Trevor Robinson
Michael Tswett
Syracuse University Syracuse 10, New York
Everywhere Michael Tswett is credited with being t,hr originator of column adsorption chromatography. However, Zechmeister ( 1 ) and ot,hers have shown that the technique had been used hefore Tslvett, aud it therefore seemed desirable to re-evaluate the work of Tslvett in order to determine the real importance of his position wit,hin the history of science. For biographical details the paper of Dh&-6 (8) is invaluable, as Dh6rit had access to personal letters of Tswet,t and other mementoes not generally available. Nevertheless D h M is incorrect about the date of Tsmett's death (5) and does not attempt a balanced, complete disrussiou of Tswett's entire scientific output,. Michael Tswett was born at Asti, Italy, on May 19. 1872, the son of a Russian father and an Italian mother. When he was young, the family moved to Switzerland, where Tswett mas educated first at Lausanne and then a t Geneva. From 1891-96 he was a student in the faculty of sciences at Geneva, studying botany with Chodat and Thury, physics with Soret, and chemistry ~ ~ i tGuye. , h He won the Davy prize in 1891 for a paper on plant anatomy and then under Thury's direction prepared his doctoral thesis, "Etudes de Physiologie Cellulaire." I11 this he proposed that tthe chlorophyll pigment is incorporated into a proteinareous substance called "chloroplastine." I n 1897 he mas teaching plant anatomy and physiology in St. Petersburg and doing research on the chemist,ryof chloroplasts. In 1901he went to the University of Warsaw as privatdocent,, becoming in 1907 professor "~~~
~~~
Wars&. These yearsin Warsaw produced his most important work until in 1915the German invasion drove him to Moscow, then to Niahni Novgorad. In 1917 he went to Vladikavkaz to recover from a serious illness, probably tuberculosis, and then was appointed professor of botany and director of the botanical garden at the University of Dorpat (Estonia). He went there late in 1917, hut the German invasion in 1918 again forced him to flee to Russia, where he died at Voronezh on June 26, 1919, a t the age of 47. In his relatively short lifetime, he was allowed only about 15 years of productive, scientific work during which he wrote some forty papers and one book. Of these only about three or four are about strictly hotanical subjects although this was the area of his training. The others are very largely of chemical int,erest and most deal in some way with the plant pigments. Presented in part at the 132nd Meeting of the American Chemical Society, New York, September, 195i. A fuller discussion of the life and works of Michael Tsaett isappearing in Chymia, 6 (1058).
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Figure 1. Photograph d Michael Trwett kindly furnished by 1. Zechmeirter, originally published in Condolleo, 10, 1943, and reproduced here b y permis3ion of the Editor.
Early Researches 1899-1906
Most of the papers of this period already show Tsmet,t's interest in the plant pigments. His helief that, native chlorophyll exists as a prot,ein 'omplex led him to attempt the purificat,ionof this complex by extraction with resorcinol followed hy adding excess water to preripitate the pigment (4, 5). By analogy with hemoglobin Tswett called the pigment-protein complex "chloroglobin" and suggested that the binding of chlorophyll to the protein was probably similar to the binding of hematin in hemoglobin. Here one sees one of the important threads xrhich go through all of Tswett's work. Trained as a biological scientist, he constantly argued that there was no a priori reason for supposing that the rhlorophyll of the chemists represented the real, native pigment of living plants. This idea goes back to his thesis of 1896 and appears in some form in much of his later work. He pointed out that knorvledge of the chemistry of the pigment was of no help in understanding the photosynthetic process and apparently hoped that understanding of photosynthesis could he advanced by obtaining chlorophyll in a more nearly native state. In 1900 Tswett (8) began his series of investigations on the isolation and chemistry of chloroplast pigments
primarily usiug solvent extraction methods which had been used by others before him. In extracting the pigment Tswett was careful to grind in the presence of calcium carbonate so as to neutraliae plant acids which might degrade the pigments. As a result he observed that except for one pigment (carotene) all mere adsorhed on the calcium carhonate. This could he washed to remove impurities and finally eluted by using a mixture of light petroleum with 10% alcohol. This fractional adsorption method was applied hy Tswett in several investigations (7, 8) and may possibly be a forerunner of the column method used later, although he nowhere indicates that one led to the other. In 1901 Tswett was ready to state his most important doctrine-the fact that not just one but two green pigments are present in leaves (7,Q). He proposed that the name chlorophyll he applied only to the tot,al mixture and used the names "chlorophyllin a" and "chlorophyllin 8" for the individual green pigments. He separated these using the solvent partition methods of Stokes (10) and Sorhy (11) and, in fact, only restated hat they had said long before regarding the duality of the pigment, hut they had simply been ignored. Tswett mas to have to fight vigorously for this idea against some of the leading chemists of his day. In 1901 (9) he also stated that the so-called "crystalliaahle chlorophyll" obtained with long alcohol extraction was actually an artifact formed during the extraction. He called it "metachlorophyllin" by analogy with methemoglobin. We know it today as ethyl chlorophyllide. In 1905 and 1906 two papers (8, 12) by Tswett appeared dewrihing his researches on pigments of the hrown a l p e . The most raluahle result of this work was thediscovery of a liexvsubstanrerelated t,o the other two chlorophyll pigments. In his terminology, higher plants contained chlorophyllins ~u aud 8,while the hrown algae have chlorophyllin a and a new pigment to he called chlorophyllin y. This is the pigment we now know as chlorophyll c, followiiig the mork of Strain and Manning (IS) as recently as 1922, t,hirty-six years after Tswett's discovery.
