HAROLD H. STRAIN
Vol. 57
PAPER CHROMATOGRAPHY O F CHLOROPLAST PIGMENTS : SORPTION AT A LIQUID-LIQUID INTERFACE BY HAROLD H. STRAIN Argonne National Laboratory, Lemont, Illinois Received March 8, 1065
The interface between two immiscible liquids, such as water and petroleum ether, exhibits selective affinity for chloroplast pigments. With water or aqueous solutions 6xed in porous filter paper, this sorption a t the interface between the liquids serves for the chromatographic separation of mixtures of chloroplast pigments dissolved in petroleum ether. With counter flow of water droplets and petroleum ether solutions of the pigments, sorption a t the liquid-liquid interface may be utilized for the continuous resolution of the mixtures into two principal fractions, the more sorbed and the less sorbed components.
Introduction The resolution of mixtures by chromatography has usually been attributed to three principal distribution phenomena. These selective distribution processes are the sorption of solutes at the interface between a solid and a liquid or gas, the partition of solutes between a fixed, dispersed liquid and a mobile solvent, and the distribution of a solute between a chemically reactive substance as an ion exchange resin and a liquid or gas. Many of these distribution processes have also been employed in paper chromatography. Paper chromatography by adsorption on the paper itself was utilized for the separation of carbon disulfidesoluble chloroplast pigments by Brown2 in 1939. It was applied to the one-way and two-way separation of water-soIuble dyes by Liesegang3 in 1943. Paper chromatography by partition of the solutes between immiscible liquids was utilized for the separation of mixtures of amino acids by Consden, Gordon and Martin4 in 1944. Reversal of the liquid phases in the paper was introduced by Boldingh6 in 1948. Mixtures of chloroplast pigments have now been resolved chromatographically by sorption on the surface of a liquid fixed in paper. The paper was either of cellulose or of glass fibers. The pigments themselves were insoluble in the fixed liquid which was modified so that it eluted the pigments from the surface of the glass or cellulose fibers. Separate experiments demonstrated that the pigments were sorbed at the liquid-liquid interface in the absence as well as in the presence of the supporting paper. With paper as the sorbent or as the su$porting medium and by suitable selection of fixed and mobile solvents, a single mixture of chloroplast pigments has now been resolved by sorption on the cellulose or glass, by sorption on a liquid and by partition between two immiscible liquids with either liquid fixed in the paper. The sequence of the pigments in the chromatograms varies with the solvent and the sorbent as already reported for analogous separations in columns.6*7 (1) H. H. Strain, Anal. Chem., 21, 75 (1949); 22, 41 (1950); 23, 25 (1951); 24, 50 (1952). (2) W. G.Brown, Nature, 143, 377 (1939). (3) R. E. Liesegang, Nalwwissenseliaflen, 31, 348 (1943); Z. anal Chem., 126, 172 (1943). (4) R. Consden, A . H. Gordon and A. J. P. Martin, Bioehem J . . 38, 224 (1944). ( 5 ) J. Boldingh, Experientza, 4, 270 (1948). (6) H.H.Strain, I n d . Eng. Chem., Anal. E d . , 18, 605 (1946). (7) H.H.Strain, J . A m . Chem. Soc.. 7 0 , 588 (1948).
Materials.-Cellulose paper was Eaton-Dikeman Grade 301, 0.03 or 0.05 inch thick. Glass paper was obtained from the Naval Research Laboratory.* For the separation of pigments by sorption on the surface of the glass or the cellulose, the paper was used without special treatment or after drying at 100". For the separation of pigments on the surface of a fixed liquid, the paper was moistened with water and with solutions of polyhydroxy compounds such as glycerol or sorbitol. The solutions ( l O ~ o )in water were poured over the paper, t.he excess solution was poured off, and the moist paper was pressed between dry paper for several hours. The paper was also moistened with glycerol or sorbitol in methanol and was then allowed to stand until all the methanol had evaporated. For a few separations, the paper was moistened with solutions containing mixtures such as glycerol plus glycine and urea, and glycerol plus methanol (10%). The solutions of the polyhydroxy compounds were utilized to prevent dehydration of the paper and also t o prevent sorption of the pigments on the fibers of the paper because the polyalcohols and acids are known to elute traces of sorbed xanthophylls from polysaccharides and from glass.@ For the separation of the pigments by partition between immiscible solvents, the paper was sprayed with 70, with 80 and with 90% methanol or dipped into these solutions and blotted. I n order to reverse the phases and to fix hydrocarbon material in the paper, a solution of vaseline (petrolatum) (about 5 % ) in petroleum ether was poured over paper which was dried in a hood. The pigment mixtures to be examined by adsorption were prepared from plant material. For preparation of the pigments of green leaves, fresh grass (10 g.) was cut into sections 1 to 2 mm. long and suspended in 100 ml. of methanol containing about 30 ml. of petroleum ether. After about 0.5 hr., the liquid was decanted through a small piece of cotton into a separatory funnel. Aqueous salt solution (about 400 ml.) was added, and after gentle swirling of the solutions, the aqueous layer was removed. The deep green, petroleum ether solution was washed once or twice with water. Owing to the lability of the pigments, this solution was protected from bright light, and was not employed after it had stood for more than a few hours. The xanthophylls neoxanthin and violaxanthin were prepared from the leaf extract by chromatbgraphic adsorption.1° The similar fucoxanthin was prepared from extracts of brown algae." Carotenes were obtained from carrots. Separations in Paper.-The mixtures were resolved in paper strips 3 X 20 cm. The solution of the mixture was placed in a spot about 7 mm. in diameter near one end of the strip which was stood in a little wash liquid contained in a covered, 2-1. beaker. Because of the rapid flow of the wash liquid into the porous paper, the separations were complete in 20 to 40 minutes. The separation of leaf pigments by sorption on cellulose paper, by sorption on glass paper, and by sorption on these papers moistened with water or with aqueous glycerol is illustrated by Fig. 1. Similar separations were obtained with paper moistened with glycerol in methanol and dried (8) T. D. Callinan, R. T. Lucas and R. C. Bowers, Naval Research Laboratory. May 1951, Washington, D. C. (9) H.H. Strain, Ind. Eng. Chem., 42, 1307 (1950). (10) H.H.Strain, W. M. Manning and G. Hardin, Bid. BUZZ., 86, 169 (1944). (11) H. H. Strain and W. M. Manning, J . Eiol. Chem., 146, 275 (1942).
