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PROBLEMS IN CHROMATOGRAPHY AKD I N COLLOID CHEMISTRY ILLUSTRATED BY LEAF PIGMENTS HAROLD H. STRAIK Carnegze Instztutzon of Washington, Diviszon of Plant Bzology, Stanford I.nwerszty, Calzfornza Recezved August 4 , 1949
Today, as never before, the fate of many a nation hinges upon the ingenuity with which man utilizes carbon compounds. Concomitant with the change from a rural to an urban population, there have been increased demands for special fuels, for varied foods, for new articles of clothing, and for tools for offense and defense. As a result of this increased specialization, the technologist has become the pivot about which much of our economy revolves. Driven by competition and often denied the blue sky and the green fields, our productive population scarcely considers the source of its wealth of carbon compounds. Technologists themselves, more frequently than not, ignore the processes taking place in the greatest of all chemical factories, the green parts of plants. Under the influence of light, chlorophyllous plants glean carbon dioxide from the air and elaborate the basic compounds from which virtually all carbonaceous matter is fabricated. Plant products are man’s primary source of carbon compounds. Autotrophic plants, ranging from the unicellular diatoms to the massive sequoias, are the master chemists in this as well as in past ages. It is about one hundred and fifty years since the r81e of the green plant in the carbon economy of nature was discovered. Shortly after this discovery, rapid developments in the science of organic chemistry made possible the determination of the atomic arrangements in most types of carbon compounds. In spite of this advance and the resultant development of new concepts regarding the properties of organic matter, very little has been learned about the photosynthetic reactions by means of which green plants utilize light energy for the production of carbon compounds (15). Pigments in green plants absorb the light energy required for the production of organic matter. As a consequence, there have been numerous investigations of the properties and possible functions of these pigments. This work has stimulated two developments that are of special interest to physical chemists. One of these is the invention and improvement of the exceptionally sensitive, chromatographic adsorption analysis, an analytical method that has now found extensive use in other fields. The other concerns the condition or itate of the pigments in the leaf. The nature and reactions of these native pigments are in need of investigation by skilled colloid chemists. CHROMATOGRAPHIC .4DSORPTION
The chromatographic adsorption method was devised in 1906 by the Russian botanist Tswett (24). When he filtered a petroleum ether extract of dried 1 Presented at the Nineteenth Colloid Symposium, which was held a t the University of Colorado, Boulder, Colorado, June 1%20, 1942.
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leaves through a tube filled with finely powdered adsorptive chalk, a series of green and yellow bands was formed. The separation of these bands from one another was enhanced if the adsorbed pigments were washed with fresh portions of the solvent. In this way, the now familiar chromatogram was developed. The leaf-pigment mixture was thus resolved into its constituents, the green chlorophylls and the yellow xanthophylls and carotenes. Several stages in the formation of a chromatogram (resulting from adsorption of diatom pigments) are illustrated in figure 1.
CH LOROF U CINE FUCOXANTHIN C FUCOXANTHIN b FUCOXANTHIN
d
NEW XANTHOPHYLL NEW XANTHOPHYLL CHLOROPHYLL
d
CAROTENE
FIG 1. Successive stages in the development of a chromatogram. Diatom pigments adsorbed on sugar from solution in petroleum ether (left) and then washed with petroleum ether containing 2 per cent 1-propanol. Y, yellow; G, green; 0, orange; B, blue.
