The Development of Chromatography - Analytical Chemistry (ACS

The Development of Chromatography ' T ' H E YEARS 1971-72 represent three important anniversaries related to the various chromatographic techniques: the 100-year anniversary of the birth of Tswett, the inventor of adsorption chromatography; the 30-ycar anniversary of the development of partition chromatography; and the 20-year anniversary of the first paper on gas-liquid partition chromatography. The purpose of this report is, on the occasion of the Seventh International Symposium on the Advances in Chromatography, to note

LESLIE S. ETTRE Encyclopedia of Industrial Chemical Analysis John Wiley & Sons, Inc., 605 Third Ave., New York, N.Y. 10016

the importance of these anniversaries and summarize the results of the pioneers that led to the techniques used today. The development of chromatographic techniques alone would assure a permanent place for the pioneers—M. S. Tswett, A. J. P. Martin, R. L. M. Synge, and A. T. James—in the history of chemistry. However, their merit is more than that; they not only described new techniques but also properly interpreted them, summarized the theoretical basis of the techniques, and showed the possibilities of practical

applications. Thus, their work is an example of how scientific work should be carried out. Twsett, Martin, Synge, and James represent the starting points in a line of distinguished scientists, all of whom contributed greatly to the advancement of chromatography. It is impossible to deal in this report with their activities; also, we cannot deal here with the development of ion exchange, thinlayer, and gas adsorption chromatographic techniques, each of which probably would merit a report in itself. However, it should be em-

REPORT FOR ANALYTICAL CHEMISTS

On the occasion of the Seventh International Symposium on Advances in Chromatography, this Report deals with the importance of the work of Tswett and the circumstances which led to the development of liquid-liquid and gas-liquid partition chromatography

phasized that the pioneers only represent the start, and without the achievements of many known and unknown chemists and scientists, we would not be where we are today. Adsorption Chromatography: The Life and Work of Tswett

For almost every invention, one can find persons who worked earlier and more or less carried out similar investigations but who are still not considered as the inventors of given techniques, processes, or machinery. Usually, the person recognized as the real inventor not only utilized or

described a phenomenon but also could interpret it and apply it knowingly for some purpose. The situation is not different in chromatography. Tswett certainly had his forerunners who described separation obtained by selective retardation on a column containing a solid substance. The most important person in this field was David Talbot Day (1859-1925), who was connected with the U.S. Geological Survey for 28 years and served as the director of its Division of Mineral Resources for 21 years. He demonstrated in 1897 that when

crude oil fractions are pressed through pulverized Fuller's earth, a certain fractionation takes place and, in the forthcoming years, investigated this phenomenon in detail. He reported on his results in 1900 at the First International Petroleum Congress in Paris and then three years later at the 43rd meeting of the U.S. Geological Society in Washington, D.C. (For details on the activities of Day and his collaborators and their publications, see ref. 1 and 2.) I t should be noted, however, that, although Day recognized the analytical potential

ANALYTICAL CHEMISTRY, VOL. 43, NO. 14, DECEMBER 1971

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Report for Analytical Chemists

of the process investigated, he and his co-workers interpreted incor rectly the physicochemical basis of the separation, calling it a capillary diffusion process. This is mainly the reason why today, Tswett and not Day is recognized as the inven tor of adsorption chromatography. Michael S. Tswett's life and ac tivities are fairly well documented (3—6) although some of the state ments in these summaries are con tradictory. His life was a fasci nating one, and a typical example of the troubled life encountered la ter by so many other European sci entists who, due to the events be yond their control, had to settle at places far from their homes or find refuge after losing everything. His scientific work shows the activity of one of the clearest and most conclu sive minds. Michael S. Tswett was born on May 14, 1872, in Asti, Piemonte, Italy, as the son of Simeon Tswett, a Russian subject, and his wife Maria Dorozza, an Italian. He grew up in Switzerland, studied there, and graduated with a P h D ; in 1896, he moved to Russia and, in January 1901, after spending a few years in minor jobs, he found a per manent position at the University of Warsaw (Poland was then part of Russia). He successively served as assistant, associate, and full pro fessor at the University, the School of Veterinary Medicine, and the In stitute of Technology of Warsaw until World War I interrupted his life. In 1915, he left Warsaw with the Institute of Technology which was evacuated to Nishi Novgorod (today: Gorkiy) before the ad vancing German troops. In 1917 he became ill and spent some time in the Caucasus recuperating, then ac cepted the chair of botany at the University of Jurjeff. [This was then the name of the city ; its Ger man name is Dorpat. After World War I, this area became Estonia, and the city's name was changed from the Russian Jurjeff to the Estonian name Tartu. When, in 1940, Estonia again became part of Russia (the Soviet Union), the Es tonian name was kept.] In the fall of 1918, the Univer sity was evacuated to Voronezh be fore the advancing German troops; he first decided to stay but, at the