PAPERCHROMATOGRAPHY OF CHLOROPLAST PIGMENTS
Oct., 1953
,Liquid front Y Carotenes
(Liquid front Y Carotenes
Y
639
Lutein + Zeaxanthin
Y
Lutein + Zeaxanthin
G Chlorophyll a Y
1
G Chlorophyll b Y Neoxanthin
3 x 20 cm. Solvent. Petroleum ether Paper: Air dried or moistened with glycerine Fig. 1.-Chloroplast pigments separated by sorption on moist or dry glass or cellulose paper: solvent, petroleum ether; Y, yellow; G, green.
/Liquid front G Carotenes + Chlorophylls
Y
T
Y Y
Y
Violaxonthin
G Chlorophyll a
Lutein t zeaxanthin
Violaxanthin Neoxanthin
3 x20cm Solvent: Petroleum ether Paper: Sprayed 80% methanol Fig. 3.-Chloroplast pigments separated by partition between methanol (80%) on the paper and petroleum ether. in air for 24 hours. I n each of these experiments, petroleum ether was employed as the wash liquid. Figure 1 shows the color of the zones, their sequence, and the degree of separation usually obtained. The resolved pigments were identified by their color, by their spectral properties, by mixed chromatogranis, and by the blue color produced by exposing the sorbed violaxanthin to the vapors of concentrated hydrochloric acid.10 Paper that had been moistened with solutions of tartaric acid or of lactic acid also yielded initial separations like that
Violaxanthin
G Chlorophyll b
t
Y
Neoxanthin
3x20cm. Solvent: Petroleum ether + 0.5% propanol Paper: Air dried or moistened with glycerine Fig. 2.--Chloroplast pigments separated by sorption on moist or dry paper: solvent, petroleum ether plus 0.5% propanol.
/Liquid
9 B
front
Y Y
Neoxanthin Violaxanthin
Y
Lutein + Zeaxanthin
G Chlorophyll b
G Chlorophyll a Y
Carotenes
3 x 20 cm. Solvent: Methanol 80% Paper: Impregnated vaseline Fig. 4.-Chloroplast pigments separated by partition b'etween petroleum ether plus vaseline on the paper and methanol (80%). shown in Fig. 1. But these acids gradually removed the magnesium from the chlorophyll forming the corresponding pheophytins which were much less sorbed than the chlorophylls themselves.10 The addition of a little propanol to the petroleum cthcr wash liquid produced separations like that in Fig. 2. This solvent mixture decreased the sorbability of the chlorophylls relative to that of the xanthophylls.'O The same effect was observed when the pigments were sorbed on dry paper or
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HAROLD H. STRAIN
on paper moistened with water or with aqueous glycerol solutions. The separation of the chloroplast pigments with methanol (80%) fixed in glass or cellulose paper and with petroleum ether as the mobile solvent is shown in Fig. 3. With 90% methanol in the paper, all the pigments migrated more slowly than with 80% methanol. Under these circumstances there was some separation of carotenes from chlorophylls. With 70% methanol in the paper, the pigments migrated fast with oor resolution. With vaseline ixed in the paper, and with 80% methanol as the mobile solvent, the pigments separated as shown in Fig. 4. With 90% methanol as the wash liquid, all the pigments except the carotenes migrated rapidly and separated incompletely from one another. With 70y0methanol, all the pigments except neoxanthin and violaxanthin migrated very slowly and were not separated. This reversal of the sequence of the chromatographic zones makes possible the complete separation of the less sorbed pigments from the more sorbed pigments as commonly effected in columns by variation of solvent.1° By separation of the pigments with one solvent fixed in paper followed by readsorption of each pigment and with the other solvent fixed in the paper, absolute separations of all the pigments from one another has been achieved.' Mixtures of carotenes with fucoxanthin were examined by means of the four methods illustrated by Figs. 1 to 4. With each method, the petroleum ether-soluble, nonsorbed carotenes were readily separable from the alcoholsoluble, strongly sorbed fucoxanthin. As indicated by the sorption sequences, the sorption of the pigments a t the liquid-liquid interface was similar to the sorption on glass, on cellulose and on powdered sugar." There was no indication of a separation of a- and P-carotenes, or of lutein and zeaxanthin. In this respect, there is a distinct difference between sorptive magnesia and the sorptive systems represented by Figs. 1 to 4, because magnesia is especially effective for the resolution of mixtures of carotenes and for the separation of mixtures of lutein and aeaxanthin.' Separations a t the Petroleum Ether-Water Interface of Droplets.