GAS CHROMATOGRAPHY

Gas chromatography: the anatomy of a ... Digital computers and the analysis of chromatographic data ... *This paper was not read at the meeting but...

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GAS CHROMATOGRAPHY IN BIOLOGY AND MEDICINE

A Ciba Foundation Symposium Edited by

RUTH PORTER

I.

& A. CHURCHILL LTD.

104 GLOUCESTER PLACE

LONDON

.

. .

1969

GAS CHROMATOGRAPHY IN BIOLOGY A N D MEDIC1NE

GAS CHROMATOGRAPHY IN BIOLOGY A N D MEDIC1NE

GAS CHROMATOGRAPHY IN BIOLOGY A N D MEDIC1NE

GAS CHROMATOGRAPHY IN BIOLOGY AND MEDICINE

A Ciba Foundation Symposium Edited by

RUTH PORTER

I.

& A. CHURCHILL LTD.

104 GLOUCESTER PLACE

LONDON

.

. .

1969

First published 1969 Containing 67 illustrations Standard Book Number 7000 1428 4 (cased) 7000 1442 X (limp)

0 1. & A. Churchill Ltd. 1969 All rights reserved. No part ofthis publication may be reproduced, stored in a retrievol system, or transmitted, in any form or by any meons, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. Printed in Great Britain

Contents P. Payne

Introduction Chairman’s opening remarks

I

A. J. P. Martin

Historical background

2

Discussion

Gas chromatography: the anatomy of a scientific revolution Lipsky, Martin, Payne, Purnell, Scott, Sjdvall, Warren

J.

S. R. Lipsky

J. H. Purnell

II 16

Design

Discussion

The main aim of chromatography: elimination of the column Brooks, Carter, Lipsky, Martin, Payne, Purnell, Scott, Sjllvall

21 31

D. W. Hill Discussion

Detectors for gas chromatography Carter, Hill, Jandk, Lipsky, Martin, Purnell, Scott, Warren

37 53

B. C. H. Warren M. G. Dalzell Discussion

The effect of water vapour on argon ionization detectors

59

Hill, Lipsky, Purnell, Scott, Warren

62

B. C. H. Warren J. E. Lovelock Discussion

The W-value detector: determination of oxygen and anaesthetic vapours in expired air A. Curry, Hill, Lipsky, Lowe, Mitchell, Payne, Purnell, Robinson, Scott, Warren

J. Janik

The gas chromatographic column as an analogue for respiratory function in the lung in man Cervenko, Hill, landk, Lipsky, Lowe, Payne, Purnell, Purves, Robinson, Scott, Sjovall

Discussion

H. L. Lowe K. Hagler

Discussion

65 69 74 79

Biological and medical applications Determination of volatile organic anaesthetics in blood, gases, tissues and lipids: partition coefficients Brooks, Cervenko, A. Curry, Geddes, Lipsky, Lowe, Payne, Purnell, Purves, Robinson, Scott

86

I04

M. J. Purves

Measurement of the gas content of blood samples using gas chromatography I I3

Discussion

Blackmore, Carter, Gray, Hill, Lipsky, Lowe, Martin, Payne, Purnell, Purves, Robinson, Scott Carter, A. Curry, Gray, Hill, Lipsky, Martin, Purnell, Scott, Sjovall

General discussion

V

I 20

I29

vi

CONTENTS

A. S. Curry

Recent developments i n the use of gas chromatography in forensic toxicology

D. J. Blackmore

The use of gas liquid chromatography in aircraft accident toxicology Blackmore, Brooks, A. Curry, S. Curry, Gray, Hill, Lipsky, Lowe, Martin, Mitchell, Payne, Purnell, Scott, SjBvall, Warren

Discussion

S. Garattini F. Narcucci

Gas chromatographic analysis of benrodiazepines*

I32 I36 145

161

E. Mussini

R. P. W. Scott

Discussion

G. B. Marson

Discussion General discussion

J. P. Payne

Trends in development Gas chromatographic and spectrometric techniques Brooks, Lipsky. Martin, Payne, Purnell, Scott, Warren

