ADSORPTION ANALYSIS : TSWETT'S CHROMATOGRAPHIC METHOD HAROLD G. CASSIDY Yale University, New Haven, Connecticut
Mixtures of organic substances which do not readily yield to ordinary treatments may often be separated by means of chromatographic adsorption. I n this process adwantage is tnken of the differences in adsorptiwe behavior of the saeral components of the mixture. This
article rainus the history of the method and its theory. Working details for carrying out the analysis are ghen and criteria to aid in the choice of adsorbents and solwents are discussed. A few examples of the application of the method are giwen. Its limitations are pointed out.
To show the remarkable separation achieved by his method Tswett drew the foliowing pleasing analogy. HE method of chromatographic adsorption was He said that in the adsorption tube the different comdevised by the Russian botanist, M. Tswett, in ponents of a pigment mixture are regularly separated the early nineteen hundreds. It is interesting from each other, much as are the light rays in a specto see how he came to the discovery. He had been trum, and may thereby be qualitatively and even studying methods of isolating plant leaf pigments such quantitatively determined. as chloro~hvlland carotene. In the course of this study he io&d that chlorophylll was readily adsorbed , After having completdy separated the zones by Passfrom petroleum ether solution by all solid substances W fresh solvent through the column, a process which is which were themselves insoluble in the petroleum ether, known as "developing the chromatogram," the column of adsorbent and adsorbed substance* o d d be pushed provided that the adsorbents were used in a Out of the tube and cut UP with a knife or spatula. finely pulverized form (to obtain a large surface area). He tried out over a hundred substances. Among these Then the separate pigments could be washed from the were dements, such as sdfUI, silicon, zinc; oxides; respective portions of the column. Tswett called this hydroxides; chlorides; oxygen-containing salts, such whole process the chromatographic method and the as chlorates,, phosphates, carbonates; organic acids, column containing its zones a chromatogram. The as tartaric, cxtnc, and picric acids; oxalates; acetates; chromatogram is also known as the Tswett column. amides; higher alcohols and carbohydrates; protein Tswett recognized that not only colored substances, materials; aniline dyes, and so forth. some of these but colorless, too, could be handled by his technic. Tswett's earliff work on this method (1, 2) was pubsubstances adsorbed not only the green chlorophyll but also the yellow carotenoid pigments which were present lished in the proceedings of the German Botanical in the solutions. Solids such as MnOz or UsOa ad- Society of 1906. In 1910 he published, in Russian, a comprehensive work on pigments of the plant and anisorbed and destroyed the pigments. In the course of this study Tswett found that an ad- mal world in which he is said (3) to have discussed the sorbed substance could be replaced on the adsorbent by usefulness of his method more fully. In the next a more strongly adsorbed substance. Thus for a given twenty years the Tswett method lay practically unadsorbent he could arrange an adsorption series of known, partly perhaps because, though of great inmore strongly and less strongly adsorbed substances. terest to chemists, it appeared in a botanical journal ~t was on this idea, he said ( I ) , that his method of and in a Russian treatise. There are some scattered evidences of use of the method during this period. chromatographic adsorption was based. He found that when asolutionof mixed plant pigments Palmer (4)worked with i t and mentioned it very favorin petroleum ether was allowed to percolate down ably in his monograph on the carotenoids. By and through a column of an adsorbent (see Figure I), the large, however, this important tool lay disused for two pigments were adsorbed from the solution in different decades. colored zones, the more strongly adsorbed ones being The present increasing use of the method dates from held a t the top, the less strongly adsorbed being about 1931 when Kuhn (5) and his co-workers used i t displaced downward. This resulted in an "adsorption in preparative studies on the carotenoids and through ranking" of all the substances in the mixture. The its agency were able to separate the isomeric carotenes. separation, he found, could be made more complete by It might be mentioned that Tswett envisioned the usethe pigment mixture with a of pure The with adsorbed material is usually named an solvent. adsorbate. HISTORICAL REVIEW
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fulness of the chromatographic method in many of the diverse fields where today it finds employment and he had great confidence in its efficacy as a tool.