last minute, followed the Univer sity group. His illness at that time was already advanced, and he died in Voronezh on June 26, 1919, prob ably of heart disease. He was buried in the cemetery around a monastery which (and this is the last intervention of history in Tswett's life) was destroyed in World War II during the fighting with the German troops, and thus, his grave cannot be located anymore. In practically his whole working life, Tswett dealt with investiga tions related to chlorophyll. To ward the end of the 19th century, many scientists showed interest in the pigments occurring in the leaves of plants, but there was no real way to separate them from each other and to check their identity in differ ent plants. Since these substances are very labile, one could not be sure whether material obtained through chemical manipulation re ally corresponded to the form exist ing in the living plant. Tswett's approach was different; he was looking for a physical method which would permit the separation of these pigments from each other and from closely re lated compounds that others felt to represent the same substances. For he was convinced that chloro phyll, as isolated by other re searchers, was not a single sub stance. In his work, he systematically checked a large number of solvents capable of extracting the pigments from vegetable matter, and more than 100 solid substances capable of selective retardation of the indi vidual pigments through adsorp tion, and he also deducted a num ber of important rules for the ad sorption phenomenon. His first paper in which his pre liminary work was summarized was presented on March 21, 1903, be fore the Biological Section of the Warsaw Society of Natural Sci ences and published in Russian in the proceedings of the Society (7) ; it is also available in English trans lation (4). The title of the paper is "On a New Category of Adsorp tion Phenomena and Their Applica tion to Biochemical Analysis," and in it, he, in essence, describes chro matography without yet naming it as such. Three years later, in his

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two remarkable papers entitled "Physico-Chemical Studies of Chlo rophyll. The Adsorptions" (H), and "Adsorption Analysis and the Chromatographic Method. Appli cation in the Chemistry of Chloro phyll" (9) published in the journal of the German Botanical Society, he very clearly defined the technique. It is worthwhile to quote from his first paper (8a) : "When a petroleum ether solu tion is filtered through a column of an adsorbent (I use mainly calcium carbonate which is tamped firmly into a narrow glass tube), then the pigments are resolved, according to the ad sorption sequence, from top to bottom, into various colored zones, since the more strongly ad sorbed pigments displace the more weakly adsorbed ones, and force them farther downward. This separation becomes practi cally complete when, after the pigment solution has flowed through, a stream of pure solvent is passed through the adsorbing column. Like light rays in the spectrum, the different compo nents of a pigment mixture, obey ing a law, are resolved on the cal cium carbonate column and then can be qualitatively and quanti tatively determined. I call such a preparation a chromatogram and the corresponding method the chromatographic method." It is generally assumed that the word "chromatography" has its ori gin in the Greek words chroma (color), and graphe (writing). However, already Tswett (8b) em phasized that: "It is self-evident that the ad sorption phenomena described are not restricted to the chlorophyll pigments, and one must assume that all kinds of colored and colorless chemical compounds are subject to the same laws." Actually, one may question whether Tswett really meant "color writing" when coming the name chromatography (10). The inter esting fact is that the surname of

Tswett, in Russian, is identical with the Russian word for color (I[ BET) and, as expressed by Purnell (11), "It would be nice to think that Tswett, whose name, in Russian, means color, took advantage of the opportunity to indulge his sense of humour." In his later papers, Tswett de veloped even further his technique —the separation of substances by retardation through selective ad sorption. Tswett did not invent adsorp tion; this had been described well before him and also explained from the theoretical point of view (e.g., in the books of W. Ostwald from 1891-95). Also, scientists before him had separated plant pigments by selective solution. Tswett's merits are in the generalization of the technique as an analytical method and in the detailed investi gations of the role of various adsor bents and solvents. As pointed out by Zechmeister (1), "Tswett's achievement is superior to Day's in two respects. First, he recognized and correctly in terpreted chromatographic pro cesses; and second, he devised a useful laboratory method that in cludes as an important feature the development of chromatograms by pure solvents. The dis tances between the individual zones are thus increased and com plete resolutions advised within minutes." Although Tswett published his most important papers in German journals (German was then the gen eral language of chemistry), his work and the importance of the chromatographic technique were probably not understood immedi ately. It is interesting to note that in 1922 in the U.S., Palmer (12) re ported on a number of chromato graphic experiments, giving credit to Tswett, but his work went largely unnoticed. Only 25 years after the publication of Tswett's two basic papers was his work taken out of oblivion by Kuhn et al. (13, 14) who applied it successfully to the separation of carotene and egg yolk xanthophyll into their isomeric compounds. From there on, the de velopment of adsorption chroma tography was straightforward.

