Evaluation of Kinetic Performance of Reversed-Phase Columns for Protein Separations by Gradient Kinetic Plots

Abstract

The increasing importance of protein biopharmaceuticals has triggered the development of new, highly efficient stationary phases for reversed-phase liquid chromatography (LC) of proteins. They typically have C4 ligands or phenyl surfaces for weak hydrophobic interactions and are based on various morphologies, such as silica monolith, sub-2-µm fully porous particles (FPPs), or superficially porous particles (SPPs). Selection of the best column based on physical parameters provided by vendors may sometimes be unequivocal. Simple performance evaluation tools, such as the gradient kinetic plot methodology reported by Desmet and co-workers, can help to shed light on this issue and allow for a more differentiated view on column performance. This article compares the performance of wide-pore silica monolithic, sub-2-µm FPP, and SPP columns. It also addresses the question of whether 1000 Å or 400 Å SPP columns are more suitable for reversed-phase LC-type protein separations and presents a kinetic performance comparison of a number of wide-pore core–shell particle columns.