I73 181

Digital computers and the analysis of chromatographic data Hifl, Lipsky, Lowe, Marson, Martin, Mitchell, Payne, Purnell, Scott Blackmore, Brooks, Carter, A. Curry, S. Curry, Gray, Lipsky, Martin, Mitchell, Payne Purneil, Scott, Sjovall

I96

Chairman’s closing remarks

207

187.. I92

Author Index

209

Subject index

210

*This paper was not read a t the meeting but contributed for publication afterwards

Membership Symposium on Gas Chromatography in Biology and Medicine held 5th and 6th February, 1969. 1. P. Payne (Chairman) Research Department of Anaesthetics, Royal College of Surgeons of England, London

D. J. Blackmore

RAF Institute of Pathology and Tropical Medicine, Home Office Central Research Establishment, Aldermaston, Berkshire

C. J. W. Brooks

Chemistry Department, The University, Glasgow

H. V. Carter

Perkin-Elmer Ltd, Beaconsfield, Buckinghamshire

F. W. Cervenko

Department of Anaesthesiology, Queen’s University, Kingston, Ontario

A. S. Curry

Home Office Central Research Establishment, Aldermaston, Berkshire

S. H. Curry

Department of Pharmacology, The London Hospital Medical College

I. C. Geddes

Department of Anaesthesia, University of Liverpool

G. M. Gray

Lister Institute of Preventive Medicine, University of London

D. W. Hill

Research Department of Anaesthetics, Royal College of Surgeons of England, London

C. Hitchcock

Unilever Research Laboratory, Coleworth House, Sharnbrook, Bedford

J. Janik

Institute of Instrumental Analytical Chemistry, Czechoslovak Academy of Sciences, Brno

G. Levis

Department of Clinical Therapeutics, University of Athens School of Medicine

S. R. Lipsky

Section of Physical Sciences, Yale University School of Medicine, New Haven, Connecticut

H. J. Lowe

Section of Anesthesiology, University of Chicago Medical School, Chicago, Illinois

G. B. Marson

Digital Systems Division, Kent Instruments Ltd, Luton, Bedfordshire

vii

...

Vlll

MEMBERSHIP

A. J. P. Martin

Abbotsbury, Elstree, Hertfordshire, and Technological University, Eindhoven, Netherlands

F. L. Mitchell

Division of Clinical Chemistry, MRC Clinical Endocrinology Research Unit, Edinburgh*

J. H. Purnell

Department of Chemistry, University College, Singleton Park, Swansea, Wales

M. J, Purves

Department of Physiology, University of Bristol

J.

S. Robinson

Department of Anaesthetics, University of Birmingham

R. P. W. Scott

Unilever Research Laboratory, Coleworth House, Sharnbrook, Bedford

J. Sjovall

Kemiska Institutionen, Karolinska Institutet, Stockholm

B. C. H. Warren

Physics Research Division, Chemical Defence Experimental Establishment, Porton Down, Wiltshire

'Present address: Division of Clinical Chemistry, MRC Clinical Research Centre,

Harrow,Middlesex.

The Ciba Foundation The Ciba Foundation was opened in 1949 to promote international cooperation in medical and chemical research. It owes its existence to the generosity of CIBA Ltd, Basle, who, recognizing the obstacles to scientific communication created by war, man’s natural secretiveness, disciplinary divisions, academic prejudices, distance, and differences of language, decided to set up a philanthropic institution whose aim would be to overcome such barriers. London was chosen as its site for reasons dictated by the special advantages of English charitable trust law (ensuring the independence of its actions), as well as those of language and geography. The Foundation’s house at 41 Portland Place, London, has become well known to workers in many fields of science. Every year the Foundation organizes six to ten three-day symposia and three to four shorter study groups, all of which are published in book form. Many other scientific meetings are held, organized either by the Foundation or by other groups in need of a meeting place. Accommodation is also provided for scientists visiting London, whether or not they are attending a meeting in the house. The Foundation’s many activities are controlled by a small group of distinguished trustees. Within the general framework of biological science, interpreted in its broadest sense, these activities are well summed up by the motto of the Ciba Foundation: Consocient Gentes-let the peoples come together.

