Chromatographic Method

CHROMATOGRAPHIC METHOD, a method for separating substances with similar properties based on their adsorption capabilities. The principle of the method was first proposed by M. Tswett in 1906-1910 for studying plant pigments. Initially it did not become widespread, but in recent years (1931-34) it has been reintroduced into biochemical preparative work and has proven to be very valuable and fruitful. The method is based on the principle of so-called chromatographic adsorption analysis: a solution of colored substances is passed through a tube filled with a suitable adsorbent substance (see figure). At this time, different substances in the solution, depending on their "adsorption affinity" for the chosen adsorbent, are retained at different levels of its column. This already allows some separation of the mixture, but it can be done even better by "development": a stream of one or another pure solvent is now passed through the tube, which elutes and gradually moves toward the lower end of the tube some substances while leaving others untouched. Thus, along the length of the adsorbent column in the tube, a series of sharply defined colored zones are obtained, each containing one specific substance from those present in the original mixture - a so-called chromatogram is obtained. The layers of the adsorbent with individual components fixed in them are successively removed from the tube (or, if its diameter is small, the adsorbent column is simply carefully pushed out) and the substances adsorbed in each separate zone are freed with a suitable solvent. Essentially, the method is based on a principle widely applied by Willstätter in isolating, separating, and purifying enzymes: the use of fine differences in the ability of substances to adsorb and elute, which cannot be detected and utilized in ordinary adsorption techniques; therefore, the chromatographic method allows the separation of substances very similar in nature and properties, in particular mixtures of individual isomers. Compared with ordinary separation methods, the chromatographic method has the great advantage that for related substances, adsorption properties depend to a much greater extent on configuration than, for example, solubility. It is possible to establish certain regularities regarding the dependence between adsorbability and the structure of the substance; the presence of oxygen in the molecule is particularly significant - the higher its content, the higher the adsorption affinity. In a series of related compounds, for example various carotenoids, those with an alcohol function are adsorbed by calcium carbonate, others with a ketone, ether function and hydrocarbons by aluminum hydroxide. By varying the adsorbent, primary solvent and developer, wide possibilities are obtained to adapt to the most different mixtures of starting substances. As adsorbents in the chromatographic method, various forms of aluminum hydroxide, calcium carbonate, calcium oxide, talc, phosphates and a number of other substances are used; for separating two modifications of chlorophyll, finely crushed cane sugar has proven particularly suitable. As solvents and developers, gasoline, petroleum ether, acetone, pyridine are used; for aqueous solutions, the chromatographic method is still little developed. The chromatographic method has found its greatest application in the chemistry of plant and animal pigments, for example in the study of carotenoids, chlorophyll, flavins, and further in the field of vitamins, since some of them apparently belong to the mentioned groups of substances; finally, in the field of enzymes, valuable results can be expected, although here difficulty is created by the circumstance that the chromatographic method is directly and conveniently applicable for separating colored substances, while for uncolored ones, the selection of adsorption and development conditions and the separation of the chromatogram into zones is possible only indirectly. Partially, a useful technique proposed by Carrer will be helpful here: observation of the fluorescence of the chromatogram in ultraviolet light.