EFFECT OF MOLECULAR-STRUCTURE AND CONFORMATIONAL CHANGE OF PROLINE-CONTAINING DIPEPTIDES IN REVERSED-PHASE CHROMATOGRAPHY

Peptides that contain proline residues may yield multiple peaks in high-performance liquid chromatography if the proline is not at the N-terminus. The phenomenon is caused by slow kinetics of isomerization that are on the same time-scale as the chromatographic separation with such peptides. In this study, the shape and number of peaks are examined qualitatively in view of the influence of isomerization kinetics on the bandspreading, and as functions of pH, temperature and flow velocity. These effects in the chromatography of alanylproline are shown in detail. Single peaks are obtained for each peptide investigated by proper adjustment of these variables in light of their effect on the pertinent rate and equilibrium constants.