o n o f the impulse response or the impulse response f u n c t i o n d i r e c t l y (2,3). Although less i n t u i t i v e l y obvious than the d i r e c t determination o f an impulse r e s ponse f u n c t i o n d e s c r i b e d above, t h i s l a t t e r technique has s e v e r a l advantages. Most importantly, the p e r t u r b i n g f u n c t i o n need not be a "spike", c o n t a i n i n g a great deal o f power at one moment i n time, but r a t h e r can d i s t r i b u t e the p e r t u r b i n g energy over a much greater time, thereby p l a c i n g less s t r e s s on the system under test. Q u a l i t a t i v e l y , t h i s l a t t e r s t o c h a s t i c method can be e a s i l y understood i f one remembers that a u t o c o r r e l a t i o n o f a waveform or c r o s s - c o r r e l a t i o n o f two waveforms r e s u l t s i n a p h a s e - r e g i s t r a t i o n of a l l frequency components present i n the o r i g i n a l waves. For a completely s t o c h a s t i c (random) f u n c t i o n , a u t o c o r r e l a t i o n y i e l d s a d e l t a f u n c t i o n or impulse (2,3). Consequently, c r o s s - c o r r e l a t i o n of a random f u n c t i o n with the response i t e l i c i t s produces the impulse-response f u n c t i o n . T h i s s t o c h a s t i c - e x c i t a t i o n approach has already found a p p l i c a t i o n i n chemistry. In NMR spectroscopy, Ernst (4) has found that a p p l i c a t i o n o f a s t o c h a s t i c radio-frequency p e r t u r b i n g funct i o n to sought-for n u c l e i , followed by c r o s s - c o r r e l a t i o n o f the response o f the n u c l e i to the o r i g i n a l p e r t u r b i n g f u n c t i o n , r e s u l t s i n a waveform i d e n t i c a l t o that obtainable by conventional pulse-Fourier-transform NMR techniques. However, Ernst noted that the s t o c h a s t i c e x c i t i n g f u n c t i o n could be a p p l i e d at much higher average power than could a conventional pulse without producing s a t u r a t i o n o f the n u c l e i under observation. In the microscopic world, the s t o c h a s t i c - e x c i t a t i o n approach 1
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8.
HiEFTjE
ET AL.
Measurement of Transient Chemical Events
121
to measurement takes on new meaning. On a microscopic s c a l e , a l l energies are quantized, and (above absolute zero) a l l species are i n constant, random motion. Consequently, most microscopic events are continuously and u n i n t e n t i o n a l l y perturbed i n a s t o c h a s t i c or semi-stochastic way, so that e x t e r n a l p e r t u r b a t i o n i s unnecessary. In such cases, temporal or k i n e t i c information can be obtained merely by a u t o c o r r e l a t i n g the s t o c h a s t i c a l l y induced f l u c t u a t i o n s . For example, Brownian motion leads to constant movement of a l l species i n any f l u i d medium. S c a t t e r i n g of l a s e r l i g h t from the r e s u l t i n g inhomogeneities i n r e f r a c t i v e index then enables measurement of the l o c a l i z e d f l u c t u a t i o n s . In t u r n , the measured f l u c t u a t i o n s can be a u t o c o r r e l a t e d to y i e l d such information as d i f f u s i o n r a t e s and net v e l o c i t i e s of species w i t h i n the medium (5_,6_>]_- See a l s o the chapter by B. R. Ware i n t h i s book). These measurements are the b a s i s o f techniques which are now e s t a b l i s h e d and have become known e i t h e doppler anemometry (6,8) Flygare (9) i l l u s t r a t e how u s e f u l such methods can be to chemistry. In other microscopic experiments, the f l u c t u a t i o n s of one component i n a r e a c t i n g mixture have been measured f l u o r i m e t r i c a l l y A u t o c o r r e l a t i o n of the measured fluorescence f l u c t u a t i o n s then enables the r a t e s of formation and l o s s o f that component to be measured, even