systems. For example, he proposed (14) that the chloroplast pigments are adsorbed on t,he substratum of the granum and this is v h y nonpolar solvents cannot extract them (except carotene) from leaves although nfter extraction they are soluble in such solvent,s as henaene or ligroin. The presence of alcohol breaks down the adsorption complex, allowing the pigment to be dissolved. He went so far as to perform model experiments showing that pigment niixtures adsorbed onto various artificial substrata behave just as they do in the leaf with regard to extraction. Then in a paper of 1906 (14), aft,er a lengt,hy discussion of adsorpt,ion phenomena, Tswett gives us the first' description of his chromatographic method: If a petroleum ether chlorophyll solution filters through a column of adsorbent ( I use chiefly calcium carbonate which is tightly tamped into R glass tube), the pigment8 separate themselves from top to hottom in different oalored zones according to tho sdsorption seri~s,in which the more strongly xdsorhed suhstances displace the more weakly held ones farther toward the bottom. This separation is precticitlly complete if one, after the passage of the pigment solution, passes a stream of solvent through the colmnn of adsorhcnt. Just like the rays of light in the spcct,rum, so are the different components of a pigment mixture soparated in an orderly way in the calcium rsrlmmte eolr~mn and thus ma," he qualitatively and also qnantit,ativelg determined in this way. Such a preparation I call s. chrorn.ztogram and the corresponding method, the chromatographic method. Needless to say, the described adsorpdion phenomena are suit,ed not only to the chlorophyll pigments. I t is to be expected that d l kinds of colored or eolorle~schemical compounds :are subject to the same laws. I11 another paper (15) of the same year he discussed the met,hod in more detail with a diagram of the apparatus used (Fig. 2). Some of his practical advice from t,his paper is well worth repeat,ing for the inst,rurtion of beginning chromatographers today:
As adsorhent m y pou-deretl mbstance insohhle in the solvent. e m serve. However, since very many substanre are not without
Chromatography and the Chlorophyll Pigments
chemical influence on the sdsarhed material, the choice of the analyst will in general fall to such a~lmtancrsas are chemicslly inert m d a t the same time may he most finel>-pondered. Suhstances which a d s o h too strongly are also t.o be avoided since the," require enormous quantit,ies of pigment in o d e r to make distinctions. Fineness of the adsorbent powder is w r y important; coarseeraitined material ~roducesindistinct chromatamzms he-
Tswett was deeply interested in the phenomenon of adsorption generally. He not only used it asan analytical tool, he also invoked it to explain certain natural
' Poliakov (3) states that the first description Ry Tswett was nrtnally puhliahed in Russian iu 1903, hut I have hpen unahlc to obtain this paper.
Figure 2. Trwett's chromdogrophic apparatus. Not. 2 a n d 3 ore clore-uo views of t h e oooarotur rhown at No. 1 , which reprerents a preswre manifold used for the onalyrir of several m o l l samples ~imultoneourly. No. 5 giver o close-up of the apparatus rhown at No. 4 ond indicates zones of repwotion for the chlorophyll and r m t h o p h y l l pigment. of ieover.
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cause adsorption and diffusion in the too-wide capillary spaces interfere. Among the adsorbents I can frequently recommend precipitated calcium carbonate, which produces the most beautiful chromatograms. Suerase also may be rather easily brought into the required state of subdivision and offers the greatest guarantee of chemical inertness. For special purposes one will choose chemically reactive adsorbents (for instances, hydrolyzing, reducing, oxidizing).
