High-performance liquid chromatography of type-III heptocarboxylic porphyrinogen isomers

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.