though the r e a c t i o n mixture was at macroscopic equil i b r i u m . T h i s l a t t e r measurement i s the b a s i s of the new and e x c i t i n g f i e l d of fluorescence c o r r e l a t i o n spectroscopy (10,11,12). In many cases, i t i s more convenient to measure the power spectrum of measured f l u c t u a t i o n s than to determine the autocorrel a t i o n or c r o s s - c o r r e l a t i o n functions themselves (13,14). Whichever approach i s employed, the r e s u l t s are e s s e n t i a l l y the same, s i n c e the power spectrum and a u t o c o r r e l a t i o n f u n c t i o n of a waveform c o n s t i t u t e a F o u r i e r p a i r . That i s , one f u n c t i o n can be r e a d i l y obtained from the other merely by a p p l i c a t i o n of a F o u r i e r transformation. S i m i l a r l y , the c r o s s - c o r r e l a t i o n f u n c t i o n i s merely the F o u r i e r transform o f the cross-power spectrum of two waveforms. The novel approach to fluorescence l i f e t i m e measurement which i s o u t l i n e d below i s s i m i l a r i n concept to the procedures c i t e d above. Because i t , l i k e the others, i n v o l v e s the a n a l y s i s of f l u c t u a t i o n s induced i n a species of chemical i n t e r e s t , and because the f l u c t u a t i o n s are measured s p e c t r o s c o p i c a l l y , we have coined the acronym FAST ( F l u c t u a t i o n A n a l y s i s Spectroscopic Techniques) to c a t e g o r i z e them. F.A.S.T. Luminescence L i f e t i m e Measurement As implied e a r l i e r , luminescence l i f e t i m e s are o r d i n a r i l y determined by methods which, i n essence, are impulse-response f u n c t i o n determinations. A c c o r d i n g l y , i t would seem to be s t r a i g h t f o r w a r d to implement such techniques with a c o r r e l a t i o n approach. An instrument f o r such measurements i s i l l u s t r a t e d i n
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
122
NEW APPLICATIONS OF LASERS TO CHEMISTRY
Figure 2. As i l l u s t r a t e d i n Figure 2, the elements of a c o r r e l a t i o n l i f e t i m e f l u o r i m e t e r would be a randomly modulated e x c i t a t i o n source, e x c i t a t i o n and luminescence monochromators, a f a s t detect o r , and e i t h e r a c o r r e l a t i o n computer or spectrum analyzer. The s p e c i f i c a t i o n s f o r each one of these u n i t s w i l l be governed by the necessary e x c i t a t i o n and luminescence wavelengths and by the time range of the luminescence l i f e t i m e . In the simplest kind of instrument, the e x c i t a t i o n source would be a free-running flashlamp, the detector could be a f a s t p h o t o m u l t i p l i e r tube, and c o r r e l a t i o n could be c a r r i e d out using any one of a number of commercial hardware c o r r e l a t o r s . Such an approach was t e s t e d i n our laboratory f o r slowly decaying luminescence s i g n a l s and has revealed the p r a c t i c a l i t y of the t e c h n i que. In these i n i t i a l s t u d i e s , i t was found that i t i s important f o r the e x c i t a t i o n source to f l a s h randomly or f o r the duration between f l a s h e s to be considerabl process being observed of luminescence decay s i g n a l s with each other makes a l i f e t i m e determination r a t h e r d i f f i c u l t . C l e a r l y , t h i s p r e l i m i n a r y kind of device cannot be employed f o r the measurement of short luminescence l i f e t i m e s . Hardware c o r r e l a t o r s simply are not capable of s u f f i c i e n t l y r a p i d response to enable c a l c u l a t i o n of c o r r e l a t i o n functions on a nanosecond time s c a l e . To achieve nanosecond time r e s o l u t i o n with t h i s approach, somewhat d i f f e r e n t instrumentation i s r e q u i r e d . Nanosecond time r e s o l u t i o n requires that the e x c i t a t i o n source f