Intensive investigations over a period of thirty-six years have revealed many of the conditions requisite for the separation of mixtures of colorless as well as of colored substances by use of this adsorption procedure. The separation of materials on the columns may depend upon adsorption, upon reversible chemical reactions, or upon partition of the solutes between two immiscible liquid phases (10, 16). Micro, macro, and industrial modifications of the method have been developed. Many of the applications of these adsorption procedures have recently been reviewed and will not be considered here (16, 17). Sensitivity and specificity of the adsorption method The chromatographic adsorption method is remarkably sensitive. For the detect,ion of extremely small quantities of plant pigments, it is desirable to
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reduce the diameter of the columns so that each pigment will yield a discrete, visible band. On a column of sugar 1 mm. in diameter, one can detect so small a quantity as 0.01 gamma of chlorophyll a. Similar quantities of chlorophyll b are detectable in the presence of large amounts of chlorophyll a (22). The specificity of the adsorption method may be illustrated by separation of the fucoxanthin isomers extracted from diatoms &s shown by figure 1. These isomers are believed t o differ only in the spatial arrangements of double bonds, or of hydroxyl groups (21). Thus far they have been separated only by chromatographic adsorption, which is many times more efficient than crystallization or distillation methods (IO).
FIG.2. Usual distribution of a pigment adsorbed on a column. Left, appearance of the band; right, schematic plot of concentration against position in column.
Shape of adsorption bands A recent theory indicates that the concentration or distribution of material in an adsorption band should conform to a normal curve of error (10). In practice, however, especially with leaf pigments, the leading edge of the band tends to become sharp, and the trailing end becomes diffuse: This is illustrated schematically by figures 2 and 3. All the causes for this effect are not understood. The diffuseness of the trailing portion is due in part to slow or incomplete elution of the adsorbed compounds. Different solvents or wash liquids have pronounced effects on the shape of a band of adsorbed material. In order to accelerate the development of the chromatogram, it is customary to use wash liquids of polarity slightly greater than that of the original solvent. These more polar wash liquids tend to elute the trailing portions of an adsorbed band. This causes an initial concentration or narrowing of the band. Finally, however, the band may become considerably wider. These effects are illustrated by figure 4.
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If, before addition of the more polar solvent, the band is washed until the upper boundary is diffuse, the use of the more polar solvent often causes a concentration of the upper portions of the band. This concentration of adsorbed material resembles formation of a new band that gradually moves through the original band. Ultimately the band becomes uniform and wider, as shown by figure 5.
FIG.3. Successive an adsorption colwni
anges in concentration of adsorbed pigment as i t is washed through
3. figure 2).
FIG.4. Successive changes in concentration of a pigment adsorbed from a non-polar solvent (left) and then washed with a more polar solvent.
If one adds to the wash liquid small but fixed concentrations of a very polar substance that tends to elute most of an adsorbed substance, the upper boundary of the band of adsorbed material will remain well defined, &s indicated by figure 6. When several substances are to be separated, addition of selected polar compounds to the developing solvent will enhance the separation of the bands. By proper selection of added substances, one can increase the specificity of the adsorption method, as illustrated by figure 7. It is believed that this effect may
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be responsible for the rapid initial separation of pigments when extracts of leaves containing colorless polar substances are adsorbed. When adsorption columns with adsorbed leaf pigments are washed with petroleum ether containing a low, fixed concentration of alcohol, separation of the colored bands proceeds rapidly. Under these conditions, the upper boundaries of the bands of the adsorbed pigments remain fairly sharp. This behavior
FIG. 5. buccessive changes in concentration of a pigment adsorbed from and washed with a non-polar solvent (left), and then washed with a more polar solvent. IX
IX
I
\ A
>A
u
LLU
FIG. 6. Successive changes in concentration of pigment A adsorbed from a non-polar solvent (left) and then washed with the same solvent containing small quantities of a quite polar substance X.
suggests that the alcohol may cause an adsorption gradient to be formed on the column, the adsorption capacity increasing toward the bottom. If this effectcould be enhanced, it might produce much greater uniformity of the bands. Sequence of bands of adsorbed compounds on adsorption columns
On the same adsorbent, bands of adsorbed substances usually occur in the same sequence. In the cme of chlorophylls and xanthophylls, however, some
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notable exceptions h a w been found. With petroleum ether a~ solvent, fuco-
xanthin a is adsorbed below chlorophylls a and b. If several per cent of alcohol is added to the petroleum ether, fucoxanthin is adsorbed above both the chlorophylls, aa illustrated by figure 8. X
