Harold H. Strain
Argonne National Laboratory Argonne, Illinois 60439 and Joseph Sherma Lafayette College Easton, Pennsylvania 18042
Michael Tswett's Contributions to Sixty Years of Chromatography
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O n l y rarely have there appeared unique reports destined to influence the course of progress in diverse fields for many years to come. One of these papers appeared just 60 years ago. It was prepared by a 34-year-old Russian botanist, M. Tswett, then Assistant at the University of Warsaw. It was published, in German, in a German botanical journal, Die Beriehte der deutschen botanisehen Gesellschaft (1). It described his chromatographic adsorption method for the separation and isolation of the green and yellow chloroplast pigments extracted from green plants. It introduced a matchless procedure for the resolution of mixtures of similar chemical substances and revealed the basic principles of the method. It also described important applications of the technique, as well as illustrating the critical steps in the chromatographic procedure that were destined to be incorporated into several analogous analytical methods. It reduced to practice an analytical approach that has become of great significance to all workers concerned with chemical substances and their reactions and introduced the nomenclature that is widely adopt,edtoday. Tswett's contributions have beeu surveyed in many reviews ($4)including one in THIS JOURNAL (6). There is, however, no consensus concerning the novelty, pertinence, and influence of his contributions. Nevertheless, his report on the invention of chromatography and its use for the separation of the chloroplast pigments of leaves has continued to stand as a landmark in these two important fields. It has now beeu selected for more critical reevaluation in the light of sixty years of subsequent experience. Tswett's Innovations in Adsorption Technique
Trained in botany and plant physiology at Geneva, Tswett was cognizant of the importance of the chloroplast pigments and of the need of methods for their separation. He was familiar with separatory methods based upon the phenomenon of adsorption, notably the use of adsorbents in columns and in suspensions for the decolorization of solutions, and he had followed the extensive studies on the capillary rise of solvents and solutes in filter paper reported by Goppelsroeder (7). For his separation of the leaf pigments, Tswett devised a novel modification of the columnar adsorption procedure. He introduced a petroleum ether, benzene, or carbon disulfide extract of green leaves into a column of the adsorbent (inulin, powdered sugar, or precipitated chalk) until the pigments formed a narrow, initial zone in the uppermost regions of the adsorbent. At this stage, he washed the adsorbed pigment mixture with
fresh solvent, whereupon the several green and yellow pigments migrated a t different rates and separated from one another forming a "chromatogram." This washing of the narrow initial zone of the mixture proved to be a novel, indispensable, and critical step in the highly selective separatory procedure. It effected the "differentiation" or development of the chromatogram, and it resolved the mixture much more extensively than filtration of the pigment solution alone. For isolation of the pigments separated in the column, the individual zones were isolated mechanically, and each pigment was eluted with a more sorbed solvent. Alternatively, the pigments were washed through the column and collected separately in the percolate. These procedures provided the individual pigments, without chemical change or alteration, for further investigation of their properties. Because the isolation of substancesin a state of high purity is essential to the investigation of many of their properties and is critical to the calibration of instruments for their determination, Tswett had originated an extremely useful technique for the investigation of various chemical substances (8). In 1903 Tswett had published a lecture, in Russian, in which he described several separatory procedures based upon adsorption. In this report, now available in English (59, he described briefly the chromatographic procedure as applied to colorless substances. In another report accompanying his chromatography article in the "Beriehte" for 1906, he described the adsorptive properties of various powdered solids (9). It was only in the report on chromatography (I), however, that he described the detailed chromatographic procedure, the nomenclature that is so widely adopted today, and the definitive resolution of the mixture of chloroplast pigments. Tswett recorded many important observations, some of which were reintroduced into the chromatographic literature many years later. He recognized the importance of the washing procedure and provided a particular term for it. He observed that strongly sorbed substances elute or displace weakly sorbed substances but that the reverse displacement is not conspicuous. He noted that two substances of similar sorbability may occur in a single zone, but that their separation could be improved by the use of various solvents or by readsorption, and that the separated substances form a chromatographic series, the sequence varying with the solvent. He observed that the adsorbed chlorophylls are extremely susceptible to photo-oxidation, and he distinguished clearly between adsorption zones in the Volume 44, Number 4, April 7 967
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columns and absorption bands in the spectrum.
Progress in Chromatography
Tswett's Characterization of Chloroplast Pigments
The chromatographic method, as perfected by Tswett, represents a prototype procedure that has been modified in many ways (19). Variation of the sorptive medium has improved the applicability, selectivity, and capacity of the columnar adsorption method. The use of inorganic and organic ion-exchange substances has created the field of ion-exchange chromatography. The use of sorptive liquids that are dispersed and fixed upon a nonsorptive support and that are immiscible with the wash liquid has provided partition chromatography and reversed-phase partition chromatography. Utilization of the washing of substances adsorbed in paper with fresh solvent has evolved into the fields of linear, radial and two-way or transverse paper chromatography, all of which may be employed as adsorption procedures or as partition procedures. Tswett's column chromatography also evolved into the analogous and convenient thin-layer chromatography, which makes possible the use of various powdered sorbents in layers suitable for lineal, radial, and transverse development. Gas chromatography, the formation of chromatograms by the flow of an inert wash gas in place of the wash liquid, was developed and instrumentized for the qualitative and quantitative examination of mixtures of various gases and vapors. These highly selective methods are applicable to the examination of all kinds of soluble and volatile suhstances (19).