ix

CHAIRMAN’S OPENING REMARKS WEowe a debt of thanks to the Ciba Foundation because there are few institutions which allow the lack of formality that is achieved in the type of meeting that this Foundation runs. I know of no other place where a small group can meet so informally and yet, at the same time, get through so much work. I am delighted that all the invited members of the group, except, unfortunately, Professor Garattini, are here, and we are especially honoured that Professor Martin, who tends to avoid meetings about gas chromatography, has been able to join us. Some of you may feel that we have not chosen the members of this group as wisely as you could have done yourselves. You may be missing your friends. But because the total number of scientists who can be invited to the Ciba Foundation’s conferences is restricted to twentyfive it is possible that had they been here you might not have been. The motive behind this symposium was the need for a multidisciplinary approach to the problems of gas chromatography. My predecessor in the Chair of Anaesthetics at the Royal College of Surgeons, Professor R. F. Woolmer, was convinced of the need for such an approach to all medical and scientific problems, and the Research Department of Anaesthetics at the College of Surgeons has followed this multidisciplinary pattern since its foundation in 1957. I was brought up in the same tradition and I have done my best to maintain it. On the grounds that it is no longer possible for any one individual to acquire the detailed knowledge necessary for the continued development even of his own subject, the case for a multidisciplinary approach is overwhelming when different fields of research overlap. Many disciplines are represented at this symposium-anaesthesia, chemistry, engineering, pharmacology, physics and physiology to mention only someand with this representation we ought to be able to sort out some of the problems which face us. We want the meeting to be as informal as possible; the papers are only a minor part of the proceedings-the guiding lines for the discussion as it were-and in the next two days we shall need all the time we can find for discussion. The object of this symposium is to promote the exchange of ideas about gas chromatography and its applications. The responsibility for this exchange now rests with all of us. 1

HISTORICAL BACKGROUND? A. J. P. MARTIN Abbotsbury, Elstree, Herlfordshire, and Technological University, Eindhmen, Netherlands

I AM going to try to present a logical account of the development of my scientific ideas. When I was a schoolboy I was exceedingly interested in chemistry and read my elder sister’s university text books. I do not remember learning any chemistry at all at Bedford School, since I was always well ahead of what I was supposed to be learning in physics and chemistry. I took great interest in distillation and was particularly impressed by distillation columns. By the time I went to Cambridge I had found a number of books describing the chemical engineering side of distillation columns, and noted that at that time (about 1930) industrial research on the preparation of good columns was much in advance of laboratory research. Eighty-plate columns turning out many tons of alcohol or petroleum were available in industry, but distillation systems in laboratories contained, at best, only a few tens of plates. At Cambridge University I became interested in countercurrent separations and plate theory, and after graduation I started some research in the Nutritional Laboratory at Cambridge. I was interested in vitamins, and decided to look for Vitamin E, unknown at that time. Various members of the laboratory were concerned with the carotenes and in 1933 Dr. A. Winterstein from E. Kuhn’s laboratory in Heidelberg visited us and demonstrated a chromatogram of a crude carotene solution on a chalk column; the carotene separated appropriately into bands of various colours. I was fascinated to see the relationship between the chromatogram and distillation columns and to realize that the processes involved in the separation of the carotenes and of volatile substances by distillation column were similar ;there was relative movement of the two phases and it was their interaction at many points that gave rise to good separations. I continued my work with Vitamin E and

This presentation was contributed by Professor Martin at the end of the symposium at the special request of the Chairman and all the members of the group. The Editor also wishes to thank Dr. A. T. James, Unilever Research Laboratories, for his help in the preparation of this material for publication. 2