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Partition Chromatography

It is rare in the history of science that somebody can claim a number of major inventions. One of those men is A. J. P. Martin. Thirty years ago, together with Synge, he invented partition chromatography and demonstrated its use with a liquid carrier (15). Three years later, together with Consden and Gordon, he described paper chromatography as a simple variant of liquid partition chromatography (16). Then, 20 years ago, in cooperation with James, he showed the validity of their prediction in the first paper on partition chromatography that a gas could be used as well as the carrier instead of a liquid (17). With this publication, the unparalleled growth of gas-liquid partition chromatography began. Today, one could not even imagine chemistry and biochemistry without partition chromatography, and its importance is best demonstrated by the fact that Martin and Synge received the 1952 Nobel prize in chemistry for their work, thus joining the few scientists who have received the Nobel prize for a development in analytical chemistry. The history of the development of the three techniques is fascinating and is described in detail by Martin (18) and James (19). As Martin explained, he early became interested in distillation columns. In 1931, when Winterstein (who, two years earlier with Kuhn and Lederer, brought back adsorption chromatography from oblivion) demonstrated at Cambridge University the separation of carotene on a chalk column, Martin realized that the processes involved in the separation are similar in both techniques. This early thinking resulted later in the expression of the efficiency of chromatographic columns by using terms established in distillation theory (HETP, number of theoretical plates). At Cambridge University, when working on the separation of carotenes, Martin developed a very complicated countercurrent extraction apparatus and continued to utilize this technique for the separation of amino acids in wool when, in 1938, he moved to the Wool Industries' Research Association where Synge became his collaborator.


However, the whole system was ex tremely complicated and difficult to operate, and therefore, he tried to develop some other technique which would do the job. As described by Martin (18) : "In 1940, it occurred to me that the crux of the problem was that we were trying to work two liq uids in opposite directions simul taneously . . . Then I suddenly realized that it was. not necessary to move both the liquids; if I just moved one of them, the re quired conditions were fulfilled. I was able to devise a suitable ap paratus the very next day, and a modification of this eventually became the partition chromatograph with which we are now familiar." In their early work, chloroform containing a small amount of alco hol was used as the mobile phase, water as the stationary phase, and silica gel as the support; they could separate the monoamino monocarboxylic acids, and, according to Martin, "One foot of tubing in this apparatus could do substantially better separations than all the ma chinery we had constructed until then." In their paper, Martin and Synge (15) emphasized that, by the selec tion of suitable mobile phase—sta tionary phase combinations, the technique can be used for many other separations, and they pre dicted that: "The mobile phase need not be a liquid but may be a vapour. We show below that the efficiency of contact between the phases (theo retical plates per unit length of column) is far greater in the chromatogram than in ordinary distillation or extraction columns. Very refined separation of vola tile substances should therefore be possible in a column in which permanent gas is made to flow over gel impregnated with a non volatile solvent in which the sub stances to be separated approxi mately obey Raoult's law." It is interesting to note that, al though this prediction clearly and unequivocally predicted the possi bility of gas-liquid partition chro matography, nobody picked it up,

and it took 10 years until Martin, then with A. T. James, proved its great potential.