With his chromatographic methods supplemented by spectral absorption observations, Tswett confirmed that leaves contain two, and only two, green pigments, namely, chlorophyllin a,now called chlorophyll a, and chlorophyllin 8, now called chlorophyll b (10). Tswett also demonstrated that there are at least three or four yellow, hypophasic xanthophylls in leaves: xanthophyll p, now usually called neoxanthin; xanthophyll a' plus a", now called violaxanthin; and xanthophyll or, currently called lutein or xanthophyll (11). Tswett confirmed the presence of the yellow, epiphasic carotene in leaves, and he separated this pigment completely from the leaf-pigment mixture. Acids converted his chlorophyllin a to a weakly-sorbed, grey pigment now called pheophytin a (10). Tswett's results concerning the number of the leaf xanthophylls were contrary to some of the early observations, which were based upon partition experiments and which indicated the presence of many xanthophylls in leaves (I$, 15). Later, a crystalline leaf xanthophyll was isolated from saponified leaf extracts by Willstatter and his co-workers, who employed partition and crystallization procedures. This crystalline preparation, called xanthophyll, was presumed to be a single pigment (14, 15). Tswett's xanthophylls were, therefore, regarded as artifacts. Consequently, Tswett's method was considered to be unreliable. Although Tswett, himself, had few opportunities to restudy these adverse criticisms, subsequent investigations by many other workers demonstrated that the saponification-partition method for the isolation of crystalline leaf xanthophyll resulted in the loss of the more-sorbed xanthophylls separated by Tswett (16). Moreover, Tswett's observations have been confirmed by numerous improved modifications of the chromatographic technique (11). Ironically, the crystalline xanthophyll isolated by the saponification-partition procedure was also shown to be a mixture of two isomeric pigments by the use of more active and more selective chromatographic adsorbents such as activated n~agnesia. The major isomer is lutein or xanthophyll; the minor is zeaxanthin (16). Chromatography and Progress in Pigment Studies
Chromatographic methods have been indispensable to current progress in studies of the chlorophylls and the carotenoids (10, 16-19). Additional chlorophylls and carotenoids have been found in species of taxonomically divergent plant groups (8, 10). Resolved in chromatographic columns, these pigments are often contaminated by various colorless substances from the plant material, from the adsorbent, or from the action of the adsorbent upon the solvent (17, 18). Fortunately, most of these colorless contaminants may be removed by crystallization of the individual pigments (10, 17, 18). Certain adsorbents convert the natural pigments into other colored derivatives that may be mistaken for normal constituents of the plant material (SO). Some chromatographic procedures also permit a single pigment to separate as two or more zones, an effect erroneously attributed to the presence of additional pigments (11). 236
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Differential Migration Methods
Chromatography is based upon the phenomenon of differential migration. The adsorbed substances are caused to migrate by the driving force of the flow of the wash liquid or wash gas. This force acts equally upon all the substances while they are in solution or free in the gas phase. Their migration is retarded by a resistive force, their selective sorption by the sorbent (8). A number of other separatory methods are now recognized as differential migration methods analogous to chromatography. These differential migration methods include zone electromigration, which is also called zone electrophoresis or electrochromatography, differential sedimentation, mass spectroscopy, continuous thermal gravitational diffusion, etc. I n this respect, chromatography has served as a model for the development and comparison of this class of separatory methods (8). One of the recent comprehensive hooks on chromatography, now in its second edition, is organized from this point of view (19). Since the publication of Tswett's fundamental paper in 1906, there has been an ever-accelerating increase in the number of publications concerning his technique. There are now dozens of books devoted to chromatography and related separatory techniques. There are hundreds of reviews concerning these methods and there are thousands upon thousands of reports devoted to the modifications and applications of his method (19). Few of these reports provide so clear an exposition of the technique and so broad a base as that evolved in Tswett's considerations more than half a century ago. The Milestone Paper
Students of chromatography have frequently asked
for key references that illustrate critical points in the evolution of chromatography. Although we have often recommended Tswett's basic article on chromatography, many students with a latent or potential interest in this subject are not sufficiently experienced in science and in German to appreciate many of the fine observations and deductions recorded in the original paper. Accordingly, it has seemed worthwhile to supplement this L'reevaluation"with an English translation of Tswett's article (21). In the translation, words commonly employed in the field are preferred to their synonyms. No explanatory notes have been added, because the descriptions of procedures and results conform so closely to current usage.
