M. Tswett: "Adsorption Analysis
Translated by Harold H. Strain
Argonne National Laboratory Argonne, lllinois 60439 and Joseph Sherma Lafayette College Eoston, Pennsylvania 18042
and Chromatographic Methods"
I
"Application to the Chemistry of the Chlorophylls"
I n the preceding part of this Journal I have reported the remarkable adsorptions which the chlorophyll pigments, in petroleum ether or carbon disulfide solution, exhibit for solid substances. It was shown there how one can obtain important separations by fractional adsorption, for example, the quantitative separation of the carotene from the other leaf pigments. Further applications of the method will be presented in later articles. Here, however, a second more significant modification of adsorption analysis, which I have designated as the chromatographic method, will be discussed. Besides, I shall compare capillary analysis with the new method with respect to its characteristics and applicability. Principles
Many colored substances (and of course also colorless substances), which are soluble in petroleum ether, benzene, xylene, carbon tetrachloride, or carbon disulfide, will he precipitated physically from the respective solutions by powdered substances, because a quantity of the dissolved substance is adsorbed (i.e., condensed) on the surface of the solid particles. The partition of the substances between the solvent and the adsorbent does not obey Henry's Law, as is known for many adsorptions (cj. van Bernrnelen), and the partition coefficient is dependent upon the concentration. For some solutes and solvents, this coefficient becomes infinitely small, and the dissolved substance is then completely carried down and cannot be washed out by the pure solvent. Truly undissociated adsorption compounds are formed. I n addition to this firmly bound quantity of dissolved material, the adsorbent is able to condense even further quantities, so that perhaps Henry's Law is applicable after all. The excess "adsorbate" can be completely removed by means of pure solvent. The adsorbed substances are set free from their adsorption compounds by alcohol, ether, acetone, chloroform, or by the addition of these liquids to the before-mentioned solvents. An adsorbent, which is saturated with a substance, is able to adsorb a small amount of a second, so that substitutions can take place. If a substance B can he dissolved out of its saturated adsorption compound by a substance A, then the reverse is impossible. There is an adsorption series by which substances can be substituted, but this depends on the solvent. 238
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From the preceding i t follows: if a solution of a mixture (for example a chlorophyll solution in CS,) is filtered through a column of an adsorbent, the pigments are held in relation to their adsorption affinity,displace one another in relation to this, and arrange themselves according to the adsorption series in the direction of the flow. Substances which do not form an undissociated adsorption compound with the employed adsorption medium migrate more or less quickly through the column. Subsequent filtration of the pure solvent ~vill, naturally, make a complete separation of the substance. But it is conceivable that two substances in one solvent exhibit the same adsorption rank. Relative concentration differences of the two substances will certainly not permit the formation of a uniformly mixed zone. Even the equal potential of two substances in different solvents can scarcely be imagined. Despite all this, even though the number of adsorption zones corresponds to the number of substances, it may happen that a zone may not be absolutely pure, as can be concluded from what was said above. By extraction of the material from a zone and readsorption, one obtains the desired purity. Consequently, we see that the laws of mechanical affinity are applicable to the most complete physical separations of substances dissolved in certain solvents. Chromatographic Apparatus
In order to obtain the composition of a solution of a pigment mixture in a few minutes, one uses an apparatus such as number 1in Figure 1. Provided with the manometer M , a 3-1 bottle R serves as a pressure reservoir, in which a certain air pressure can be produced through the tube D by means of a rubber bulb P.
The paper here presented in translation from the original Gennan is one of the true milestones in chemical literature. Not only does it describe the essential techniques of chromatographic analysis, but it also introduced the nomenclature that is now universally accepted. For the translation, we acknowledge gratefully the permission of Gehruder Borntrager, Berlin, West Germany, publishers of Berichte der deulscha botanischen Gesellschajt, where this paper first appeared in Vohune 24, page 384 (1906). The original bears the data "Submitted July 21, 1906."
Figure 1 .
Chromotogrophis apporotu. for use with pressure, 1-3;
and suction, 4; with typical chromatogmm, 5.
For explanation, see the text.