process goes on in the upper layers of the tube. Where less tenaciously adsorbed substances had been retained in the first rush of adsorption these are now displaced by more strongly adsorbed substances brought by the COMPARISON OF ORDINARY A D S O R ~ I O N WITH TSWETT'S flowing liquid. The displaced materials wander down in the solvent stream to lower regions, to fresh adMETHOD sorbent, where they are again held, only to be again Zechmeister and Cholnoky (3) have drawn a graphidisplaced until all the most surface-active material is cal comparison which clearly shows the relation befixed. The next most active substance then is fixed tween ordinary adsorption and Tswett's method and which points the difference between the two: Method
Shaking with the< adsorbent
Separate Phases Oblaimble Total adsorbate
Unadsorbed material (filtered off in solvent) -- , Component with strongest gadsorption alikity
Tswett's chromatographic method
3
Component with second strongest adsorption atfinty Component with third strongest adsorption sty, and so nadsorbed component, 'ggfiltrate through the column: pass-
It can readily be seen from this diagram that Tswett's method is superior to the other method when fine separations are desired. THEORY
The chromatographic method separates the substances in a solution on the basis of their different surface or interfacial activities. According to the Gibbs Adsomtion Law anv substance that lowers surface tension or interfacial tension will concentrate a t interfaces. If a number of substances are compared with respect to their surface activities in one solvent and toward one type of interface, then these substances may be ranked, say, in descending order of their interfacial activities, or, if the interface is liauid-solid, of their adsorbabilities. TGS is exactly what happens i n the Tswett column. The substances in a common solvent are separated by their different affinities for a single adsorbent. It should be oointed out that adsomtion affinities are greatly influenced by changes in solvent, by the presence of other adsorbable substances, and by the nature and state of subdivision of the adsorbent. These effects will be discussed later. Consider what happens when a solution of mixed pigments is poured into the tube (refer to Figure 1). The solution cannot come into contact with all the adsorbent at once, only with the top layer. Nor can the adsorbent-solution interface draw from all the solution but only from the narrow lower region within its immediate vicinity. The adsorbent then "withdraws" by adsorption to the surface of its particles a mixture of pigments, perhaps with the more easily adsorbed oncs predominating. As the solution percolates downward a dynamic &
FIGURE1.-PORTION OF TUBE SHOWING COLUMN OP ADSORA , WrrH CHROMATOGRAM, B AND DEVELOP ME^. ARROWS INDICATE SPREADING OF ZONGS
BEm
DURINGDEVEWPMENI.
in its turn, displacing its less well adsorbed companions until in this way a series of zones is formed. These zones are the result of the previously mentioned adsorption-ranking of the components of the solution. This adsorption-ranking is probably not fully complete a t this stage. Each zone probably contains some of the other components. The chromatogram is now "developed" with fresh solvent. This development completes and clarifies the adsorption-ranking. As pure solvent is passed through the tube the dynamic processes a t the surfaces of the granules continue, with the following results: in the system solution-adsorbate there is a partition of
dissolved substances between the solution and the adsorbing surface. This partition obeys an equilibrium law. Also adsorption is relatively greater the more dilute the solution. As the solvent passes over a zone of adsorbed substance it picks up some of the substance to the extent permitted by the equilibrium. This it retains as long as the surface over which it passes is in equilibrium with it. However, as soon as the solution comes to a region "undersaturated with respect to the substance, adsorption begins and the solute is finally completely fixed on the adsorbent in a series of more and more dilute layers. The pigment content of the "developer" solvent has, as Tswett put it, started a t zero (pure solvent poured in), risen to a limiting value in the passage through the zones and finally fallen to zero as the substance was successively removed. This process reoccurs all along the adsorption tube. "Each unit of volume of the wash liquid fractionally washes out the pigment and transports i t to a lowerlying region of the column. The integral of all the partial processes sums up to a slow downward wandering of all the pigment components" (3). In general the more weakly adsorbed substances move downward more quickly, and the chromatogram is seen to expand, the zones separating and becoming more sharply delineated. When a sufficient degree of separation has been achieved the development is stopped. At this point the zones are clearly distinguishable. There is no well elaborated theory which predicts the behavior of an adsorbent toward different substances in different solvents. There are, however, a few general rules which are applicable (6). Among these may be mentioned that adsorption is generally greater from liquids of higher surface tension than from those of lower surface tension. Substances are usually adsorbed better from solutions in which they are less soluble, provided