Prediction of peptide retention times in reversed-phase high-performance liquid chromatography II. Correlation of observed and predicted peptide retention times factors and influencing the retention times of peptides

We have assessed the accuracy of a set of amino acid residue retention coefficients by applying them to the prediction of the retention times of 58 peptides under linear gradient elution conditions (solvent A = 0.1% trifluoroacetic acid in water, and solvent B = 0.1% trifluoroacetic acid in acetonitrile). These coefficients were determined by examining the retention times of synthetic model peptides in reversed-phase chromatography. The high degree of correlation (0.98) between predicted observed retention times not only indicated good predictive accuracy for our coefficients but was also further evidence that composition is generally the major factor affecting peptide retention time. For optimum accuracy in retention time prediction on any single column, it was essential to include an internal peptide standard in each run to correct for run-to-run deviations and column aging. The resolution of five commercially available synthetic peptide standards was found to improve with increasing flow-rate and decreasing gradient steepness. Increasing temperature resulted in a decrease in peptide retention times and slightly improved resolution. Rules for retention time prediction are presented which not only enable the experimenter to correct for instrument and column (length, diameter, n-alkyl chain length and ligand density) specifications, but also allow the prediction of peptide retention times at any gradient steepness, flow-rate and temperature.