HISTORICAL BACKGROUND

3

Dr. T. Moore and I started separating carotene by distribution between two solvents using separating funnels. I was sufliciently mathematically inclined to work out the extent of separation that can be obtained in this way, and was appalled to find how small this was with a single extraction. So I set up chains of separating funnels, moving upper and lower layers countercurrently, but found that even when one has such a small number as, say, six funnels just shaking and separating the layers becomes a full-time job. I had always been interested in engineering processes and so I started to devise machines to do the countercurrent extractions. The first machine was designed for the first stage in the separation of Vitamin E;vegetable oils were saponified and the soaps extracted with ether. This was a tedious, smellyjob. So I put one twenty-litre aspirator bottle on the floor outside the laboratory and another on the flat roof (the laboratory was a single-storey building). I filled the top bottle with soaps and the bottom bottle with ether and joined the bottom of the top bottle to the top of the bottom bottle with half-inchbore tubing. By this means the ether and soaps changed places over a period of hours (over night, in fact). I found that ten feet of tubing gave about eight theoretical plates and I obtained very efficient extraction in this way. This method was satisfactory for extracting a particular substance from one liquid to another but much more was needed to separate two or more substances of closely similar partition coefficients. It was by no means obvious how one could duplicate the performance of a batch distillation column. Devising a still to evaporate the liquid leaving the column, and continuously dissolving the residue in the other liquid phase was not easy. Ican stillremember the delight of realizing (while walking home to lunch) that all that was necessary was to inject the substance to be separated into the centre of the column and fix the ratio of the flow rates of the liquids so that they equalled the reciprocal of the partition coefficient. The liquids flowing in at the ends of the column then carried the wanted substance back to the centre of the column and allowed it to escape only very slowly. But other substances, with higher or lower partition coefficients, left more or less rapidly at one end or the other. It was not worth-while trying to make an apparatus with less than about two hundred theoretical plates, and so I amplified the type of apparatus I had used for the ether extraction of the soaps. Forty-five half-inch tubes, each about five foot long, were stacked vertically in a rack. A pair of narrow tubes ran up between the top of one tube and the bottom of the succeeding tube and this was repeated for the whole series. When a pulse of liquid was pushed through this apparatus the heaviest liquid, which had collected at the bottom of the tube, was forced to the top of the next tube in the series,

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A. J . P. M A R T I N

and a ball valve prevented the liquid from dropping back. Similarly the lightest liquid, which had collected at the top of the tube, was pumped down to the bottom of the next tube (on the reverse stroke) and so on. These successive strokes produced a circulation between the bottom of one tube and the top of the next and made available, in effect, a continuous 200-foot column for separation purposes. The 90 ball valves rattling on their seats made a noise like the sea on shingle! I used cyclohexane and methanol, or various petroleum ethers with methanol and water mixtures, as solvents. Small drops or saucers, about one millimetre in diameter filled the tubes when the apparatus was working. The machine was completed with diaphragm pumps (the diaphragms were protected from the solvents by mercury) which pumped the liquid through the tubes and from the top and bottom of a single external reservoir at any desired rate ;an evaporation system collected the separated substances as they flowed from the ends of the column and returned the solvents to the reservoir. It was a considerable effort to make this machine and when it was finished it was a month before I could summon the courage to try it out. But apart from some fires and difficulties when tubes broke, the machine did ultimately work. I was able to separate Vitamin E into several obviously different and distinct fractions, for the first time. I have always had difficulty in writing up my results and I never published this work (although it does appear in my Ph.D. thesis) or pursued it further, but it left me with a profound interest in countercurrent problems. In 1937 my chief, Sir Charles Martin, introduced me to R. L. M. Synge who was working on a scholarship from the International Wool Secretariat at the Biochemical Laboratory at Cambridge. Synge was trying to improve the methods for the analysis of proteins. He had measured the partition coefficients of acetylamino acids between chloroform and water and thought that a separation method could be based on this sort of method. But his technique with separating funnels was not good enough. Sir Charles suggested that my apparatus be used. But chloroform and water were not suitable phases for it, so we designed another completely different machine in which these two compounds could be used. This was made in Cambridge for Synge to my design, but it didn’t work, and I took it and Synge to the Wool Industries Research Association, Leeds, where I had moved in 1938. We were eventually successful in using this machine to separate, and measure fairly accurately, the monoamino, monocarboxylic acids in wool. It was a fiendish piece of apparatus, we had to sit by it for a week for one separation; it had 39 theoretical plates and filled the room with chloroform vapour. We used to watch it in 4-hour shifts. We had constantly to adjust small silver baffles