the liquids being compared are themselves about equally adsorbed. Also adsorption is greater from liquids which are themselves least adsorbed granting that their solvent powers for the dissolved substance are about the same. A useful generalization is the Traube Rule which states that within limits adsorption (from water) increases regularly as one ascends in an homologous series, such as the fatty acids. The reverse of this rule holds for adsorption from liquid hydrocarbons, such as toluene. One should note that the same adsorbent and solvent will always yield the zones in the same order. However, the place taken on the column will depend on the nature of the other substances present and their relative amounts. Much of the work done with Tswett columns is still perforce of an empirical nature. MATERIALS AND METHODS
For chromatographic adsorption one does not want an adsorbent which is too e5cient. One wants an adsorbent which will retain the required substances yet
have equilibrium relations with the solvent such that efficient development of the chromatogram is possible. The adsorbent must, of course, be insoluble in the solvent. Substances differ markedly in their adsorption behavior, and with a given substance the adsorptive power can be changed within rather wide limits by treatments such as drying or changing the particle size. In general the smaller the particle size (and hence the greater the surface area per unit of weight) the greater will be the adsorptive power of the adsorbent. A powder which is too coarse yields an indistinct chromatogram. The most commonly used adsorbents are aluminum oxide, which may be used in watery or organic solvents, calcium oxide, hydroxide or carbonate, and magnesium oxide. Many other substances such as gypsum, silica gels, carbon, anhydrous sodium sulfate, talcums, fuller's earth, sugars of various kinds are used for special purposes. Mixtures of adsorbents have been found useful in many cases. They may either be mixed homogeneously, the weaker "diluting" the stronger, or they may be placed one above the other in the tube, the weaker being above the stronger. The most generally used solvents are water and petroleum ether. Water is used, for example, with watersoluble dyes. Its high surface tension probably is responsible for aiding adsorption from it. Petroleum ether or light petroleum is the most commonly used of the organic solvents. Carbon disulfide or benzene have also beeu employed with much success as solvents. The column of adsorbent is prepared in the following manner. A glass tube of suitable size, depending on the amount of substance to be chromatographed, is closed a t the bottom with a perforated disc (Gooch or other disc), or fitted with a ground connection, or closed with a perforated cork, and so forth (Figures 1 and 2). Above the disc or constricted portion is placed a mat of cotton or glass wool of sufficient thickness to retain the adsorbent. The adsorbent is poured into the tube in small amounts, each addition being carefully tamped down with a glass or wooden rod, with a flattened end almost as wide as the tube itself. It is most important that the adsorbent be packed as homogeneously as possible. The regularity of the zones in the chromatogram depends to a considerable extent on this careful filling of the tube. It is a good idea to place a thin layer of cotton a t the top of the column so that when liquid is poured into the tube the adsorbent is not stirred up. A suitable height for the column is best determined by trial. Great length prolongs the filtration, but with too short a column i t may happen that only one pigment is retained, a single colored zone going throughout the column. After the column has beeu built up, the tube is mounted on a suction flask and mild suction is applied while the outside of the tube is gently tapped along the length of the column. This causes further settling of the adsorbent. Pure solvent is now poured
into the tube while the suction pump is still running and is allowed to wet the column. If the column is well packed the front edge of the solvent will advance down the column in a regular ring. Some workers prefer to suspend the adsorbent in a little of the pure solvent. The suspension is poured into the tube, under suction, whereupon a well packed column free from gas bubbles is usually obtained. As an alternative method, the tube may be partly filled with solvent and the adsorbent sifted in so that it settles to form an homogeneously packed column. After the column has been wet down with the solvent it is ready for use. It is important now not to allow the to^ of the column to become dried. The level of solven't or solution must not be permitted to fall below the top of the adsorbent for this will cause channeling or the development of air bubbles and cracks in the column which may prevent satisfactory zone formation. The test solution is poured onto the column and sucked down until it has almost disappeared into the column. The pure solvent is then drawn through until the column is sufficiently developed. At the end of the development the solvent is sucked through the column so that the cylinder of adsorbent is partially dried. When the column is pushed out of the tube it should retain its shape. If it is too dry it crumbles and is very bard to separate into zones. Of course it should not be too wet. It is convenient to have a glass or ~orcelaind a t e on