High-performance liquid chromatography of amino acids, peptides and proteins. CXXXIII. Peak tracking of peptides in reversed-phase high-performance liquid chromatography.

A peak tracking algorithm for peptide analysis has been developed based on a normalised spectral overlay method which directly compares the UV spectra of any two chromatographic peaks. Additionally, the algorithm compares the spectrum of each peak in the first chromatogram with the spectra of every peak in the second chromatogram to determine the best cross-match. The sensitivity of the technique was further enhanced by incorporation of the primary and secondary derivative spectra for cross-match normalisation. The utility of the software was demonstrated by its application to the analysis of tryptic digests of porcine growth hormone. Peptide solutes could be identified and tracked in chromatograms generated with various column types, gradient times, mobile phase types and temperatures. These results therefore constitute the initial stages of development of a more robust approach to the optimisation of the resolution, detection and characterisation of peptides and proteins separated by HPLC techniques.

Molecular definition of the retention parameters of peptides separated by RP-HPLC.

Different physicochemical properties of the constituent amino acid side chains have been employed in order to investigate the molecular basis of the experimentally derived retention parameters, S and log k0, of peptides separated by reversed-phase high performance liquid chromatography (RP-HPLC). The results demonstrate that the S-value is strongly related to the total surface area of a peptide but correlates poorly with the corresponding predicted hydrophobicity based on the summated amino acid group retention coefficients. In contrast, the experimentally derived log k0 value correlates well with both the total surface area and the predicted affinity values derived from the incremental free energy of transfer calculated from the summated amino acid retention coefficients. Since the S and log k0 values are parameters useful to the interpretation of peptide and protein chromatographic behavior in RP-HPLC, the results of the present study document the physicochemical relationships that exist between these retention parameters and peptide structure.