High-performance liquid chromatography of amino acids, peptides and proteins. LXVI. Investigations on the effects of chromatographic dwell in the reversed-phase high-performance liquid chromatographic separation of proteins

Polypeptide and protein retention on chemically modified hydrocarbonaceous surfaces such as n-alkylsilicas is generally assumed to be mediated by weak solvo-phobic effects with relatively rapid kinetics of adsorption and desorption. Recent studies have, however, indicated that multi-zoning effects can be generated for many proteins under certain chromatographic conditions. These effects can lead to asymmetric or multiple peaks for an apparently homogeneous polypeptide or protein. The present study examines retention models for the reversed-phase liquid chromatographic separation of polypeptides and proteins undergoing conformational changes in the mobile phase and at the stationary phase surface. In addition the influence of the chromatographic dwell or residence time on resolution and recovery is examined. Analytical expressions for the change in retention and peak shape have been used to evaluate the role of secondary dynamic effects which may lead to broad asymmetric peaks or multiple peaks for an apparently homogeneous protein chromatographed on n-alkylsilicas under defined chromatographic conditions. Methods of classifying protein retention behaviour in reversed-phase systems according to the characteristics of these multiphasic transitions are suggested.