which to extrude the column. If the substances being separated on the column are colored, i t is relatively easy to know where the zones begin and end, and hence where to cut. Many ingenious methods have been devised to carry the usefulness of the method over to colorless materials. There are five or six methods used in working with colorless materials. Four of these indicate the region of the zones with certainty. Perhaps the most striking method is that devised simultaneously by Karrer (7) and Winterstein (8). When working with substances which fluoresce in ultra-violet light these authors prepare their columns in quartz tubes, or tubes of special glass permeable to some ultra-violet. When the development is in process i t can be followed by examining the column under a quartz mercury arc lamp with a suitable filter to remove all except the ultraviolet radiation, for the different zones fluoresce brightly, often with different colors. When the column is extruded i t is placed under the ultra-violet lamp. The fluorescence of the different zones is an excellent guide to sectioning. Another method involves addition to the tube of a dye whose position on the column relative to that of the unknown substance has been sufficiently well determined. Brockmann (9) was aided in his isolation of one of the vitamins D by the use of an indicator which was adsorbed on his columns to the same extent as the vitamin was. The zones may be made visible by means of a color reaction For example, the extruded column may be streaked with a brush bearing the test reagent. At
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the region of zone formation the color reaction will appear. Zechmeister (10) and others have used this very effectively. Strain (11)devised still another way out of the difficulty. He changed his colorless compounds into brightly colored products before separation. He was working with colorless carbonyl compounds and by converting these into 2,4-dinitrophenylhydrazones he obtained brightly colored compounds which would form readily visible zones on the column. If the substance being studied is colorless, will not fluoresce, does not yield to the use of an indicator, gives no color reactions, and is not readily changed to a
)I 1
colored derivative, there are yet one or two expedients remaining. The column may be prepared and developed empirically, then cut up arbitrarily into different parts. Each section may then be eluted and the eluent solutions examined for differences in content. This is the empirical method, and on the face of i t one can see that it should be avoided whenever possible. Actual trial of the method lends point to this conclusion. A rather tedious method employs the "flowing chromatogram." Here the columh is continually washed with fresh solvent until the zones from the lowest up in turn are successively washed through the column. The percolate is examined from time to time as it comes through and is "cut" each time i t changes in properties. This type of chromatogram is usually necessary when the column is of carbon, say, on which colors cannot easily be observed. I t has the disadvantage that i t requires quite a long period of washing, but was found very useful by Holmes and his students in work on vitamin A (12). After the column has been developed and cut into separate portions with a spatula the adsorbed substance must be removed from its fixed position: it must be eluted, or desorbed. The best eluents are
usually solvent mixtures containing one or two per cent. of ethanol, methanol, or acetone. Thus petroleum ether containing one to two per cent. absolute methanol, or ether containing methanol are frequently used. Perhaps the presence of polar substances such as alcohols in the eluting solvent have the effect of displacing the adsorbed substance, its position on the adsorbent being taken by the alcohol. In the watersoluble realm dilute alcohol, or water containing pyridine, or slightly acid or basic watery solutions may give best results. Sometimes i t is necessarv to heat the mixture to obtain best elution. Adsorption usually decreases with rise in temperature. RELATION BETWEEN CHROMATOGRAM AND CONSTITUTION
In the discussion of theory i t was implied that position of adsorption on the column bears a relation to molecular constitution (as, for example, in Traube's Rule). There is indeed a very strong relation. It has been studied in relatively few cases, however, and wide generalizations cannot as yet be made; nor has there been a detailed study of how closely related two substances may be and yet remain separable by the Tswett method. Alpha- and beta-carotene, Wering only in the position of one double bond which is no longer conjugated, are separable on the column. Also cis- and trans-bixin have been successfully fractionated. On the other hand compounds are known which differ in structure to quite an extent, yet are not chromatographically separable. The relation between position on the chromatogram and structure can be shown very weU by two examples which will be given below. This does not exhaust the field on which data are available and many other examples are brought forward together with discussion of the whole matter in Zechmeister and Cbolnoky's excellent book (3). When the following diphenyl polyenes, which have the general formula CBH~-(CH=CH),-C~HF,, are separated on a column theftake the positions sh& in Table 1.
