A Method of Separating Small Quantities of the Coproporphyrin Isomers 1 and 3

Abstract

Quantitative separation of the naturally occurring coproporphyrin isomers (1 and 3) has hitherto been impossible. Crystallization has usually permitted identification of the porphyrin predominating in any given mixture, such as obtained for instance from urine and feces. 1-4 This, however, has required that relatively large amounts of porphyrin be available. The present investigation was undertaken with the purpose of finding a means by which mixtures consisting of as little as 5-10 γ of total coproporphyrin could be resolved quantitatively.

We have found that the methyl esters of coproporphyrins 1 and 3 are quantitatively adsorbed on Brockmann's A1₂O₃ ∗ under the conditions noted in the following. The ester of coproporphyrin 3 may be eluted quantitatively with 35% acetone in water while that of copro-1 remains adsorbed, and is later removed by elution with pure acetone. The various steps in the procedure are as follows: (1) Esterification of the total, free porphyrin mixture in methyl alcohol saturated with HC1 gas. (2) Dilution with equal volumes of distilled water, followed by neutralization of the HC1 with a saturated aqueous solution of sodium acetate, which is added drop by drop with constant stirring until the solution no longer turns Congo paper blue. Ten percent NH₄OH is then added drop by drop until the mixture becomes pink to phenol red. (A few drops of an aqueous solution of the latter indicator having been added to the entire mixture.) (3) The faintly alkaline solution is at once run through the column of Brockmann's A1₂O₃, designated “a” in the accompanying diagram (Fig. 1). The column is next washed with 15-20 cc of distilled water. (4) The copro-3 ester is then removed by repeated washing with 35% acetone, as long as any red fluorescence is visible at b (Fig. 1).