Isolation of porphyrins from biological materials by partition chromatography.

The present investigation was undertaken because of the need for a more practicable procedure for isolating pure porphyrins from biological materials. The existing methods, based primarily on solvent fractionation, on adsorption chromatography with calcium carbonate, talc, aluminum oxide, or other adsorbents, or on a combination of the two procedures, have failed in our hands to give an entirely satisfactory resolution of porphyrins from mixtures. Therefore, other possible methods of isolation were sought, and, since partition chromatography with hydrated silica gel had proved so successful in the separation of organic acids in this laboratory (l), this procedure was investigated and was found to be highly satisfactory, not only for the separation of the porphyrins, but also for their subsequent isolation and crystallization. While our work was in progress, a paper (2) appeared describing the successful use of partition chromatography on filter paper for the separation of the unesterified porphyrins, thus independently demonstrating the applicability of partition chromatography to the separation of the porphyrins. The filter paper procedure, however, does not as readily lend itself to the isolation and subsequent crystallization of the porphyrins as does the present method. The present procedure involves the preliminary separation of porphyrins from other pigments and biological materials by adsorption on talc, conversion to the methyl esters, and finally the isolation of the methyl esters on a column of hydrated silica gel at approximately pH 4.5; a mixture of chloroform and ligroin in varying proportions was employed as the developing and eluting solvent. It was found necessary to convert the porphyrins to the methyl esters before elution because the free porphyrins were held so tenaciously by the silica gel that elution was extremely difficult. Fractions of the eluate containing the methyl esters of the individual porphyrins are allowed to evaporate at room temperature and the porphyrins separate as crystals visible even to the unaided eye. Crystals of the