HISTORICAL BACKGROUND

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to keep the apparatus working properly. One of the effects of 4 hours of chloroform intoxication was that when our partner arrived to take the next shift he was invariably sworn at by the one who had been watching the machine. Another curious effect of the chloroform was that when I went into the fresh air, it smelt peculiar. This was my first experience of the interesting phenomenon of negative smell and may have been partly responsible for my current interest in the physiology of the sensation of smell. I continued designing new machines that I hoped would be more satisfactory, but although I worked out some dozens of ideas none of them produced a machine that was sufficiently cheap and easy to seem worth making. In 1940it occurred to me that the crux of the problem was that we were trying to move two liquids in opposite directions simultaneously. Equilibrium had to be established rapidly or the experiment took far too long, but this meant converting the liquids to very fine droplets and if the droplets were too small they would not settle out or move in the required direction within any reasonable period of time. This meant that the machine was bound to be a compromise unless I could either introduce centrifugal force to speed up the movement of the droplets or think of a completely different system. Then I suddenly realized that it was not necessary to move both the liquids; if I just moved one of them the required conditions were fulfilled. I was able to devise a suitable apparatus the very next day, and a modification of this eventually became the partition chromatograph with which we are now familiar. Synge and I took silica gel intended as a drying agent from a balance case, ground it up, sieved it and added water to it. We found that we could add almost its own weight of water to the gel before it became noticeably wet. We put this mixture of silica gel and water into a column, put the acetylamino acids on to the top and poured chloroform down the column. We wondered how we should know where the amino acids were in the column and when to expect them to emerge at the bottom of the tube. By the end of the first day there was no sign of them. To h d out what was happening in the column we added methyl orange to the liquid on the silica gel and thus were able to see the acetylamino acids passing down the column as a red band. One foot of tubing in this apparatus could do substantially better separations than all the machinery we had constructed until then. Normal chloroform contains about 1 per cent of ethyl alcohol as a stabilizer. The first experiment we did, with chloroform straight out of a bottle in the laboratory, gave the results we expected, and we separated acetylalanine and acetylleucine. We next used carefully distilled chloroform and were surprised to find that the amino acids did not move from the top of the

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P. MARTIN

column. The reason for this, of course, was increased absorption due to the absence of ethyl alcohol. When we added alcohol to the chloroform our bands could move down the column again. But it was difficult to produce a satisfactory colour change; we needed large amounts of acids with our original silica1 gel-methyl orange system. So we experimented with different ways of making precipitated silica, and eventually developed a process of stirring hydrochloric acid into sodium silicate. This process reliably produced material that behaved as we wished in the columns. But this work was more magic than science; we never understood in detail what we were doing. Later we changed the indicator; at one time we used pelargonidin which we extracted ourselves from various flowers. In spite of all our efforts we could separate only the monoamino, monocarboxylic acids. We could not make the system work for the dicarboxylic or basic amino acids. So we looked for materials other than silica to hold the water, and our first choice was paper. I had seen paper chromatograms of dyes and was familiar with the uptake of water by cellulose, so paper was an obvious choice. Dr. A. H. Gordon, who was now working with us at Leeds, looked through Beilstein’s Handbuch der Organischen Chemie to find a colour reaction that would reveal our amino acids on the paper ; he found ninhydrin which proved admirable for our purpose. Our first paper chromatograms were circles of paper in a Petri dish containing water and water-saturated butanol fed by capillarity to the centre by a tail on which a drop of amino acid solution had been placed. When the butanol reached the edge, the paper was dried and sprayed with ninhydrin in dry butanol. Later, we used strips of paper in test-tubes and more suitable containers-boxes in which the air was kept saturated with water-with troughs containing the mobile solvent into which the tops of the strips could dip. Several boxes were needed since it was characteristic of the method that though it was not particularly quick, very little work was needed to run many strips simultaneously. An important step was running the chromatogram in two dimensions. The first solvent spread the amino acids in a line near one end of the paper from a spot near the corner; then, after drying, we turned the paper through a right angle and spread the line of spots into a two dimensional pattern by using a different solvent. (See Consden, Gordon and Martin, 1944.) Our next problem was to deal with the curious fact that in some, but not other, solvents the purple amino acid spots had a pink “beard” underneath them; and as they ran further down the paper the purple colour showed less and the pink more. The purple-coloured spots of leucine and phenylalanine, for example, had almost vanished before they got to the bottom of the paper,