An introduction to the significance of Tswett's contribution would not be complete without brief reference to some of the circumstances that influenced his life and his scientific efforts (2-6). These include his heritage, his scientific training, his experience with several languages, his Russian citizenship, his positions in Russian universities, his membership in German scientific societies, and his aspirations to a position in Germany (3). Finally, there was the calamity of World War I, which disrupted so much of the academic life in Russia. Born in Asti, Italy, on May 19, 1872, Tswett was registered Michele De Zevett, son of Simone De Zevett, a Russian subject, and Maria (De Doroaza) who was but twelve years older than her son and whose lineage has not been established. Neither of the parents had been residents of Asti and apparently did not remain there. Michael Tswett remained a Russian subject (S). Tswett received his secondary education a t College Gaillard, Lausanne, and a t College de St. Antoine, Geneva, and took his university degree and his doctorate in botany at the University of Geneva (1891-96). He obtained a second doctorate in botany from the University of Warsaw (1910). Tswett first accepted a teaching position at St. Petersburg. Later, he became Assistant at the University of Warsaw (1901-1908), Professor of botany and agronomy at the Veterinary Institute (1907), and Professor of botany and microbiology a t the Polytechnical Institute (1908). He is credited with 56 publications, 15 in Russian, 13 in French, and 28 in German (3). In 1915 Tswett's work was interrupted by the occupation of Warsaw by German troops. There followed
several years of moving about in Russia, a period of poor health, and finally death, a t Voronesh, on June 26, 1919, at the age of 47 (3). His death has also been reported as 1920 (2, 4). Like many visionary and creative individuals, he never enjoyed the rich harvest from the imaginative seed provided so generously for the benefit of all. Three pictures of Tswett, as a doctoral candidate and a t the ages of 23 and 40 years, were presented in the review by DhBr6 (2). The second of these pictures has already been reproduced in THIS JOURNAL (6) and in other reviews (5). The third picture has also been reprinted (5). Acknowledgments
The authors are indebted to the U S . Atomic Energy Commission for support and for a Resident Research Associateship for J. Sherma. Mr. Norman P. Zaichick collected some of the historical material. Literature Cited (1) TSWETT, M., Ber. d. deut. bolan. Ges., 24,384 (1906). (2) DHBEB,C., Candolka (Geneva), 10,23 (1943). (3) HESSE, G., AND WEIL, H., "Michael Tswett's First Paper on Chromatography," (Editor: WOELM,M.), Esehwege, Germany, 1954. L., Isis, 36, 108 (1946). (4) ZECHMEISTER, (5) ZECAMEISTEE, L., "Chromatography," (1st ed.), (Edibr: E.), Reinhold, New York, 1961, p. 3. HEFTMANN, (6) ROBINSON, T., J. CAEM.ED., 36,144 (1959). F., Verhandl. der Naturf. Gesellseh. Basel, (7) GOPPELSROEDER, 14 (1901). (8) STRAIN,H. H., "Chromatography," (1st. ed.), (Editor: E.), Reinhold, New York, 1961, p. 11. HEXTMANN, (9) TSWETT, M., Ber. d. h t . botan. Ges., 24,316 (1906). (10) STRAIN,H. H., AND SVEC,W. A,, in "The Chlarophylls," (Editors: VERNON, L. P., AND SEELY,G. R.), Academic Press, New York, London, 1966, p. 21. (11) STRAIN,H. H., SEERMA,J., BENTON,F. L., AND KATZ, J. J., Bkchim. Biophys. Ada, 109.1, 16, 23 (1965). H. C.. Proc. Rou. Soc.. 21.442 118731. 112) SORBY. L. ' S., "cakenoids 'and ' ~ e l a k dPigments," (13j PALM& Reinhold, New York, 1922. R., AND MIEG, W., Liebiv's Ann., 355, 1 (14) WILLSTATTEE, (14M) ,.
(15) WILCSTATTER, R , AND STOLL,A,, "Untersuchungen iiber Chlorophyll. Methoden und Ergebnisse," J . Springer, Berlin, 1913. (16) STRAIN,H. H., "Leaf Xanthophylls," Carnegie Institution of Washineton. Publ. No. 490. Washineton., D.C.. 1938. ~~-~ (17) STRAIN,H. k,THOMAS, M. R.; AND KATE,J. J., ~iochim. Biophys. Acta, 75, 306 (1963). (18) STELIN, H. H., THOMAS,M. R., CRESPI, H., AND KATZ, J. J., Biochim. Biophya. Acta, 52, 517 (1961). (19) HEFTMANN, E. (Editor), "Chromatography," (1st ed.), Reinhold, New York, 1961, (2nd ed., in press). J., unpublished observations. (20) STRNN,H. H., AND SHERMA, 44,238 (1967). (21) See THIS JOURNAL,
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