Technique
Too strongly adsorbing substances should not he used, because they require enormous quantities of pigments in order to provide differentiations. Fineness of the adsorptive powder is very important; coarse-grained materials yield indistinct chromatograms, because in them diffusion and adsorption interfere in the large capillary spaces. Among the adsorbents, I currently recommend precipitated CaC03,which yields the most beautiful chromatograms. Also, sucrose is rather easily powdered and offers the greatest guarantee of chemical passivity. For special purposes, however, one may turn directly to chemically active (hydrolytic, reducing, oxidative) adsorptive materials. More about it elsewhere. I n order to develop its full adsorption power and to establish a uniform diiusion of substances, the adsorbent should be as dry as possible. Generally, I dry my CaCOa for two hours a t 150°C and preserve it in tightly closed bottles. A thick plug of wadding is first pressed to the base of the adsorption tube; then the powdered adsorbent is poured in and carefully tamped with a tight-fitting glass or bone rod. The homogeneous texture of the adsorbing column is very important, otherwise the various adsorption zones are formed so very irregularly that their mechanical separation is very difficult. When the adsorption column has reached the desired height2-I use at most 20-30 mm for t,he
With the appropriate solvent, every insoluble, powdered substance can serve as an adsorption medium. Because very many substances are not without chemical effect on the adsorbed substances, the choice of the analyst will in general be those solids which are chemically indifferent and which can be finely powdered.
The head of the Freudenreich culture vessels employed in bacteriology may also be used. Great heights retard the filtration. With too little height, however, (if much pigment solution is filtered through) a single pigment zone may be carried through; hence the height, besides other conditions, should be strictly controlled.
By means of the pinchcock Q, P is closed off from the rest of the apparatus. Tube D serves as a pressure distributor; it is provided with a number of tube-shaped attachments to which the actual filtration vessels are to be attached. Number 2 of the figure represents such an apparatus.' This consists of a cylindrical or amphora-shaped reservoir (r), which extends into a cylindrical part f (3040 mm long, 2-3 mm diameter). The attachment j is slightly constricted at the lower end so that the opening narrows and forms a support for the adsorbent that is to be added through the top. The small filtration funnel is connected with the pressure distributor D by means of a tight-fitting stopper, which is provided with a glass tube and rubber tube in a suitably movable connection (number 3). Pinchcock q allows each small funnel to be isolated from the rest of the apparatus. This apparatus is very suitable for the rapid separation of small quantities of pigment solutions. If larger quantities of the pigments are to be obtained in the form of adsorption compounds, in order to study each pigment separately, a diierent experimental arrangement is preferred. A larger adsorption filter (10-20 mm diameter), which is inserted into the neck of a vacuum flask, is now used (number 4).
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small columns and 40-50 mm for the large--a second plug of wadding is pressed in and only a little of the solvent to he used for the adsorption of the solution is poured in, thus facilitating the saturation of the adsorption column. This protective wadding is then removed. If this described saturation is not utilized, it often happens that the further addition of the liquid causes the upper layers of the adsorptive powder to lift u p probably due to the action of the liberated Pouillet heat of wetting-causing air bubbles to form and stir up the adsorbent, thus interfering with the regular course of the chromatography. For filtration of the solution under examination, I employ a pressure of 250-300 mm; for work with a large adsorption tube, I employ the full suction of a water aspirator. If there is a certain quantity of liquid to he percolated through, a stream of the pure menstruum is arranged so that the various zones spread out a little and yield their final maximal differentiation. The nonadsorbed substances are completely washed through, and substances which form appreciably dissociated adsorption compounds with the utilized powder migrate slowly through in the form of rings, and can be recovered separately at the mouth of the filtration tube. When the chromatogram (one is generally dealing with colored substances) is finally differentiated, it is freed from excess solvent by means of pressure or vacuum and pushed out of the tube without loss of form and fractionated with a knife. Application to Chlorophyll Analysis
The green pigment of leaves, chlorophyll, is recognized as a pigment mixture of varying complexity as estimated by different investigators. Chromatographic analysis is qualified to finally determine the degree of this complexity. I t is related to other methods as spectral analysis of a colored substance is related to analysis with colored glasses. The following procedures are suited to the preparation of appropriate solutions: 1 . Extraction of the materid, whieh has been rubbed with the finest emery and neutralized with some MgO and CaCOa, by means of alcohol-petroleum ether (1:10), and removal of the alcohol by careful washing with distilled water (cf. Tswett 111). This washing must be especially thorough, otherwise the remaining traces of alcohol (and water) form separate phases on the surface of the adsorbing particles, and one obtains indistinct chromatograms. 2. Extraction of leaves, which are first boiled for several minutes in water and triturated, with pure petroleum ether. Some decomposition products are formed. 3. Extraction of triturated and neutralized leaves by meansof Cslls, CCls or pure C&. All pigments are extracted. CS1 is usually employed. 4. Extraction of triturated and neutraliaedleaves with alcohol, acetone, ether, or chloroform; evaporation of the menstruum in vacuum; dissolution of the residue in petroleum ether or CR. Chemical decompositions are difficult to avoid. The pigments can be transferred from the alcohol (by the addition of water) directly to petroleum ether. Subsequent extraction with water.