l u c t u a t e or be modulated on a sub-nanosecond time s c a l e and at a f a i r l y large amplitude. In a d d i t i o n , f o r maximum s i g n a l to-noise r a t i o , the f l u c t u a t i o n s should occur continuously and should not be separated i n time as would the pulses from a f l a s h lamp. In p r e l i m i n a r y i n v e s t i g a t i o n s , s e v e r a l p o t e n t i a l sources were t e s t e d f o r t h i s a p p l i c a t i o n and a continuous-wave l a s e r was found to be most s u i t a b l e . In a d d i t i o n , because nanosecond-scale c o r r e l a t i o n was required, we found i t more expedient to employ spectrum a n a l y s i s than software or hardware c o r r e l a t i o n . The r e s u l t i n g instrument and i t s performance have been described i n a recent p u b l i c a t i o n (13), and w i l l only be b r i e f l y and q u a l i t a t i v e l y o u t l i n e d here. In the new instrument, l a s e r mode noise i s used as a pseudorandom fluorescence e x c i t a t i o n f u n c t i o n . Mode n o i s e i s j u s t the r a p i d f l u c t u a t i o n i n l a s e r output amplitude which r e s u l t s from "beating" (mixing) of the l a s e r o s c i l l a t i o n modes with each other. Because l a s e r modes occur at d i s c r e t e frequencies (wavelengths) (15), they produce v a r i a t i o n s i n the l a s e r ' s output which are a l s o at d i s c r e t e frequencies, as revealed by the comb-like f l u c t u a t i o n power spectrum of the l a s e r ' s output (16,17,18) . These d i s c r e t e frequency f l u c t u a t i o n s occur i n i n t e r v a l s of c/2£ (c = speed of l i g h t ; I = l a s e r c a v i t y length) up to frequencies as high as 4 GHz f o r an argon-ion l a s e r , [ i . e . , up to the Doppler width of the emission p r o f i l e of the a c t i v e medium (Ar i o n s ) ] . For a t y p i c a l
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
HiEFTjE
ET AL.
Measurement of Transient Chemical Events
System
o Inputs
-o
ο
ίο be
-o
o
Tested
-o
Outputs
Figure 1. Conceptual view of a system whose time response is electronic network, fluorescing molecule. corresponding response might then he, respectively, the RC time con stant, the chemical reaction rate, or the fluorescent lifetime.
Excitation Monochrom.
Random
•
Fluorescence Cell
Emission
Modulator
Monochromator cross-correlation < signal
PMTl High-Speed
Photodetector
Correlation Computer Spectrum Analyzer
Figure 2. Diagram of an instrument to measure luminescence lifetime using a randomly varying light source
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
124
NEW
APPLICATIONS O F LASERS T O
CHEMISTRY
rare-gas i o n l a s e r , with a 1 meter c a v i t y , c / 2 £ = 150 MHz. Essent i a l l y , the l a s e r output f l u c t u a t e s i n power at a l l these d i s c r e t e frequencies (150 MHz, 300 MHz, 450 MHz, etc.) simultaneously, thus causing f l u c t u a t i o n s i n e x c i t a t i o n o f the i l l u m i n a t e d sample at the same f r e q u e n c i e s . However, because the e x c i t e d s t a t e of a fluorophore e x h i b i t s a f i n i t e l i f e t i m e , f l u c t u a t i o n s i n the induced luminescence cannot occur at the highest frequencies present i n the v a r y i n g l a s e r output. Therefore, the power spectrum o f luminescence f l u c t u a t i o n s i s attenuated at h i g h e r f r e q u e n c i e s . From t h i s a t t e n u a t i o n , the luminescence l i f e t i m e can be c a l c u l a t e d . For example, luminescence from a fluorophore having an upper s t a t e l i f e t i m e o f 1 ns ( i . e . , a frequency response o f 1 GHz) would be able to f o l l o w the lower frequency l a s e r f l u c t u a t i o n s ( i . e . , at 150, 300, 450 MHz, etc.) but would not be able to f a i t h f u l l y follow the highest frequency f l u c t u a t i o n s h i g h e r frequencies woul luminescence v a r i a t i o n s . Mathematically, the luminescence l i f e t i m e can be found from the envelope o f the d i s c r e t e - f r e q u e n c y peaks i n the luminescence f l u c t u a t i o n power spectrum. A f t e r deconvolution, t h i s envelope i s L o r e n t z i a n , r e v e a l i n g the exponential time-domain p r o f i l e o f luminescence decay. Deconvolution i t s e l f i s s i m p l i f i e d i n t h i s approach, and merely i n v o l v e s a d i v i s i o n , s i n c e data are already i n the frequency (Fourier) domain Q2,3) . The advantages o f t h i s new approach are numerous. For one, the technique i s capable o f measuring luminescence l i f e t i m e s as short as those a c c e s s i b l e with a mode-locked l a s e r . In f a c t , the power spectrum measurement i n v o l v e d i n t h i s technique i m p l i c i t e l y c o r r e l a t e s the l a s e r f l u c t u a t i o n s and thereby emulates the l a s e r s performance when mode locked (19) . However, mode l o c k i n g i t s e l f i s not necessary, so that l a s e r o p e r a t i o n i s rendered both simpler and more r e l i a b l e . A l s o , t h i s method r e q u i r e s no large amplitude output p u l s e from the photodetector, thereby reducing the l i k e l i h o o d of s a t u r a t i o n . T h i s new method has drawbacks as w e l l . Most important o f these i s the need to perform a spectrum a n a l y s i s of the induced f l u o r e s c e n c e f l u c t u a t i o n s . This a n a l y s i s must be performed with the a i d o f a high frequency spectrum analyzer, whose cost i s subs t a n t i a l . From a p r a c t i c a l standpoint, i t would be f a r more a t t r a c t i v e to use less expensive instrumentation. One p o s s i b i l i t y would be to s t a t i o n fixed-frequency bandpass detectors (such as UHF t e l e v i s i o n tuners) at each o f the d i s c r e t e f l u c t u a t i o n f r e quencies. Of course, the exact l o c a t i o n of each o f these f r e quencies i s dependent on the p a r t i c u l a r l a s e r used, and the bandpass frequencies would have to be adjusted i f l a s e r sources were exchanged. Another l i m i t a t i o n i n the system as now c o n f i g u r e d i s the a c c e s s i b l e wavelength and time r e s o l u t i o n range. Although a r a r e gas i o n l a s e r emits only at a number of d i s c r e t e wavelengths, i t would be d e s i r a b l e to have a v a i l a b l e u l t r a v i o l e t 1
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8.
HIEFTJE ET AL.
Measurement of Transient Chemical Events
125
r a d i a t i o n or r a d i a t i o n tunable over a broad wavelength range. In a d d i t i o n , mode noise from such a system extends only t o approxi mately 4 GHz, l i m i t i n g time r e s o l u t i o n t o approximately 0.1 ns. Presumably, both these l a t t e r objections could be overcome through use o f a continuous-wave dye l a s e r . With frequency doubling, such a l a s e r would be usable over most o f the wavelength range commonly employed f o r luminescence e x c i t a t i o n . Moreover, the l a s e r should e x h i b i t mode n o i s e t o frequencies as high as 100 GHz, making de t e c t o r speed the l i m i t i n g device i n the measurement o f u l t r a - s h o r t lifetimes. Literature Cited 1. Ippen, E. P., and Shank, L. V., Opt. Commun. (1976) 18, 27. 2. Bendat, J . S., and P i e r s o l A G. "Rando Data Analysi and Measurement Procedures" NY, 1971. 3. Bracewell, R., "The F o u r i e r Transform and I t s A p p l i c a t i o n " , McGraw-Hill, New York, NY, 1965. 4. E r n s t , R. R., J. Mag. Resonance (1970) 3, 10. 5. Gabler, R., Westhead, E. W., and Ford, N. C., Biophys. J. (1974) 14, 528. 6. She, C. Y., and Wall, L. S., J. Opt. Soc. Amer. (1975) 65, 69. 7. Brown, J. C., and Pusey, P. N., J. Phys. D. (1974) 7, L31. 8. LeBlond, J., and El Badawy, E. S., Appl. Opt. (1975) 14, 902. 9. Ware, B. R., and Flygare, W. Η., Chem. Phys. L e t t . (1971) 12, 81. 