Simulation of pH elution in high-performance affinity chromatography using non-porous adsorbents

Abstract

Abstract Alteration of the pH of the mobile phase is the most common method to perform elution of the bound components from an affinity column. The values of the adsorption rate constant and the dissociation constant between the protein to be separated from a mixture and the immobilized ligand vary during the chromatographic process. A mathematical model used to predict the performance of pH elution on a high-performance affinity chromatographic column, packed with non-porous adsorbents, has been solved numerically with the application of the finite element method. The effects of binding capacity, sample load, adsorption rate constant and equilibrium dissociation constant under adsorption and elution conditions on the split-peak phenomenon and the shape and spreading of the elution peak were investigated. Simulation results showed that the time required to elute all bound solutes is mainly determined by the dissociation constant which is adjustable by altering the pH of the mobile phase. The dispersive effect on the elution peak is largely influenced by the rate constant under adsorption and elution conditions, while for the non-retained fraction (split-peak), it is governed by the rate and dissociation constants under adsorption conditions only. Influence of sample loading on the non-retained fraction and the elution peak also is significant, especially for columns with small binding capacity. As the affinity column is employed for the purpose of analyzing, a calibration graph based on the height of the elution peak is easily obtained provided that the effects of adsorption rate constant and dissociation constant are all taken into account.