Ideally, the chromatography must take place in very weak light, especially when working with CeHa or CSz solutions. From a CS2 solution, the following chromatogram was obtained: I . Uppermost zone. Colorless. The substance or mixture comprising this zone is hypophasic in the Kraus partition procedure. It remains predominantly in the lower phase. 240
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Journal of Chemical Education
I I . Z a e , especially poorly separated from the following. Yellow, due to xanthophyll @ . 3 This pigment is hypophasic in the Kraus partition procedure. Characteristic absorption bands of the alcoholic solution: 475-462 and 445430 mp. The alcohol solution quickly turns blue with a little HC1. The pigment can be isolated from the others by passing a stream of one percent alcohol in petroleum ether through the chromatogram. All pigments except xanthophyll ,3 are quickly washed out; the latter is removed by 10% alcohol-petroleum ether. III. Zone. Dark olive-green. Chlorophyllin @. Epiphasic in the Kraus partition procedure. The principal absorption of the petroleum ether solution a t 450465 m p becomes 46M75 mp in alcoholic solution. A second absorption a t 640-650 mp (petroleum ether) ; a third at 580-600 mp. Chlorophyllin B was discovered by Sorhy (1873) and not by Marchlewski and C. A. Schunck, as is stated erroneously by some recent authors4 (cf. Czapek; Kohl, I., 139; Strasburger, 656). Sachsse (p. 332) (who worked with Kraus' method) bad also observed the red absorption band of this chlorophyllin, but he attributed it to the xanthophyll. For my part, I checked Sorby's work in 1901. (Tswett 11.). Sorby's reasoning was correct; Marchlewski and Schunck have only repeated his experiments as well as Hartley's chemical experiments. They were unlucky concerning Sorby's work in that they did not obtain sufficiently pure substances, and the true absorption relationships of chlorophyllins u and 9, in the blue-violet part of the spectrum escaped them. A second established error concerning chlorophyllin p is that this pigment occurs in very small amounts and exerts no noticeable influence on the absorption spectrum of an unrefined chlorophyl: solution (cf. Marchlewski and Schunck, II., 258; Kohl, I., 139). A glance a t my chromatograms shows that no small amounts of chlorophyllin @ accompany chlorophyllin or. On the other hand, the I . absorption band in the blue-violet half of the spectrum of a chlorophyll solution is chiefly due to chlorophyllin 6, as shown by Preyer's alkali test (p. 50) and Hagenbach's elegant fluorescence experiments. If the blue absorption maximum of chlorophyllin 0 in alcohol lies a t 460-475 mp, one may assume that in the living leaf it will occur at a wavelength corresponding to line F. The second assimilation maximum at line F, established hy Engelmann and more recently by Kohl (III.), must be attributed to this chlorophyllin & not to carotene. I V . Zone. Dark blue-green. Due to chlorophyllin or (Sorby's blue chlorophyll). Contaminated with a little 8 My xanthophyll 0 (apparently identical with Sorby's "yellow xsnthophyll") has nothing to do with the similarly but unsuitably named pigment reported by Kohl (I., 140), because the latter d w r not I~eloz.gto the ~ n t h o p h y l ymup l I,ut repre.cl.r.i a w31crsoluldr sniinrt of the type
