Mechanistic model of retention in protein ion-exchange chromatography

Abstract

A mechanistic model is developed to describe the retention of proteins in ion-exchange chromatography, as a simplified version of a more elaborate colloidal model within which retention is related to protein and stationary-phase structural and functional parameters and eluent composition. The protein parameters are the size and net charge, while incorporation of stationary-phase properties, namely the surface charge density and a short-range interaction energy, allows a more mechanistic interpretation of stoichiometric displacement model (SDM) parameters as well as prediction of retention on different stationary-phase materials. Experimental exploration of the model capabilities was performed on two different PEI-based carboxylic acid cation exchangers. Isocratic experiments using lysozyme were used to estimate the stationary-phase parameters for each material. Predictions of isocratic experiments on chymotrypsinogen A correctly captured the Z slope of the data, along with reasonable absolute retention times. In addition, the correct trends and reasonable quantitative results were predicted for gradient elution of a set of small globular proteins. The mechanistic basis for the model, particularly the explicit inclusion of stationary-phase properties, makes it a powerful tool to use in the selection of materials and optimization of operating conditions.