More GH-CH=CH-CH=CH-CH=CH-CH= strongly adsorbed diphenyl octatetraene
CH-&HI
C~H~CH=CH-CH=CH-CH=CH-C~HS diphenyl hexatrime
t
CsH8-CH=CH-CH=CH-&H5 diphenyl butadiene CsHsCH=CH-CbHs stilbene
Less strongly adsorbed
CsHsCsHa diphenyl
Each homolog takes a sharply defined place which may be clearly seen under the ultra-violet. The strength of adsorption is evidently correlated with the increase in number of conjugated double bonds.
From a chromatographic point of view the carotenoids, a naturally occurring group of polyene pigments, constitute one of the best studied series. Indeed, the chromatographic method has been the most valuable single tool in the investigation of these important substances. The following table (Table 2) shows an adsorption series among the carotenoids adapted from data collected by Winterstein and others.
Pigment Fornula Groups More Flavoxanthin CoHsaOl 3 --OH strongly Zeaxanthin C4~HsbO22 -OH adsorbed Lutein C,,HaaOz 2 -OH Kryptoxanthin CloHs10 1 -OH Rhodoxanthin Cl0HsoO2 diketone
/
C,?Hl1O4 ester Physalene (Zeaxanthin dipalm~tate) Lycopene C.OH~
Less 7-carotene strongly adsorbed 8-carotene a-carotene
CaoHsa CdIss C4~Hsa
Double Bonds 11, conjugated 10, conjugated 11, conjugated 12 carbon and 2 carbonvl all conjugated
11 conjugated, 2 isolated 11 conjugated, 1 isolated 11 conjugated, 0 isolated 10 conjugated. 1 isolated
There are a great many more ckrotenoids which may be included in a table such as this. However, i t is of adequate size to illustrate the following rules which are derived by Zechmeister and Cholnoky (3). With otherwise analogous molecular structure the adsorption affinity is stronger and the relative position in the tube is higher: 1. When the number of double bonds is larger (example: lycopene and 7-carotene). 2. When with the same number of double bonds all are conjugated (example: @- and a-carotene). 3. When in the case of identically unsaturated systems hydroxyls are present in the molecule (example: kryptoxanthin and 8-carotene). 4. When the number of OH groups increases (example: flavoxanthin and zeaxanthin). It is apparent too from the table that when an hydroxyl is esterified its activity is almost completely nullified. Thus physalene, a dipalmitate of zeaxanthin, falls far below zeaxanthin in activity. The position of an ester is, of course, influenced to some extent by both the acid and alcohol parts of the molecule. From the examples given and from a great deal of other data it is also derivable that adsorbability and light-absorption are correlated. Thus the absorption maximum of the carotenoids is displaced toward the red end of the spectrum with increase in the number of conjugated double bonds, and it is the substance with the longest waved absorption band which, other things being equal, is adsorbed most strongly: lycopene 548 m&,p-carotene 521 mp. In the case of the aromatic polynuclear compounds linear arrangement seems to impose a greater strain on the bonds, with concomitant appearance of color,
The chromatographic method is amenable to quantitative handling in its various applications. A word or two of warning is necessarv with regard to . . . . AA chromatographic analysis As yet it Gas not been de(orange-red).over cbrysene veloped to the point where it can be used advantage'4'4 ousl; in place ofthe recognized chemical methods where (colorless) (13). these are applicable. But if fractional distillation, Over the range these there is evidence fractional crystallization, solvent partition, and so forth, of a conformity to law. However, the law cannot be fail to effectthe separation of a mixture, then chromacompletely stated as yet for it must take into account tography ~houldby all means be tried. However, in not only the substance adsorbed, but also the solvent with unstable substances it is very importaut and the