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Paper Chromatography

Liquid partition chromatography, as described originally by Martin and Synge, had superior separation power. However, in their original work, using water as the stationary phase and silica gel as the support, they were unable to separate the dicarboxylic and basic amino acids. They realized that the problem lay in the adsorptive power of silica gel and thus, were looking for some other support. As Martin ex plained, he had seen a "paper chromatogram" of dyes before and thus their first choice was paper. First, they used circles of papers in a Petri dish containing water ; a drop of the amino acid solution was placed in the center of the paper, and water-saturated butanol was fed to the center of the paper; when it reached the edge, the paper was dried and sprayed with ninhydrin, a substance found by Gordon to give proper color reaction with the amino acids, enabling the detection of their spots. Later, they used pa per strips in boxes, in an atmo sphere saturated with water, and the edge of the paper was dipped into the solvent (the moving phase). They also learned to run the paper in two dimensions. This is how paper chromatography, which revo lutionized biochemical analysis, was born (16). Gas-Liquid Partition Chromatography

As mentioned earlier, Martin and Synge predicted, in their original paper, the possibility of using a gas as the moving phase in partition chromatography. However, no body thought to test experimentally this prediction at that time al though gas adsorption chromatogra phy underwent an important devel opment in the 1940's. (See, e.g., the works of G. Hesse, E. Cremer, S. Claesson, E. Glueekauf, and C. S. G. Phillips ; for a listing of their pa pers, see Bibliography, ref. 20). In 1948, Martin moved to the Na tional Institute for Medical Re search, where A. T. James, who had previously been working with Synge, joined him. They were en gaged in research work which did

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not result in any success, and D r . J a m e s became so discouraged t h a t M a r t i n suggested t h a t they switch to a project t h a t surely must work— to test the prediction of the possibility of gas-liquid partition chromatography. A request to t r y to develop a more advanced method for fatty acid analysis came as a good model for the investigations. Their work succeeded fairly rapidly, and soon the new technique was born; their paper was submitted for p u b lication on J u n e 5, 1951, and p u b lished in the first p a r t of 1952 in Biochemical Journal {17). I n their original work, J a m e s and M a r t i n added the sample by a pipet a t the front of the column and determined the eluted fractions by titration, and it was fairly difficult to record the "chromatogram." In the same year, D . H . Desty of British Petroleum, N . H . R a y of I.C.T., and R. P . W. Scott of Benzole Producers Assn. contacted M a r t i n about the separation of hydrocarbons, and then the possibility of using a thermal conductivity detector (applied at t h a t time by Claesson) in gas adsorption chromatography was raised. In the next three years, the applicability of the new technique to a wide variety of problems was shown, and in 1955, the first commercial instrument appeared on the market. T h e rest is history which those of us who "joined the club" in its early period will never forget. Sometime ago I read about life in the Spanish ports in the years after Columbus' return from the New World, where every ship brought something new, interesting, and exciting in treasures, people, and tales about new discoveries. W e who have participated in the development of gas-liquid partition chrom a t o g r a p h y since its beginning, probably felt similarly; every new issue of the journals, every meeting we have attended has brought something new and interesting t h a t everybody wanted to t r y out in his own laboratory the next day. I t is proper to quote D r . Lipsky who finished one of his lectures {21) by paying tribute to D r . M a r t i n : " H e has twice made outstanding contributions t o this field—in his discovery of partition chromatog-


r a p h y and in his pioneering work on gas chromatography. H e has thus altered, for the better, the lives of m a n y of us. We, his scientific colleagues, t h a n k him for allowing us to share with him this wonderful adventure." Interaction of Chromatographic Techniques

I t is interesting to note t h a t , until fairly recently, some artificial classification barriers have been dividing the various types of chromatography, and scientists rarely deserted their own fields, M a r t i n being a rare expection. A few years ago, however, these artificial b a r riers started to corrode. Scientists who, during the genesis of gas chrom a t o g r a p h y became identified with it, started to be active in the other branches of chromatography. As a result of this healthy development, chromatography is treated t o d a y more and more by a unifying a p proach. This happening is a n a t u r a l one, and the proper w a y development of a complex method is carried out. I t starts in different channels which, for some time, look like separate techniques with nothing in common. But, sooner or later, it becomes clear t h a t these channels are not parallel but approach each other and, a t a given time, converge.

F r o m this point on, they are developed together, benefiting from each other's achievements. T o d a y , chromatography is the most widely used analytical technique, and there is practically no laboratory in the world where it is not practiced. I t permits results which otherwise would be impossible. P r e s e n t - d a y chemists who, in less t h a n 30 min, can separate a complex multicomponent mixture and establish its concentration often do not realize t h a t , until fairly recently, the solution of this problem would probaby have required weeks of hard work. All of us who are active in this field should therefore appreciate the genius of the pioneers of this technique.

