Correlation of protein retention times in reversed-phase chromatography with polypeptide chain length and...

The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time behaviour on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption holds up well enough for small peptides (up to ca. 15 residues), it is now recognized that polypeptide chain length must be taken into account when attempting to equate retention time behaviour of larger peptides and proteins with their overall hydrophobicity. In the present study, we have examined the reversed-phase retention behaviour of 19 proteins of known sequence on stationary phases of varying hydrophobicity and ligand density. From the observed protein retention behaviour on C4, C8 and C18 stationary phases under gradient elution conditions, we have been able to correlate the observed retention times of proteins ranging in molecular weight from 3500 to 32,000 dalton and in chain length from 30 to 300 residues with their overall hydrophobicity (based on retention parameters derived from small peptides) and the number of residues in the polypeptide chain. The retention behaviour of the proteins on the C4, C8 and C18 columns was also compared to that obtained on supports containing lower ligand densities (phenyl ligands). The maintenance of native or partially folded protein conformation on the phenyl columns, resulting in lower retention times than would be expected for fully denatured proteins, underlined the importance of efficient protein denaturation for satisfactory correlation of protein retention times with protein hydrophobicity. In addition, the effectiveness of increasing temperature and/or ligand density of the stationary phase in denaturing proteins was also demonstrated.