I n the same paper he also described application of the method to pigment analysis, stating that two chlorophyllins, four xanthophylls, and carotene are present in leaves. The second chlorophyll pigment (his chlorophyllin 0) was generally believed by the chemists of the day to be either an artifact or present in insignificant amounts. Marchlewski in particular was a leader in this field, and Tswett's contention that chlorophyllin 0 was a native pigment present in considerable quantity touched off a feud between these two workers which lasted for many years and produced some anite dramatic (and to us todav. " , unbelievable) polemics. The chemistry of chlorophyll a t this time was being studied by the application of various acid and base degradations. Interpretation of these experiments also brought Tswett into conflict with Marchlewski and other leading chemists. Acid treatment of "chlorophyll" clearly yielded two products, "phylloxanthin" and "phyllocyanin." These were thought to represent different stages in the breakdown of the molecule. However to Tswett, who had separated chlorophyll itself into two components, it was evident that one of these products must be formed from his chlorophyllin a and the other from chlorophyllin 8. Accordingly he proposed the names chlorophyllan a and 0 for these compounds (16). Using chromatographic separations he showed that it was possible to purify the two chlorophyll pigments first and then convert each one separately to its related derivative or else to convert L'chlorophyll" to a mixture of the derivatives and then separate them from each other. The information on acid derivatives was further complicated by the appearance of Willsttitter's work in which he gave the name "phaeophytin" to the first acid derivative, ignoring Tswett's work on the duality of chlorophyll. The objections made by the chemists of Tswett's day seemed to center in an emotional reaction to this new technique of chromatography which seemed t,o supply answers too easily, bypassing the tedious chemical separations which were familiar. Marchlewski (17) complained that Tswett had not put himself to the trouble to isolate and purify the substances he had described, and that one could not with the help of a " filtration experiment" swing himself to the he~ghtof a reformer of chlorophyll chemistry. Willstiitter argued (18)that chromatography was not suitable as a preparative method. Tswett's replies were clear and to the point (19, $0):
". .
The assertion that mv ureosrations are i r n ~ u r because e I have not olnninrd rhr pigment in s A d form is m t i r d v uujuatitird. n The rhl~rt,phyllnl.sn e w ia,l:~trdI y m w n s of ~ d w r p t i ~:11_1dwi9 which is precisely a purification method and in many respects is superior to precipitation or crystallization methods. I t will probably offer no special difficulties to apply the adsorption methods t o the preparation of larger quantities of pigment suitable for analysis. One could, for example, as Dr. Werner Magnus kindly suggested t o me, use whole batteries of wide ad-
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sorptlon tubes. In any case, adsorption analysis is already now applicable as a powerful control method; and no pigment preparation can be asserted to be a defined, pure substance if it does not show itself as unitary in the chromatographic test also.
In retrospect Marchlewski was right-no one could hope to reform chlorophyll chemistry merely by applying a simple, new-fangled method. Willstitter's heantifully detailed, painstaking applications of standard procedures to kilogram quantities of chlorophyll finally convinced everyone that Tswett had been right all along; or, rather, everyone accepted Willstltter as the founder of chlorophyll chemistry, adopted Willstatter's nomenclature, and forgot that Tswett had said the same things years before-indeed had s a ~ dthem when Willstatter had been arguing the c ~ n t r a r y ! ~ Tswett's views on chlorophyI1 chemistry, summarized in 1908 (el), are generally quite acceptable today although the nomenclature of Willsta.tter has replaced that of Tswett. Other Researches
Returning to 1908, Tswett published in this year two papers ($2, $3) on the coloring of autumnal leaves in which he distinguished two phases, the first with the leaf still living, retaining semipermeability, and producing anthocyanin pigments. The second phase involved death of the leaf with concomitant hringingtogether of chromogens and enzymes which oxidize them to brown pigments. Using adsorption chromatotography, he purified the pigments of yellowed leaves in the first, living, phase and showed that the chief component was not the normal pigment of green leaves merely unmasked by the disappearance of chlorophyll: rather, it was a newly synthesized substance which he named "herbstxanthophyll." I n 1910 Tswett published his only book, "The Chromophylls in the Animal and Vegetable Kingdoms." Unfortunately this was never translated out of Russian, and even the Russian edition is practically unobtainable. I n view of Tswett's shorter papers, it seems likely that this may be a treasure trove of valuable observations and insights. The author deeply regrets that he has been unable to see it. Following this, Tswett published a half dozen short papers on miscellaneous subjects and then, in 1914, his last two papers (84, $5) which are again rather important ones and unfortunately neglected. In these he turns his interest in pigments away from chloroplast pigments to the water-soluble anthocyanin pigments, which he introduces with one of his beautifully illuminating generalizations: I t is a natural inclination of our spirit to give special notice to anything colored and to want to explain the colored substances in living nature with finality for no other motive. For this reason many apparent problems arise. No reason exists t o vindicate special physiologicel or ecological functions for a substance only because it appears tinted to the human eye. Regarded objectively, every substance is indeed "colored," protein, sugar, or water just as much as anthocyanin or chlorophyll. Only, the strongest abmrptions of radiant energy lie in another part of the wavelength soele. Now, whether the absorbed color is purposeful (i.e., necessary or required for preservation of the living being) is to be determined empirieslly in each ease. I n and of itself, therefore, the subjective coloration has no significance. The best summary of Willstitter's work is the classic "Untersuchungen iiber Chlorophyll," R. WILI.ST~TERA N D A. STOLL, Springer Verlsg, Berlin, 1913.
