It is a fact, already long known, that different organic liquids that serve as solvents for chlorophyll are very unequally suitable for the extraction of leaf green from leaves. While alcohol or ether gives intensive dark green extracts, other solvents, such as the aliphatic or cyclic hydrocarbons and carbon disulfide, give more yellowish extracts, much poorer in chlorophyll even when extraction of dry material is carried out. The most characteristic in this respect are petroleum ether and petroleum benzine, which when brought into contact with fresh leaves or those dried at low temperature give mostly more or less pure yellow extracts, colored by carotin, on which property Arnaud based his process for preparation of this pigment from leaves.
If the chlorophyll itself is completely soluble in petroleum ether, as is generally accepted, why is it not removed from fresh or dried leaves in this solvent? Why is only a yellow portion dissolved?
This problem, no less important for the question of the physical constitution and chemical composition of the chlorophyll apparatus, has remained unsolved up to now. Because of the brilliant development of the chemistry of chlorophyll derivatives, many questions of broad physiological interest have been neglected. The few investigators who have turned their attention to the phenomena with which we are concerned have offered different and contradictory explanations, among which not one can be said to stand on a broad experimental basis.
Thus far the literature dealing with our problem. When I turn to the experimental treatment of this question, it has first of all seemed worthwhile to me to test more closely in situ the effect of various indifferent organic solvents on the leaf pigments.
Among the numerous materials used I found especially suitable Plantago species and Laminum album. The softness of the Laminum leaves and the approximate neutrality of their tissue juice marked them as especially suitable objects.
Their behavior toward the leaf pigments permits us to divide the solvents tested into three groups:
1. Alcohols (methyl, ethyl, amyl), acetone, acetaldehyde, ether, chloroform. These solvents, acting on fresh (ground) or dry leaves, dissolve all the pigments equally and abundantly.
2. Petroleum ether and petroleum benzine. Fresh leaves finely ground with sand or emery and again ground under the solvent give more or less pure yellow extracts that are chiefly colored by carotin, but also contain traces of the other pigments. The carotin can be completely extracted in this way. Dried leaves (dried at low temperature!) likewise give their carotin to the solvent, and in somewhat purer condition. Tissues cooked or warmed at higher temperatures, however, when ground with the solvent give a green extract, a fact which will be explained later.
3. Benzene, xylene, toluene, carbon disulfide. They have an action on leaf pigments intermediate between the solvents of the first groups.
As mentioned, petroleum ether dissolves only traces of the other pigments besides carotin. However, it is enough to add some absolute alcohol (10 per cent for fresh leaves, 1 per cent for dry ones) in order to obtain a richly colored, beautiful green solution. Acetone or ether has an analogous action.
The total chlorophyll can be extracted with petroleum ether containing alcohol. What is the significance of this "solubilizing" action of the alcohol? Since, with pure petroleum ether, one component of the chlorophyll, carotin, is very well extracted, we cannot believe that the solvent does not reach the chlorophyll. A chemical action of the alcohol is excluded here, as the following investigation shows. Fresh leaves were ground with emery and the resulting homogenate was treated with about 40 per cent alcohol. If the material was then immediately treated with petroleum ether, a green solution resulted, but if it was dried at 45°, petroleum ether gave only the usual yellow carotin solution. The alcohol must therefore act simply by its presence physically, and not chemically. Actually, the pigments, recognizably soluble in pure petroleum ether, after alcohol extraction can again become insoluble in the solvent.
My first study (1901, III) in this direction was as follows. Alcohol-petroleum ether solutions of chlorophyll were digested in a flask with several strips of filter paper and the solvent was distilled off in a vacuum; by this treatment the pigment was taken up by the paper. The dry green paper now behaved toward solvents exactly like the green leaves, and pure petroleum ether took up only the carotin, while the addition of alcohol produced decolorization of the paper at once.
The phenomena mentioned at the beginning of this paper, which still remained puzzling, therefore depended on adsorption of the pigments, on the mechanical, molecular affinity of the substances for the chloroplast stroma which could indeed be overcome by alcohol, ether, etc., but not by petroleum hydrocarbons. However, if the pigments were removed from the sphere of molecular forces, as, for example, by cooking or warming the tissues, which, as is well known, forces little green droplets from the chloroplasts, then these pigments dissolved easily in petroleum ether and the dark green extract was obtained.
