Characterization of the influence of displacing salts on retention in gradient elution ion-exchange chromatography of proteins and peptides

Abstract

It has been shown earlier that the choice of displacing salt has a large effect on the retention in ion-exchange chromatography of proteins and peptides. The influence of different displacing salts cannot be predicted or quantitatively explained, owing to the current lack of an adequate theoretical framework. In this work a general characterization is made by using a considerable number of proteins and peptides and all displacing salts found feasible. Principal component analysis is used to interpret the large amount of data that is generated. The results of the analysis indicate that most of the retention variations are due to non-specific effects and can be explained by changes in the apparent gradient slope, i.e., the increase in elution strength per unit volume, and the elution strength of the starting buffer. This differs from the interpretation given earlier, where the selectivity changes were attributed to specific effects of the salts. However, as it is impossible to test all existing proteins and peptides, specific effects are still possible, but they might be less common than previously considered.