Preparation, high-performance liquid chromatographic separation and characterization of hexacarboxylic porphyrinogens

A simple method for the preparation and reversed-phase high-performance liquid chromatographic separation of hexacarboxylic porphyrinogen isomers is described. Uroporphyrin I or III was partially decarboxylated in 0.5 M hydrochloric acid at 150 degrees C. Unreacted uroporphyrin and the hepta-, hexa- and pentacarboxylic porphyrins formed were esterified and then group-separated by thin-layer chromatography. After hydrolysis, the porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) sodium amalgam. The hexacarboxylic porphyrinogens were separated on an ODS-Hypersil column by elution with acetonitrile-methanol-1 M ammonium acetate, pH 5.16 (8:12:80, v/v/v) as mobile phase. Isomers were identified by high-performance liquid chromatography of the characteristic mixture of two pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers. Except for the two type I isomers, resolution of the hexacarboxylic porphyrinogens was superior to that of the corresponding porphyrins.