Evaluation of Stationary and Mobile Phases for Reversed-Phase High Performance Liquid Chromatography of Peptides

Abstract

A series of silica-based, chemically-bonded, reversed-phase supports were evaluated for reversed-phase high performance liquid chromatography of peptides. Chromatograms of hydrophilic and moderately hydrophobic peptides, ranging from 5 to 51 amino acid residues in length, were compared on octadecyl, hexyl, phenyl, and cyanopropyl phases with and without secondary silanization (end-capping); an octadecyl phase of moderate carbon loading was found to provide optimal retention and selectivity for most separations. Comparison of a variety of mobile phase conditions indicated that aqueous buffers of low pH and high ionic strength containing alkylamine or alkylammonium compounds were most effective in eluting retained species. Increased retention of peptides at high concentrations of organic modifier above 50% was observed, suggesting limitations on gradient elution of multicomponent peptide mixtures. Peptide recovery was better than 80% when amounts of 0.5μg or more were injected.