HISTORICAL BACKOROUND

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leaving only a faint pink blob. This unsatisfactory colour change was particularly marked with papers on which the chromatogram had been run in two directions. We could not understand the reason for this. We first tried running in one direction in phenol and in the other in collidine, which can distinguish between the acidic, basic and neutral compounds. (The first two-directional separation that I did was with electrophoresis in one direction in an acetate buffer and paper chromatography in the other. The tract for the electrophoresis was isolated from the rest of the column by saturating the paper with paraffin wax on either side. But chromatography turned out to be more satisfactory than electrophoresis at that time, so I did not work with electrophoresis again until 1944.) Eventually we found the cause of these pink beards. Phenol was used as one of the solvents and when the separation was run in an atmosphere of ammonia so as to increase the pH, the paper became covered with black spots. We identified the cause of these spots as copper from the fans used for drying the papers in the laboratory; these fans had a badly sparking commutator which filled the room with copper. The large amounts of copper in the Leeds atmosphere also contributed to the copper on our papers. We finally discovered that the pink beards were caused by a copper complex of the amino acid that formed on the paper. The black colouration, due to the catalytic oxidation of phenol by copper in the presence of ammonia, indicated that copper was present. The formation of these copper complexes could be suppressed by including a complexing agent for copper-cyanide for example-in the coal gas we put into the atmosphere in the box. This technique was gradually developed into the paper chromatographic systems that are used today for the separation of amino acids. We were pleased to find that our system worked equally well for peptides and surprised to find that it could separate almost every other group of compounds it was tried on-carbohydrates (Partridge, 1946) and flower petal anthocyanin (Bate-Smith, 1948a, b) are examples. F. H. Pollard (at Bristol) worked with metals and found that they also could be separated by paper chromatography. Synge and I were busy with the amino acids and peptides, but we were visited by many scientists who were interested in our technique. They came and looked at the paper chromatograms and then went away and used paper chromatography for their own separations. Paper chromatography was amazingly applicable to the separation of widely different groups of chemicals. This work was eventually published in 1941 (Martin and Synge, 1941). In this paper we noted that the mobile phase could just as well be a gas as a liquid. We also predicted that, if the stationary phase were a liquid, very

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refined separations of various kinds of compounds would be possible. Although this paper was widely read by chemists in the petroleum industry, no one thought this prediction worth testing experimentally until, nine years later, in 1950, Dr. A. T. James and I started to work on gas liquid chromatography (James and Martin, 1951,1952,1956). Synge and I, in our first paper on partition chromatography (Martin and Synge, 1941), had evolved a theory relating the speed of the zones to the partition coefficient. Further, by introducing the concept of the theoretical plate for chromatograms, a prediction could be made about the shape of the zones and their rate of broadening. Later, after work with peptide paper chromatograms, I found it possible, by assuming that the free energy of transfer of a compound from one phase to another was an additive function of the free energies of individual atoms or groups of atoms, to forecast with reasonable accuracy the partition coefficient and chromatographic behaviour of peptides and many other substances. In 1948 I moved for a short time to the Lister Institute of Preventive Medicine in London and then to the National Institute for Medical Research, Mill Hill, where Tony James later joined me (he had previously been working with Synge). James and I tried to separate our materials using crystallization on a column-what is now known as zone-refining. This project looked hopeless for a few months, and James became more and more discouraged; we could do much better with a couple of beakers than with all the complicated apparatus we had constructed. So (to improve James’ morale) I suggested that we study gas chromatography; I was sure this would work. Professor J. Popjak had asked me for a more refined method than paper chromatography for separating fatty acids and I thought that gas chromatography might be able to do this. So we spent our first week waiting for the bands to come out of a gas chromatograph: in fact, they had all come out in the first few seconds. We used quarter-inch-bore glass tubing, about 15 inches long, packed with Celite (which had been found to be the most convenient material to use with liquid-liquid columns). We passed nitrogen in at one end of the column, the other end of which was provided with a capillary that dipped into a test-tube containing indicator solution. A small conical flask, instead of a burette, held the titrant. The flask had a doubly bored stopper, one hole carrying a tube that passed from the bottom of the flask to a jet just above the level of the liquid in the test-tube, while the other hole had a piece of valve rubber attached that could be milked between finger and thumb to express a drop of titrant from the jet. James sat with a stop-watch and a piece of graph paper and timed and plotted the drops while I watched the test-tube and put in a drop of titrant whenever the colour of the indicator changed. Plotting the number of the

