Effect of pH changes on water release values in hydrophobic interaction chromatographic systems

The effect on pH on protein binding in HIC systems was investigated. Isocratic experiments were carried out to determine the capacity factors of various proteins as a function of temperature, pH and salt type. This paper presents a framework based on the Maxwell linkage function for estimating the number of released water molecules during the adsorption/desorption process due to a change of buffer pH. This approach also enables one to predict the effect of pH change on the water released values upon binding at any temperature condition. The results indicate that the total number of released water molecules (Δν) for a pH change increased more on aromatic surfaces (phenyl Sepharose) than on aliphatic resins (butyl Sepharose). The results also indicate that the total number of released water molecules (Δν) for a pH change increased with salt concentration and when changing from chaotropic to kosmotropic salts. The (Δν) values also increased as the buffer pH approached the protein's pI, and decreased away from its pI. This work helps to establish a framework for the investigation of pH effects on protein selectivity in HIC systems.