Normal-phase high-performance liquid chromatography of free acid dicarboxylic porphyrins and hematoporphyrin derivative on silica.

The isocratic separation of dicarboxylic porphyrins (hematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin) and their isomers by normal-phase high-performance liquid chromatography is described. The stationary phase is unmodified silica and the mobile phase consists of acetone—ethyl acetate (1:1, v/v) mixtures containing water and inorganic acids. Retention (capacity factor, k′) was found to decrease exponentially with the mole fraction of water (NH2O) and to increase linearly with the concentration of hydrochloric acid, following the relation k′ = A [HCl] NH2O−4.85, where A is a constant characteristic of the porphyrin. The effects of the concentration and the nature of the acid used strongly suggest that retention involves a form of the porphyrin in which the inner nitrogens are protonated. The retention is thus partly determined by the basicity of the inner nitrogens, which depends on the electron-donating power of the porphyrin side-chains. Good resolution of the various components of hematoporphyrin derivative was obtained. In comparison with reversed-phase chromatography this method shows a different retention mechanism, appears to yield results of comparable reproducibility and provides complementary information. Possible retention mechanisms based on partition or adsorption equilibria are discussed.