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drops against time yielded a series of steps. The height of the steps denoted the quantity of acid emerging, and their position on the time axis showed the retention time. We first separated the methylamines, since they would run at room temperature. Later, using a steam jacket for the column, we separated the first members of the fatty acids series. Initially we used a fatty, oily material, but this gave very distorted bands. I had enough experience with chromatography by this time to realize that non-linear absorption creates tailing. But on these plots we had the reverse of tailing-a long front and a sharp tailwhich we eventually realized was due to the association of the fatty acid to dimers; in other words, dimerization was a considerable problem to us for six months or more. By adding a soluble acid (such as stearic acid) in excess to the stationary-phase liquid, we were able to sort out this difficulty and obtain reasonably shaped bands. This technique worked very well for fats and oils and equally well for amines. We obtained our first useful results six weeks after starting the experiment. This was the beginning of gas chromatography for us. The details still had to be worked out, but it is really astonishing how closely similar this first column was to many columns still in use today. We wanted to illustrate the technique by using it to separate some natural mixtures, so we tried to identify the amine responsible for the fishy smell of stinking goose-foot (Chenopodium vulvarium). We found trimethylamine in this plant and were able to separate the three methylamines and ammonia quite readily from it. We next made an automatic titrating machine. This was a rather HeathRobinson arrangement. The titration was recorded by using the eye to detect colour changes in the indicator and the whole thing was operated by manual drive. This was a most demanding machine; if one looked away for a second a kink in the curve appeared! We next incorporated a motor with a photocell, to drive the machine automatically. In 1952 Dr. R. P. W. Scott, from the Research Laboratories of Imperial Chemical Industries Ltd, consulted James and me about the separation of hydrocarbons. We suggested using a thermal conductivity cell because S . Claesson (1952) had already described displacement of gas chromatography on charcoal, using a conductivity cell. In 1953, Dr. N. H. Ray told us about his results with the thermal conductivity cell (Ray, 1954), but his method was not completely satisfactory for us. I tried to devise a better method and developed the gas density balance (James and Martin, 1956), a good detector in its day. That is really my only contribution to gas chromatography; since 1955 I have worked in other areas although I am now again working on electrophoresis. Tony James and I studied enough different systems to show the kinds of relationships that can occur in liquid chromatography, that is, we

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found that there is a log relationship between retention volumes and the numbers of carbon atoms in a molecule and we probably laid the theoretical foundations for gas liquid chromatography reasonably well at that time. Partition chromatography has developed much further than Synge and I originally expected, perhaps most surprisingly in connexion with the quantity of material needed for analysis. Accepted methods of amino acid analysis before we began our work required half a kilogramme of protein and about six months’ work for a monoamino, monocarboxylic analysis. The silica partition columns require a few milligrammes and paper chromatographs only a few microgrammes of protein. Now gas chromatographs with modern detectors can work with nanogramme quantities; these methods are indeed the most sensitive that are available so far for the analysis of many substances. Thus, within a thirty-year period, the quantity of sample needed has been reduced by a factor of 10l2. I am hopeful that methods I hope to work on, either myself or through others, will reduce this quantity by a further 103-106without loss of accuracy. REFERENCES BATE-SMITH, E. C. (1948~).Nature, Lond., 161, 153. BATE-SMITH,E. C. (19486). Nature, Lond., 161, 835-836. CLAESSON, S . (1952). Ark. Kemi. Miner. Geol., 23A, 1. CONSDEN, R., GORDON, A. H., and MARTIN, A. J. P. (1944). Biochem. J., 38, 224. JAMES,A. T., and MARTIN,A. J. P. (1951). Biochem. J., 48, PVII (abstract). JAMES,A. T., and MARTIN,A. J. P. (1952). Biochem. J., 50, 679-690. JAMES,A. T., and MARTIN,A. J. P. (1956). Biochem. J., 63, 138-143. MARTIN, A. J. P., and SYNGE,R. L. M. (1941). Biochem. J., 35,1358. PARTRDGE,S . M . (1946). Nature, Lond., 158, 270-271. Fhy, N.H. (1954). J. appl. Chem., 4, 21, 82.