10. Magde, D., E l s o n , E., Webb, W. W., Phys. Rev. L e t t . (1972) 29, 705. 11. E l s o n , E. L., Magde, D., Biopolymers (1974) 13, 1. 12. Magde, D., Elson, E. L., Webb, W. W., Biopolymers (1974) 13, 29. 13. H i e f t j e , G. M., Haugen, G. R., and Ramsey, J. Μ., Appl. Phys. L e t t . (1977) 30, 463 14. Chu, Β., "Laser Light S c a t t e r i n g " , Academic Press, New York, NY, 1974. 15. Lengyel, Β. Α., " I n t r o d u c t i o n t o Laser Physics", John Wiley and Sons, New York, NY, 1966. 16. Casperson, L. W., Opt. Commun. (1975) 13, 213. 17. Bridges, T. J., and Rigrod, W. W., IEEE J. Quantum E l e c t r o n . (1965) QE-1, 303. 18. Sedel'nikov, V. Α., S i n i c h k i n , Y. P., and Tuchkin, V. V., Opt. Spectrosc. (1971) 31, 408. 19. Weber, H. P., and Danielmeyer, H. G., Phys. Rev. A (1970) 2, 2074. RECEIVED August 7, 1978.
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9 Laser Applications in Photoelectrochemistry 1
1
S. P. PERONE , J. H. RICHARDSON, B. S. SHEPARD , J. ROSENTHAL , J. E. HARRAR, and S. M. GEORGE 1
General Chemistry Division, Lawrence Livermore, Laboratory, Livermore, CA 94550
Photoemission S t u d i e s . The U V - v i s i b l e i r r a d i a t i o n of an e l e c t r o d e / s o l u t i o n i n t e r f a c e can stimulate any of s e v e r a l i n t e r e s t i n g e l e c t r o d e processes and the e l e c t r o d e i s a e l e c t r o n s may occur, with the formation of solvated e l e c t r o n s and initiation of r e a c t i o n s with a v a i l a b l e scavengers. I f the s o l u t i o n absorbs the r a d i a t i o n and p h o t o l y s i s occurs, e l e c t r o a c t i v e p h o t o l y t i c intermediates and products may be detected at an i n d i c a t o r e l e c t r o d e by t h e i r e l e c t r o l y s i s currents under pot e n t i o s t a t i c c o n d i t i o n s . I f the e l e c t r o d e i s a semiconductor and absorbs r a d i a t i o n s u f f i c i e n t to promote e l e c t r o n s through the band gap, o x i d a t i o n or r e d u c t i o n process may be induced which would not occur i n the absence of r a d i a t i o n . I f a dye absorbed on a semiconductor e l e c t r o d e i s e x c i t e d by the i r r a d i a t i o n , the e x c i t e d state may undergo an o x i d a t i v e or r e d u c t i v e e l e c t r o n t r a n s f e r step which would not occur with the ground state s p e c i e s . These are examples of some of the more i n t e r e s t i n g r a d i a tion-induced e l e c t r o d e processes which have been studied. Much of the i n t e r e s t has been stimulated r e c e n t l y by the prospects f o r d i r e c t s o l a r - t o - e l e c t r i c a l energy or s o l a r - t o - c h e m i c a l energy conversion which might be p o s s i b l e with photoelectrochemical ce l i s . ( 1 - 3 ) The work reported here was designed to demonstrate the u t i l i t y of l a s e r sources for these photoelectrochemical s t u d i e s ; we have focused our a t t e n t i o n on those phenomena u n i quely r e l a t e d to the c h a r a c t e r i s t i c s of l a s e r i r r a d i a t i o n . In p a r t i c u l a r , we report here the r e s u l t s of our studies of l a s e r - i n d u c e d photoemission processes and l a s e r - induced p h o t o l y s i s . The former study was undertaken to i l l u s t r a t e the e f f o r t s of wavelength, source power, and i n t e n s i t y on photoem i s s i o n - r e l a t e d process; whereas the l a t t e r study was designed to i l l u s t r a t e the c a p a b i l i t i e s f o r photoelectrochemical quantum y i e l d measurements on t r a n s i e n t p h o t o l y t i c species made p o s s i b l e with a l a s e r source. 1Current address: Department of Chemistry, Purdue University, West Lafayette, IN 47907 This chapter not subject to U.S. copyright. Published 1978 American Chemical Society
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
PÉRONÉ ET AL.
Laser Applications in Photoelectrochemistry
127
Photoemission of e l e c t r o n s from mercury e l e c t r o d e s i n t o e l e c t r o l y t e s o l u t i o n s has been studied e x t e n s i v e l y i n recent years.(4-10) O c c a s i o n a l l y other metals have been used as a source o f photoemitted electrons,(11) but the dropping mercury e l e c t r o d e (DME) i s g e n e r a l l y recognized to have d e s i r a b l e chara c t e r i s t i c s . (12.) I n t e r e s t i n photo-related currents has not only been i n c h a r a c t e r i z i n g the emission process but a l s o i n studying the r e a c t i o n s o f the r e s u l t i n g hydrated e l e c t r o n s with various s u i t able scavengers.(13-16) T h e o r e t i c a l studies have a l s o been i n i t i a t e d both with respect to the k i n e t i c s o f scavenging i t s e l f (17_-18) and t r a n s i e n t e f f e c t s i n the e l e c t r o c h e m i c a l detect i o n o f the photorelated phenomena.(21) Many previous experimental studies have been c a r r i e d out with continuous or choppe steady state (DC)(22) o under p o t e n t i o s t a t i c c o n d i t i o n s . Most o f the more recent work has used pulsed xenon flashlamps. ( 5 .6 .10 .13 .14.24.25 ) Detect i o n o f photo-related phenomena was by p o t e n t i o s t a t i c ( 1 0 , 2 4 ) or coulostatic(13,14,17) monitoring of the e l e c t r o d e process. Very l i t t l e work has involved the use o f l a s e r s as the r a d i a t o n source. The few laser-induced photoelectrochemistry studies which have appeared mostly used s o l i d - s t a t e l a s e r s (ruby or neodymium) with one or two l i n e s and a slow r e p e t i t i o n rate (sometimes s i n g l e - s h o t only) . ( 13 ,26-28) A recent report(J29) used the n i t r o g e n l a s e r at 337.1 nm. The work reported here had the general o b j e c t i v e o f studying the e f f e c t s o f very intense l a s e r sources on e l e c t r o d e photoemission processes. Thus, the s p e c i f i c goals of t h i s work were t h r e e f o l d : 1) to develop appropriate i l l u m i n a t i o n and measurement instrumentation f o r both pulsed and cw l a s e r sources i n conjunction with a conventional DME assembly; 2) to determine the e f f e c t o f source c h a r a c t e r i s t i c s on e l e c t r o d e photoemission processes i n the presence o f s u i t a b l e hydrated e l e c t r o n scavengers; 3) to evaluate the general u t i l i t y o f l a s e r sources f o r photoelectrochemical s t u d i e s . In order to achieve the above goals we have used both a pulsed n i t r o g e n pumped dye l a s e r and a cw argon i o n l a s e r . The dye l a s e r i s continuously tunable from 258 to 750 nm, the output c o n s i s t i n g o f 10 nsec pulses at a r e p e t i t i o n rate o f 1 to 50 Hz and peak powers o f s e v e r a l k i l o w a t t s . The argon ion l a s e r was a cw source, u s u a l l y chopped around 1 kHz. I t was operated on one o f four f i x e d wavelengths, with output powers up to 6 W. A conventional DME polarographic assembly was i l l u m i n a t e d under c o n t r o l l e d p o t e n t i a l conditions with c a p a b i l i t i e s f o r both polarographic steady state and t r a n s i e n t (boxcar i n t e g r a tor and o s c i l l o s c o p e ) current measurement c a p a b i l i t i e s . Scavengers used i n these studies included N2O, NO3", s e v e r a l d i v a l e n t
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
NEW
128
2 +
2 +
2+
APPLICATIONS OF LASERS TO CHEMISTRY
2 +
2 +
2 +
2 +
