Effect of peptide chain length on peptide retention behaviour in reversed-phase chromatography

The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time and/or the elution order 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 generally agrees well for small peptides (up to ca. 15 residues), the retention times of increasingly larger peptides are less than expected from a simple summation of retention coefficients. In the present study, we report the synthesis of four series of peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues, Ac = acetyl): Ac-(G-L-G-A-K-G-A-G-V-G)n-amide (n = 1-5), Ac-(G-K-G-L-G)n-amide (n = 1, 2, 4, 6, 8, 10), Ac-(L-G-L-K-A)n-amide (n = 1, 2, 4, 6, 8, 10) and Ac-(L-G-L-K-L)n-amide (n = 1, 2, 4). From the retention behaviour of these peptide polymers on C4, C8 and C18 stationary phases under gradient elution conditions, we have clearly established the effect of polypeptide chain length and hydrophobicity on peptide retention. This, in turn, has enabled us to extend the utility of retention time prediction for peptides containing up to 50 residues by introducing a peptide chain-length correction.