References

(1) L. Zcchmeister, "Historical Introduction," in "Chromatography," E . Heftmann, Ed., 2nd éd., pp 3—10. Reinhold, New York, N.Y., 1967. (2) V. Heines, Chem. Tech., 1, 280-5 (1971). (3) C. Dhéré, Condollea (Geneva), 10, 23-73 (1943). (4) G. Hesse and H. Weil, Michael Tswett's First Paper on Chromatography, M. Woelm, Eschwege, Germany, 1954. (5) L. Zechmeister and L. Cholnoky, "Die chromatographische Adsorptionsmethodc," Springer, Vienna, 1936 ; English éd., "Principles and Practice of Chromatography," Wiley & Sons, New York, N.Y., 1941.

Leslie S. Ettre is executive editor of the 18-volume "Encyclopedia of Industrial Chemical Analysis," published by John Wiley & Sons, Inc. Mr. Ettre graduated in 1945 from the Faculty of Chemical Engineering of the Institute of Technology, Budapest, Hungary. He became interested in chromatography in 1957 while employed at LURGI-Companies in Frankfurt am Main, Germany. From 1958 to 1968, he was working in gas chromatography at Perkin-Elmer Corp., serving in the last years as chief applications chemist. He has over 70 publications, mostly in the field of gas chromatography, is author of the book, "Open Tubular Columns in Gas Chromatography," and editor of the books, "The Practice of Gas Chromatography," (with A. Zlat-

(6) K. Sakodynsky, Chromatographia, 3, 92-4 (1970). (7) M. S. Tswett, Proc. Warsaw Soc. Nat. Sci. Biol. Sect., 14, minute No. 6 (1903). (8) M. Tswett, Ber. Deut. Bot. Ges., 24, 313—26 (1906); the two quotations are from ρ 322 and 323. (9) M. Tswett, Ber. Deut. Bot. Ges., 24, 384-93 (1906). (10) L. S. Ettre, Chromatographia, 3, 95-6 (1970). (11) H. Purnell, Gas Chromatography, ρ 1. Wiley & Sons, New York, Ν.Ϋ., 1962. (12) L. S. Palmer, "Carotinoids and Re lated Pigments," Chemical Catalog Co., New York, N Y . , 1922. (13) R. Kuhn and E. Lederer, Ber. Deut. Chem. Ges., 64, 1349-1357 (1931). (14) R. Kuhn, A. Winterstein, E. Lederer, Hoppe Seyler's Z. Physiol. Chem., 197, 141-60 (1931). (15) A. J. P . Martin and R. L. M. Synge, Biochem. J., 35, 1358-68 (1941). (16) R. Consden, A. H. Gordon, and A. J. P. Martin, ibid., 38, 224-32 (1944). (17) A. T. James and A. J. P . Martin, Biochem. J., SO, 679-90 (1952). (18) A. J. P. Martin, "Historical Back ground," in "Gas Chromatography in Biology and Medicine," R. Porter, Ed., pp 2-10, J. & A. Churchill Ltd., London, England, 1969. (19) A. T. James, "The Development of an Idea," in "Gas Chromatography." H. J. Noebels, N . Brenner, and R. F . Wall, Eds., pp 247-54, Academic Press, 1961. (20) R. L. Pecsok, Ed., "Principles and Practice of Gas Chromatography," Wiley & Sons, New York, N Y . , 1959, pp 154-6. (21) S. R. Lipsky, "Gas Chromatogra phy ; The Anatomy of a Scientific Revolution," in "Gas Chromatography in Biology and Medicine," R. Porter, Ed., pp 11-16, J. & A. Churchill Ltd., London, England, 1969.

kis) and "Ancillary Techniques of Gas Chromatography" (with W. H. McFadden). He is a member of the editorial advisory board of the J o u r n a l of Chromatographic Sci ence, one of the regional editors of Chromatographia, and serves as chairman of the Subcommittee on Nomenclature of ASTM Committee E-19 on Chromatography. Mr. Ett re is a member of ACS, ISA, the New York Academy of Sciences, the British Society for Analytical Chemistry and Gas Chromatogra phy Discussion Group, and a fel low of the American Institute of Chemists. Leslie Ettre has cooperated with Albert Zlatkis in the organization of the International Symposia on Ad vances in Chromatography since their beginning.

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The Development of Chromatography - Analytical Chemistry (ACS

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