It follows from the foregoing that it is impossible for chlorophyll to be enclosed in the chloroplasts in the form of microscopically definable grana and it must be that the grana themselves possess an insoluble adsorbing substrate. Moreover, the grana theory is not well grounded micrographically.
It was mentioned above that the chlorophyll pigments (except for carotin) bound to the filter paper from the petroleum ether were held firmly by an adsorption force. As expected, these pigments were taken from the petroleum ether solution by the filter paper. However, not only cellulose but all solid bodies insoluble in petroleum hydrocarbons adsorb chlorophyll and, if used in finely powdered condition, decolorize petroleum ether partly or completely. From this point of view I have studied more than a hundred substances belonging to different chemical systems and always with essentially the same result. I will give here a short summary of the substances tested.
Simple substances (S, Si, Zn, Fe, Al, Pb, Sb); oxides (SiO2, MgO, MnO2, PbO, Sb2O3, Fe2O3, Ag2O, HgO, U3O8); hydroxides (B(OH)3, NaOH, Ba(OH)2, Al(OH)3); inorganic chlorides (Na, K, NH4, Ca, Mg, Al, Fe, Co, Cu, Hg); chlorates (K, Ba); potassium bromide, potassium iodate; nitrates (K, Ca, Ba, Pb, Ag, Cu, U); phosphates (K, Na, NH4, Fe); sulfides (K, Hg); sulfite (Na); sulfates (K, Ca, Mg, Ba, Zn, Fe, Mn, Cu); carbonates (Na, K, Ca, Mg, Fe); silicates (K, asbestos); ammonium molybdate, potassium permanganate, potassium ferricyanide and potassium ferrocyanide, oxalic acid, tartaric acid, citric acid, quinic acid, tannic acid, uric aid, picric acid, phenolphthalein; oxalates (NH4, Mn); acetates (Pb, Cu); amides (urea, asparagines); higher alcohols and carbohydrates (mannite, dulcite, sucrose, galactose, inulin, dextrin, amylose); proteins (egg, albumin, peptone, hemoglobin); trioxymethylene, chloral hydrate, hydroquinone, resorcin, pyrogallol, aniline dyes (gentian violet, chrysoidin, etc.); finally a series of chemically undefined substances (bone meal and blood meal, soil, kieselguhr).
Some of these substances can also carry down carotin from its petroleum ether solution (HgCl2, CaCl2, PbS, etc.). Many bodies decompose the pigments adsorbed on them. Some, for example (MnO2, KMnO4, U3O8), destroy the chlorophyll completely, obviously by oxidation. Others act on the chlorophyllines in the well-known manner of acids; these include the acids mentioned, acid salts, and many neutral salts whose water solutions can acquire an acid reaction by hydrolysis. This is not the place to discuss more fully the type and manner of these chemical reactions. We shall speak more fully of the method of adsorption and its analytical use. In order to obtain a petroleum ether solution, the best procedure is the following. Fresh leaves (best from Lamium) were ground in a mortar with fine emery and again extracted with petroleum ether containing alcohol (10 per cent). The green solution was repeatedly washed with twice the volume of water in a separatory funnel.
The alcohol had a greater affinity for water than for petroleum ether and so it could be separated practically completely from the petroleum ether in this way. The washed green solution, usually somewhat cloudy, was not clarified by centrifuging or filtration and was suitable for adsorption studies.
The most suitable adsorptive materials were precipitated calcium carbonate, inulin, or sucrose (powdered).
If the petroleum ether chlorophyll solution was then shaken with the adsorptive material, the latter carried down the pigment, and with a certain excess of this, only the carotin remained in solution, escaping adsorption. In this way a green precipitate and a pure yellow, fluorescence-free carotin solution were obtained (test for fluorescence in my luminoscope). This carotin solution showed a spectrum with absorption bands at 492-475 and 460-445 μμ. If it was shaken with 80 per cent alcohol, the lower alcohol-water phase remained completely colorless.
The green precipitate was then brought onto a filter and carefully washed with petroleum ether to separate the last traces of carotin. The filtered yellow liquid could be immediately regenerated with bone meal. Then the precipitate was treated with petroleum ether containing alcohol, which completely decolored it and gave a beautiful green solution which could then be separated by 80 per cent alcohol by the method of Kraus. The petroleum ether phase, colored blue-green, contained chiefly the chlorophyllines, while the lower yellow phase contained chiefly the xanthophylls.
