75 Years of Chromatography (Journal of chromatography library)

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 17

75 years of chromatographya historical dialogue

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

Extraction Chromatography edited by T. Braun and G. Ghersini

Volume 3

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

Volume 4

DetecJors in Gas Chromatography by J. SevElk

Volume 5

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

Volume 6

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

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

Chromatography of Steroids by E. Heftmann

Volume 9

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

Volume 0

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

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

Affmity Chromatography by J. Turkovg

Volume 13

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

Volume 14

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

Volume 15

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

Volume 16

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

Volume 17

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

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 17

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

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

ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam - Oxford - New York 1979

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

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

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

Library of Congress Cataloging in Publication D a t a

Main entry under t i t l e :

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

QV79.C4%8

ISEN c-44t-417544

544'.92

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

78-23917

'

Elsevier Scientific Publishing Company, 1979

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

V

CONTENTS

Introduction Contributors

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

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

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

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

. . .

. .

. .

.

.

.

VII XI

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

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

G . Schomburg

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

VII

INTRODUCTION

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

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

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

VIII This statement gave us the impetus for this book.

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

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

This Page Intentionally Left Blank

XI

CONTRIBUTORS

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

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

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

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

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

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

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

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

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

Romagnosi M A , R o d , Italy.

XI11

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

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

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

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

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

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

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

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

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

1

EDWARD R. ADLARD

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

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

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

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

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

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

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

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

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

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

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

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

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

4

5

6 7

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

11

HENDRIK BOER

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

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

13

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

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

14

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

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

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

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

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

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

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

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

19

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

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21

ERIKA CREMER

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

....

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

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

.

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

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

25

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

C02,

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

26

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

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

.

27

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

28

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

START

FRONT

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

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

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

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

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

z.

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

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

31

DENIS H. DESTY

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

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

.

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

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

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

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

35

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

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

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

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

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

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

40

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

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

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

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

42

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

43

GREULT DIJKSTRA

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

44

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

45

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

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

46

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

47

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

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

48

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

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

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

49

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

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

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

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

The Historical Lesson

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

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

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53

LESLIE S. ETTRE

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

54

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

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

*

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

56

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

57

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

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

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

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60

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

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

62

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

63

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

64

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

65

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

66

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

67

PER FLODIN

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

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

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

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

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

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

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

72

Interferometer

reading

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

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

73

A* 500

700

ELUTION

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

1100 ML

VOLUME

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

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

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

75

CHARLES W. GEHRKE

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

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

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

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

j

AMINO ACID STANDARD

1

VJVq#i

TY ,o/&c R

IIILJU 80 0

5

100

120 10

140

15

160

180 20

200'w220230-lsotC 25

30

35MIN

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

79 ;

1

NOTHYDROLYZED

h

\

E/

2 -o_ 55

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

6 91

127

163

----

24 33 MIN 199 2040 Is0 -204 oc

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

80

x PROCEDURAL BLANK, HYDROLYZED

APOLLO 17. 72501.62, UNHYDROLYZED

E APOLLO 17.72501.62. HYDROLYZED

--v-(

::

long AMINO ACID STANDARD

ASP ;THRnSER ,

GLY fl +LA

I

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

.

APOLLO 17.70011.37. UNHYDROLYZED

APOLLO 17,70011.37, HYDROLYZED

2ng AMINO ACID STANDARD

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

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

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

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

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

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

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

84

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

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

85

I

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

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

1

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

om VIV cnan

A.

o.

45% V

cnm

~ V

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

FLOW

DETECTOR

EX. Y O nin EM. 480 nm

TEMP.

0

10

-BUFFER

20

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

30

A

40

50

0

MINUTES +BUFFER

!

70

80

B-4

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

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

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

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

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

87

J. CALVIN GIDDINGS

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

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

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

.

+

.

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

Year

-+

1.

Statistical theory

2.

Nonequilibrium theory

3.

Plate height & column parameters in GC

4.

Basic paper chromatography ( & TLC

5.

Eddv diffusion

6.

Programmed temperature GC

7.

Optimization and separation

&

h955 I 5 6 I57 I58

models

8. Physicochem. constants from chrom.

9. Non-Gaussian zones, kinetics I

0.

Electrophoresis

1. Misc. general

& theoretical chromatography

2.

Preparative scale GC

3,

Theory of high efficiency LC

4.

High pressure & supercritical GC

5.

Steric exclusion chromatography

6.

Field-flow fractionation (1-phase chromatography

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

l

l

(13

0

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

92

Nonrquili brium disploccmr nt,

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

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

93

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

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

94

5hr\

J

i

i

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J 4 6 Reduced Ddrtoncr. y =

e

I

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

95

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

96

I

I

-0.4 -0.2

I

0

I

I

1

I

0.2 0.4 0.6 08 X

I

I

J

1 . 0 1.2 1.4

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

97

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

-

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

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

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99

EUGEN GLUECKAUF

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

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

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

101

BREAKERSEAL GAS SAMPLE TUBE

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

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

102

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

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

103 r BUFFER GAS

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

-

d f2(c1c2)

c2

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

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

105 n

n

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

106

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