A Study of the Solvent Composition Effects on the Separation of Seven Clinically Important Porphyrins on Cyclodextrin Bonded Phases

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Abstract
The isocratic separation of the seven polycarboxylated porphyrins of heme biosynthesis that was previously thought to be difficult has been demonstrated using cyclodextrin bonded phases via a novel mobile phase. The multi-component mobile phase contained 0.28 g of 18-crown-6 ether in 0.06 M phosphate buffer: acetonitrile: pyridine (5: 50: 30, v/v/v, pH 6.37). The seven porphyrins included mesoporphyrin, uroporphyrin, heptaporphyrin, hexaporphyrin, pentaporphyrin, coproporphyrin and protoporphyrin. The solvent composition effects on the retention characteristics of these porphyrins were studied with some common HPLC solvents. The solvent selectivity of the mobile phase was discussed. The elution order of these porphyrins on the cyclodextrin column resembled that on the reversed-phase C18 column. The retention of the porphyrins increased as the number of side-chain carboxylated substituents decreased. However, there was no correlation between the two parameters.