adsorbent, and no complete, unifying generali- to make that the processes of adsorption and zation of this sort exists. There lies here a fertile field elution have not produced changes in the substance. for research. The catalytic effect of surfaces often shows up to a FIELD OF APPLICATION marked degree in the column. Thus Gillam and El The chromatographic method has a wide field of use- Ridi (16) have been able to change p- and or-carotenes fulness. In the laboratory it may be employed to indi- into other carotene forms by this procedure. cate whether or not a substance is pure, for in passage SUMMARY through a column impurities are often concentrated A brief review of chromatographic adsorption analyuntil they are plainly visible. This idea has been extended and is used by Kuhn to test the identity of sis has been given. The method lay unused for twenty two compounds. The compounds are mixed and years but is now beginning to be extensively employed. The theory of chromatography has been very briefly passed into a column. If they yield only one zone they are probably identical. This is called a "mixed sketched. chromatogram" and is analogous to a mixed melting The method of preparing a column has been discussed and several hints obtained as to criteria for point. From a preparative standpoint the method of chroma- choosing adsorbents and solvents. Some correlations have been drawn between adtography is also of great service. It may be used to concentrate a substance from a large volume of liquid, sorbability and chemical constitution, but it has been since a large amount of solution may be passed through made evident that a wide and fertile field yet remains for investigation here. a small tube. The method has found its way into several books on A very few of the applications of the method have organic laboratory technic (14, 15). A good discus- been mentioned, and warning has been given that unsion, with experiments, is found in A. A. Morton's stable substances are subject to alteration during book (15). chromatography. than angular junction, and i t is the linearly arranged compounds which are better adsorbed: naphthacene
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( > C a
LITERATURE CITED
TSWBTT, M., "Physikalisch-chemische StudienTuberTdas
Chlorophyll. Die Adsorptionen," Bcr. deut. botan. Ges.. 24, 316-23 (Ju!y, 1906). Tswsrr, M.. "Adsorpt~onsanalyseund chromatographische Methode. Anwendung auf die Chemie des Chlorophyll~," ibid., 24, 384-93 (Aug., 1906). ZEC~MEISTER, L. and L. "ON CAOLNOKY, "Die Chromatographische Adsorptionsmethode. Gmndlagen, Methodik. Anwendungen," Julius Springer, Vienna, 1937. A new, expanded edition of this excellent book is now available, and, according t o Dr. Zechmeister, it is being translated into English. PALMER, L. S., "Carotenoids and related pigments. The chromolipaids," The Chemical Catalog Co. Inc., New York City, 1922. KUHN, R. AND E. LEDERER, "Zedegung des Carotins in seine Komponenten. (Uber das Vitamin des Wachstums. I . Mitteil.)," Ber., 64, 1349-57 (June, 1931). HOLMES, H. N., "Laboratory manual of collo~dchemistry," 3rd ed., John Wiley and Sons, Inc. New York City,
.
n2"
-7-.
KARRER, P. AND K. SCAOPP,"Filtrationen bei tiefer Temperatur. Chramatographische Analyre farbloser Subs t a n z a (Ultrachramatogramm)," Helm. Chim. Acta, 17, 693 (1934). WINTERSTEIN. A. AND K. S C H ~ N "Fraktionierung , und Reindarstellung organischer Substanzen nach dem Prinzip der chromatographischen Adsorptionsanalyse. 111
Mitt.: Gibt es ein Chlorophyll c?' Z. physiol. Chem., 230, 1 3 9 4 5 (1934). H.. "Die Isolierung des antirachitischen Vita(9) BROCKMANN, mins aus Thunfischleberiil," ibid., 241, 104-15 (1936). AND E. UJHELYI, (10) ZECWIEISTER, L.. L. VON CAOLNOKY, "Contribution A la chromatogaphie de substances incolores," Bull. soc.