-Pigments such as neoxanthin and fucoxanthin, that are insoluble in water, that contain many hydrophilic hydroxyl groups, and that contain a large hydrocarbon skeleton with affinity for petroleum ether, have a marked affinity for the interface between water and petroleum ether. When a solution of these pigments in petroleum ether was shaken with water, the pigments accumulated on the surface of the water droplets and were removed from the petroleum ether. When the water droplets coalesced, the pigments were liberated t o the petroleum ether. When droplets of water from an atomizer were allowed to fall through solutions of neoxanthin or of fucoxanthin in petroleum ether contained in a narrow tube 1 to 2 m. tall, the pigments accumulated on the droplets and were carried to the bottom of the solution. There they were liberated as the droplets coalesced, and under favorable conditions, the liberated pigments were concentrated so that they separated from the solution. I n this way, the xanthophyll was removed from the petroleum ether in the tube. Carotenoid pigments without hydrophilic groups, as for example the carotenes, were not sorbed a t the petroleum ether-water interface and were not removed from the petroleum ether solution with water droplets. Chlorophylls were weakly sorbed and were slowly removed from the solution. This selective sorbability of the polyhydroxyxanthophylls on water droplets served for the continuous separation of two substances such as carotene and fucoxanthin that differed with respect to their sorbability. As an ex@
Vol. 57
ample, a solution of fucoxanthin and carotene in petroleum ether was allowed to flow upward through a long tube. Water droplets falling downward through the solution collected the fucoxanthin and liberated it as the drops coalesced. The separated fucoxanthin was then removed mechanically or by various supplemental flow methods. Both the water and the petroleum ether could be recycled to make the process continuous. With these petroleum ether-water systems, the sorbent, which is the interface, is formed, destroyed, and regenerated over and over again. For substances that are sorbed at a liquid-liquid interface these sorption procedures may be made the basis of practical separations. NOTEADDED MAY13, 1953.-A photograph of the paper chromatogram of chloroplast pigments prepared by Brown in 1939 has recently been reproduced.12 Additional separations of chloroplast pigments by adsorption on paper with various organic solvents have also been reported.18
DISCUSSION IRAKLwx.-Concerning
the separation of the neoxanthin type compounds by separation a t a petroleum etherwater interface, could not one get a similar type of separation by a solvent-solvent type of extraction, as, for example, petroleum ether-methanol? Is there any advantage to your extraction method which a t first glance would appear to be more cumbersome, tedious, and less efficient?
J. L. SHERESHEFSKY.-The suggested variant Of chromatographic method consisting of sending a spray of immiscible liquid through a solution of several com onents is based on the preferential adsorption of one of t t e components will be separated first, may be predicted from the relative lowering of the surface tension of the solutions of each component in the given solvent. CHARLES G. Dom.-The author has emphasized the similarity between adsorption at an extended liquid-liquid interface formed by water droplets passing through a petroleum ether solution and adsorption within cellulose or glass paper a t water interfaces restrained as films surrounding cellulose or glass fibers. Apparently water is held in an essentially bulk phase enveloping each fiber if the paper were moistened previously with glycerol or glycols. I should like to ask the author if this corresponds with his concept of the intermolecular geometry of the systems such as those in Figures 1 and 2 of the paper? Is this the manner in which the extended interface is developed?
HAROLDH. STRAIN.-In the moist paper the a ueous phafie is presumed to envelop each fiber so that thefiquid forms a porous, rather than a continuous, bulk phase. The separations a t the liquid-liquid interface (Fig. 2) were more effective and more ronvenient than the separations based upon the solvent-solvent extraction or partition (Figs. 3 and 4). The experiment with falling droplets was introduced t o show that the surface of the droplets is an effective sorptive medium even in the absence of the paper. Falling particles of sorptive solids and falling droplets of liquids already had been utilized for the partial resolution of mixtures of solutes. These procedures serve primarily for the segregation of the solutes into two principal fractions, the more sorbed and the less sorbed components. Where applicable, continuous extraction by the sorption of a solute on the surface of droplets is a convenient and economical separatory procedure. (12) H. H. Strain, Technion Year Book 1952-53, American Technion Society. New York, 1953, P. 85. (13) L. Bauer, Naturvlissenechaflen,39, 88 (1952).