The most important part of these papers is that, concerned with a discussion of the substances we now call "Ieucoanthocvanins." Tswett remarked that for many years people had ohserved certain plant parts t,o turn red when heated with acid, but apparently he &-as the first to carry on det,ailed investigations of this phenomenon. He purified the red pigment and studied its properties, hot,h chemical and physical, concluding it t o be very similar t,o the natural anthocyanins. He also isolated t,he precursors of the red pigmeut and showed them to he insoluble in most organic solvents, colloidally soluble in water, and precipitated by egg white. If aldehydes were present during the heating with acid, the pigments that were formed were more violet-red than the pure red ones formed with acid alone. All in all, these compounds seem to have been investigated with great thoroughness by Tswett, aud even today it would be possible to initiate some research in this area taking up directly where he left off. However, it is Rosenheim who is geuerally given the credit for being the first worker t,o give much attention to leucoanthocyanins on account of his paper (26) published in 1920. Rosenheim does deserve credit for proposing the name "leucoanthocyanin," but Tswett's work is fully as detailed and appeared six years earlier. Conclusion
Whether or not me regard Tswett as the originator of the chromatographic method, it seems clear that he deserves an honored place in t,he pantheon of science, if not for the discovery of the method, a t least for powerful and valuable applications of it as well as for other important discoveries. However, as important ns any single discoveyy mas the over-all attitude which Tswett, hrobght to biological rhemistry. I n contrast to Willstatter, the organic rhemist, who said (27): I t was possible without investigating chlorophyll itself, to deduce the peculiarities of its constitubion from the consideration
of the derivatives which are obtained by the reactions with acid and alkali
. . .'
we see Tswett's guiding idea (28), one which biochemists are only recently coming t,o appreriate: I t is too often forgotten, especially on the chemical side, that living tissues itre not mere mixtures of compounds remaining in chemical equilibrium, but are organized structures, where, as a result indeed of osmotic boundaries, the mast varied reactive bodies stand next to each other and can exercise their mutual chemical potentiality only after the disorganization of protoplasm concomitant with the extraction procedure. Literature Cited
ZECEIMEIGTER, L., Ann. N . Y . Acad. Sci., 49, 145 (1948). DHBRB,C., Candollea, 10, 23-73 (1943). POLIAKOV, J. A., Isis, 38, 243 (1948). TSWETT,M., Compt. vend., 129, 607-10 (1899). TSWETT,M., Bolan. C a t r . , 81, 81-87 (1900). TSWETP,M., Compt. ? a d . , 131, 8 4 2 4 (1900). TSWETT,M., Botan. Cent?., 89, 1 2 0 3 (1902). TSWETT, M., Ber. deut. hotan. Ges., 24, 2 3 5 4 4 (1906). TSWETT,M., Compt. ?end., 132, 149-50 (1901). STOKES.G. G.. Proe. R o z d Soe. ilondoni. 13. 144-5 (18641. SORBY, C., RO& Soc. ondo don),'^^, i42-83 (1873j. TSWETT,M., Bolan. Z., 63, 273-8 (1905). STRAIN,H. H., AND MANNING, W. M., J . B i d . Chem., 144, 625-36 (1942). TSWETT,M., Ber. dmt. hotan. Ges., 24, 3 1 6 2 3 (1906). TSWET~,M., B w . deut. bolan. Ges.,. 24,. 384-93, Plate XVIII (1906). TSWER, M., Biochem. Z., 5 , 6 3 2 (1907). MARCHLEWSKI, L., Biochem. Z., 7 , 282-5 (1908). WILLST~LTPER, R., Ann., 390, 2 6 9 3 3 9 (1912). TSWETT,M., Biochem. Z . , 10, 404-13 (1908). TSWETT,M., Be?. deut. bolan. Ges., 26A, 214-20 (1908). TSWER, M., Biochem. Z., 10, 4 2 6 2 9 (1908). T s w ~ r r M., , Ber. deut. hotan. GPS.,26A, 88-93 (1908). Ibid., 94-101 (1908). TSWETT,M., Biochem. Z., 58, 225-35 (1914). TSWETT,M., Ber. deut. bohn. Ges., 32, 61-68 (1914). ROSENHEIM, O., Bioehem. J., 14, 178-88 (1920). WILLST~~TTER, R., J. Am. Chem. Soe., 37, 323-45 (1915). TSWETT,M., Biochem. Z., 10, 414-25 (1908).
k. me.
(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28)
a This is not to disparage Willstittter, whose fine work was of course necessary and of funditmental importance, but only to point out that there was s difference in approach.
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