If the petroleum ether solution of the chlorophyll was treated with the adsorption material not in excess, but in portions until the fluorescence vanished, then along with the carotin the xanthophylls also remained in solution. These could be freed from carotin by again treating the decanted solution with the adsorption material and liberating the pigment from the resulting adsorption compound with petroleum ether containing alcohol. The solution of xanthophyll mixture thus obtained shows the following absorption spectrum: 480-470 and 452-440 μμ. If it was shaken with 80 per cent alcohol, the pigment remained almost completely in the alcoholic phase.
The physical interpretation of the adsorption phenomena that we have considered will be discussed elsewhere. However, here we can mention some related regularities and the resulting applications. The adsorption material saturated with a pigment can still take up another member of a certain mixture and hold it firmly. Substitution can also occur. For example, the xanthophylls can be partly displaced from their adsorption compounds by the chlorophyllines, but not the reverse. There is a definite adsorption series according to which the substances can substitute. The following important application comes from this rule. If a petroleum ether solution of chlorophyll filters through a column of an adsorption material (I use chiefly calcium carbonate which is firmly pressed into a narrow glass tube), the pigments will separate according to the adsorption series from above downward in differently colored zones, and the more strongly adsorbed pigments will displace the more weakly held ones which will move downward. This separation will be practically complete if, after one passage of the pigment solution it is followed, by a stream of pure solvent through the adsorbing column. Like the light rays of the spectrum, the different components of a pigment mixture in the calcium carbonate column will be separated regularly from each other, and can be determined qualitatively and also quantitatively. I call such a preparation a chromatogram and the corresponding method the chromatographic method. In the near future I will give a later report on this. It is perhaps not superfluous to mention here that this method in its principle and also in the exceptional ease of carrying it out has nothing to do with so-called capillary analysis.
Up to now we have considered only the adsorption of chlorophyll pigments from petroleum ether solutions. However, adsorption also occurs from benzene, xylene, toluene, and carbon disulfide solutions. From benzene indeed almost the chlorophyllines alone are adsorbed, in much less degree than from petroleum ether. It seems, however, that adsorption from a CS2 solution obtained simply by treatment of the ground leaves with the solvent is especially suitable for chromatographic analysis. In CS2 the different pigment zones have a much brighter color than in petroleum ether, especially the xanthophylls which in it are very sharply differentiated from each other. Carotin passes through as a rose-colored solution.
It is obvious that the adsorption phenomena described are suitable not only for chlorophyll pigments, and it can be assumed that any sorts of colored or colorless chemical compounds will follow the same rules. Up to now I have studied lecithin, alkannin, prodigiosin, sudan, cyanin, and solanorubin as well as the acid derivatives of chlorophyllines with positive results.
If a mixed solution (e.g. a chlorophyll solution in CS2) filters through a column of adsorbent, the pigments precipitate in the manner [similar] to adsorption, mutually repel each other however and arrange themselves according to the adsorption series in the direction of the stream. Substances which do not enter into any undissociable adsorption compounds with the adsorption medium used wander away more or less quickly through the column. Subsequent filtration of the pure solution medium will understandably make the separation of the substances more complete. It can however be supposed that two substances in a solvent might be adsorbed to the same degree. Relative differences in concentration of the two substances would however not allow the formation of a unitary mixed zone. Also the equal potency of two substances in different solvents can scarcely be imagined. In spite of all this, although the number of adsorption zones will correspond to the number of substances, it can happen that any one zone is not absolutely pure, as is to be concluded from what is said above. By extraction of the substance of one zone and renewed adsorption one will reach the desired degree of purity.
We see thus that the laws of mechanical affinity may be used for the most complete physical separation of the substances soluble in certain fluids.
As means of adsorption any one of the powdered substances insoluble in the solute concerned can serve. As however very many substances do not remain without chemical effect on the adsorbed substances, the choice of the analyst will fall in general on such bodies as are chemically indifferent and at the same time can be brought into as fine a form as possible... Amongst the adsorption means I can provisionally recommend precipitated CaCO3 which gives he most beautiful chromatograms.
The green pigment of the leaves, the chlorophyll, is known to be a mixture of pigments, the complexity of which was differently estimated by different investigators. Chromatographic analysis is called upon to settle finally this degree of complexity. Compared to the other methods, it behaves like spectral analysis of the colour of a substance in comparison with analysis by means of tinted glass specimens ...
The chromatograms obtained from a CS2 solution have the following form: