High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Evaluation of the use of hydrophobicity coefficients for the prediction of peptide elution profiles.

The gradient elution behaviour of five synthetic decapeptide analogues has been investigated using an octadecylsilica stationary phase and trifluoroacetic acid-water-acetonitrile mobile phases. The influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides was assessed using quantitative expressions derived from linear solvent strength theory and general plate height theory. Linear relationships between logarithmic median capacity factors, log k, and the mole fraction of organic solvent modifier, phi, were observed over the experimental range of conditions used. The slopes of these plots were different for all peptides, which indicates that divergences will occur in the prediction of peptide retention times due to conformation dependent changes in hydrophobic contact area occupancy at the stationary phase surface. However, the differences in S values (tangent to the curve obtained in a plot of log k versus phi) for these peptides were not substantial enough to seriously affect the prediction of peptide retention times at one gradient slope from those observed at another. In addition, significant differences existed between experimental and theoretical peak capacity data of these peptide analogues of similar molecular weight and overall polarity, particularly at lower flow-rates or longer residence times. These results once again demonstrate that additional diffusional and interactive processes occur during the reversed-phase separation of peptides and proteins which are not yet adequately formalized by current chromatographic theory.