High-Performance Liquid Chromatography of Amphibian Peptides. Selectivity Changes Induced by pH

Abstract

The effect of pH on the retention behavior under reversed- phase liquid chromatography conditions of a series of peptides was examined. Isocratic conditions were used with either methanol or acetonitrile as organic modifiers. The intrinsic hydrophobicity of the peptides was altered by changes in the pH of the eluent mixture. Increased retention at pH 7 relative to pH 4 was correlated with the presence of a histidine residue in a hydrophobic environment. An experimental parameter, α_pH, was defined as the positive quotient of capacity factors at pH 4 and pH 7 for a given eluent. These α_pH values are interpreted as reflecting changes in peptide hydrophobicity introduced by variations in solvent and pH. Identical α_pH values were obtained for homologous peptides, particularly histidine containing peptides. This approach to selectivity effects yielded diagnostic conditions for the analysis of bombesin, a peptide touted as a potential marker for human small-cell lung carcinoma.