GAS CHROMATOGRAPHY: THE ANATOMY OF A SCIENTIFIC REVOLUTION S. R. LIPSKY Section of Physical Sciences, Yale University School of Medicine, New Haven, Connecticut

INthe preface to the first edition of one of the earliest books on chromatography, Principles and Practice of Chromatography (Zechmeister and Cholnoky, 1943) there is written: “Every scientific advance is an advance in method.” It is also stated in this prefcice that “the invention of a new specialized laboratory procedure brings about rapid conquests in new fields of science and technology, finally it exhausts itself and is replaced by a still more practical method. The method of chromatographic adsorption invented by the talented Russian botanist, Professor M. Tswett makes possible spatial separation of components of a mixture. It is just now at the beginning of a bullish development: it offers a simple experimental procedure to the investigator especially in the fields of both pure and applied organic chemistry, of biochemistry and of physiology.” Undoubtedly, today, much of the content of this statement would be appropriate to the most powerful technique ever devised-gas chromatography. One could dwell at great length on all the developments that have contributed to make this so. Moreover it would be difficult to think of any scientific discipline which has not been affected by this discovery. The unique feature of the method that made repeated breakthroughs possible was the fact that the sample remained in the gas phase and the resistance to mass transfer in the mobile phase was relatively small. Since the original contributions of Professor Martin (Martin and Porter, 1951 ;James and Martin, 1951, 1952) we have learned much about the transfer processes that occur in gases, and much of our present theory and column and detector technology have evolved with remarkable speed because of this. With the maturing of the field of gas chromatography, and the inevitable diminution of the number of solid scientific advances in this sphere with the passage of time, a few theoreticians and experimentalists continued to look 11

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for new horizons. Naturally they turned their energies to an area that had been with us since the beginning of this century-liquid chromatography. The problems were easily defined, but their solutions have proved more elusive. In essence the main problem was this: Could those molecules or even their derivatives which are not readily analysed by gas chromatography (compounds with high molecular weights or the highly polar materials that do not possess sufficient vapour pressure to be volatilized without thermal rearrangement or degradation) be separated in the liquid phase by techniques possessing the same versatility, speed, resolution and sensitivity that we have come to expect in gas-phase analysis? If this were so, it would bring about a magnificent era characterized by challenges of unparalleled magnitude. And it would profoundly influence a large number of different scientific disciplines. For example, in biochemical research, the consequences would be revolutionary if we could rapidly separate and detect picogramme quantities of all the differentforms of insulin, each having a slightly different amino acid sequence and each easily synthesized by the solid-phase method of Merrifield (1963). This would be a daring step forward for the scientifically oriented physician. He would at last be in a position to precisely correlate the relationship between chemical structure and physiological function of the small active peptides and proteins. Professor Howard Purnell wants to take steps to eliminate the column from gas chromatography and perhaps introduce a new form of spectrometry. I would like, via the concept of a different sort of column, to travel in a rather similar direction. 1 hope that these two routes may lead to the same desired goal-the easy and rapid separation and detection of picogramme quantities of all chemicals-without meeting too many insoluble problems on the way. Recently, scientists were awed by the synthesis of ribonuclease, one of the most important enzymes known to man, by the Rockefeller (Gutte and Merrifield, 1969) and Merck (Denkewalter et al., 1969; Strachan et al., 1969) groups in the US. This enzyme, made up of 124 amino acids, controls the composition and distribution of vital organic compounds in cells. Now that the barriers to synthesis have finally been overcome, scientists will eventually be able to make an infinite variety of ribonucleases containing a full range of differences (from subtle to profound) in amino acid sequences. Each ribonuclease thus produced will need purification and precise structural identification before being assessed for mysiological function in a variety of systems. The solution of this problem is one of the most important challenges of our time. Those of us with long experience in gas chromatography, where