Hydrophobic interaction chromatography of proteins. II. Solution thermodynamic properties as a determinant of...

A general thermodynamic relation was derived to correlate protein solubility to retention in hydrophobic interaction chromatography (HIC). This relation is built on a thermodynamic formulation presented previously by Melander, Horváth and co-workers in the context of the solvophobic theory, but the final result is independent of this model framework. The relation reflects an increase in protein retention in HIC under conditions that promote precipitation or crystallization, consistent with early descriptions of HIC. To examine the contribution of protein solubility to retention in HIC, isocratic elution experiments were performed with four different commercially available agarose media and four model proteins (ribonuclease A (RNA), lysozyme (LYS), myoglobin (MYO), and ovalbumin (OVA)). A wide variety of retention trends were observed as a function of protein, adsorbent type, salt type and concentration, and pH. In general, however, the results show that solubility, or its surrogate, the second osmotic virial coefficient, which reflects solution thermodynamic properties, correlates well with HIC retention in many cases; this includes correctly predicting reverse Hofmeister effects, which cannot be explained by retention models based on the solvophobic theory and preferential interaction theory. However, solution properties could not explain retention behavior under some conditions. In those cases, effects such as protein-surface interactions or conformational change could be important determinants of protein adsorption.