Size-exclusion high-performance liquid chromatography of peptides. Requirement for peptide standards to monitor column performance and non-ideal behaviour.

A series of five synthetic peptide polymers with the sequence Ac-(G-L-G-A-K-G-A-G-V-G)n-amide, where n = 1-5, was employed to assess the resolving power of high-performance size-exclusion columns in peptide separations. The peptide standards showed great versatility in monitoring both ideal (no interactions of solutes with the column material) and non-ideal (hydrophobic and/or ionic interactions of solutes with the column material) size-exclusion behaviour in volatile and non-volatile mobile phases. The effectiveness of adding salts or organic solvents to overcome non-specific interactions of solutes with the column materials was well illustrated by the standards. In addition, the advantageous use of non-ideal size-exclusion behaviour was highlighted. The ability to predict the position and/or elution order of peptides during size-exclusion chromatography (SEC) requires peptides to be separated by a pure size-exclusion process. Although the peptide standards demonstrated similar ideal size-exclusion profiles in non-denaturing medium on all the columns studied, this study suggested that, if the conformational character of a peptide-protein mixture in a particular mobile phase is uncertain and ideal size-exclusion behaviour is required, SEC should be carried out under highly denaturing conditions.