Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems.