Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary...

Peptides are routinely separated with reversed-phase high-performance liquid chromatography using increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. While these separations may be improved by adjustments of gradient slope or substitutions of different solid-phase chemistries, many mixtures would benefit from systematic optimization of mobile phase components. Tryptic digests of cytochrome c from various species were separated on Waters Delta-Pak C18. The effects of varying pH as well as the concentration and type of ion-pair reagent were examined. In addition, low pH, ion-suppression/ion-pairing chromatography was inverted using a polymeric reversed-phase column at high pH with alkyl amine ion pairing. Finally, a tryptic digest of cytochrome c was resolved by ion-exchange chromatography with a strong cation-exchange high-performance liquid chromatography column. These data suggest a framework for dramatically changing the selectivity of peptide separations, leading to more satisfactory peptide mapping.