Reversed-phase high-performance liquid chromatography (RPC) is currently the method of choice for the rapid estimation of the homogeneity of synthetic peptides. The possibility of using RPC “retention coefficients” for the prediction of peptide retention times is based generally upon the assumption that peptide retention is due primarily to the amino acid composition of the peptide. 1, 7 We have shown, however, that the use of amino acid retention coefficients to predict peptide retention times can be unreliable. This appears to be due to the induction of secondary structure in the peptides by the hydrophobicity of the n-alkyl or hydrocarbon chains of the solid support, or by the increasingly organic component of the mobile phase, 8 - 12 although we believe the latter to be less likely. Previous investigators, using sets of unrelated peptides, have found correlations as high as 0.99 between predicted and observed retention times. 1 - 5, 13 - 15 Deviations have been attributed to anomalous conformational effects and/or stationary phase interactions which, as yet, have not been well defined. We believe that these variations from theory can be better understood through the systematic study of closely related peptides to narrow the range of conformational possibilities.