cations (e.g., C o , F e , Mn , N i , C u , C d and P b ) , and Co(NH ) . The photo-related current was studied as a f u n c t i o n of wavelength, e l e c t r o d e p o t e n t i a l , and i n t e n s i t y f o r a l l the s c a vengers, using both sources and a l l three d e t e c t i o n c a p a b i l i ties. D e f i n i t e scavenging of e l e c t r o n s was not observed with the metal c a t i o n s , i n contrast to NO3" and N2O. A h i g h l y non l i n e a r photo-effect was observed with the high peak-powered, pulsed l a s e r system. This e f f e c t was p a r t i c u l a r l y n o t i c e a b l e with the metal c a t i o n s . To our knowledge such a pronounced e f f e c t has not been observed before. Our work i l l u s t r a t e s some of the l i m i t a t i o n s as w e l l as advantages of using l a s e r sources with e l e c t r o c h e m i c a l d e t e c t i o n of photoemission c u r r e n t s . 3 +
3
6
Experimental Laser P h o t o l y s i s - Quantum Y i e l d Studies. E l e c t r o a c t i v e species can be q u a l i t a t i v e l y and q u a n t i t a t i v e l y c h a r a c t e r i z e d by chronoamperometric measurements at a microelectrode i n the p h o t o l y s i s cell.(30-33) I f the f a r a d a i c current f o r p h o t o l y t i c species i s d i f f u s i o n - c o n t r o l l e d and uncomplicated by k i n e t i c e f f e c t s , the C o t t r e l l equation a p p l i e s :
i
= nFAD
1 / 2
C°/(ut)
1 / 2
(1)
where i is the f a r a d a i c current at time t, η i s the number od e l e c t r o n s t r a n s f e r r e d , F i s the Faraday, A i s the e f f e c t i v e e l e c t r o d e area, and D i s the d i f f u s i o n c o e f f i c i e n t of the e l e c t r o a c t i v e s p e c i e s . Thus, a p l o t of i versus 1/ t should be l i n e a r with a slope p r o p o r t i o n a l to C°, the bulk concentra t i o n of e l e c t r o a c t i v e species i n s o l u t i o n . A t h e o r e t i c a l expression has been derived(30) d e s c r i b i n g the i n i t i a l concentration of intermediate, R, produced when a pulse of l i g h t i s passed through a s o l u t i o n containing a photor e a c t i v e s p e c i e s , 0,
Cj(b) = 4Q [aC°]exp[-abC°]
(2)
o
where C R i s the concentration of intermediate as a f u n c t i o n of pathlength (b), the quantum e f f i c i e n c y (Φ), the instantaneous i n i t i a l quanta of monochromatic l i g h t per u n i t area ( Q ) , the absorption c o e f f i c i e n t of the photoreactive species (a), and the concentration of the reactant before the f l a s h (Cq) . This r e l a t i o n s h i p assumes i d e a l conditons where C Q does not change Q
In New Applications of Lasers to Chemistry; Hieftje, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
PÉRONÉ ET AL.
129
Laser Applications in Photoelectrochemistry
during the f l a s h , the l i g h t pulse i s c o l l i m a t e d and monochromat i c , and no other absorbing species are produced during the flash. Replacement i n t h i s work of the p r e v i o u s l y used xenon flashlamp with a pulsed dye l a s e r e x c i t a t i o n source extends the v a l i d i t y and, consequently, the usefulness o f t h i s expression. The pulsed l a s e r s u p p l i e s c o l l i m a t e d , monochromatic l i g h t p u l ses o f high i n t e n s i t y and very short d u r a t i o n , and, t h e r e f o r e , allows measurements to be made at s e l e c t e d wavelengths and on shorter time s c a l e s . The present study involved l a s e r f l a s h p h o t o l y s i s e x p e r i ments using the F e ( l I I ) oxalate system. Both the photochemist r y (34-J37.) and the photoelectrochemistry(38 ,
