Prediction of peptide retention times in reversed-phase high-performance liquid chromatography I. Determination of retention coefficients of amino acid residues of model synthetic peptides

We have examined the contribution of individual amino acid residues to peptide retention on reversed-phase (RP) columns by measuring their effect on retention of a model synthetic peptide: Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X is substituted by the 20 amino acids found in proteins. Consistently similar resolution of the 20 peptides on several RP columns enabled the determination of empirical sets of retention coefficients, describing the hydrophobicity of amino acid residues at pH 2.0 and pH 7.0. The much superior resolution and selectivity obtained with acetonitrile, compared to 2-propanol and methanol, confirmed its value as the best organic eluent for most practical purposes. The necessity of using peptides rather than alkylphenones as internal standards for peptide retention prediction is demonstrated and underlined by the predictive accuracy of our coefficients when applied to the resolution of a mixture of five commercially available synthetic peptide standards on several RP columns. Rules for retention time prediction for linear elution gradients, employing our hydrophobicity parameters, of peptides of known composition are presented and enable the researcher to correct for: (a) instrument variations, (b) varying length or diameter of RP columns, (c) varying n-alkyl chain length and ligand density of RP packings and (d) column aging.