Hydrophobic interaction chromatography: Displacement modeling, process development, selectivity investigation, and displacer design

Hydrophobic interaction chromatography (HIC) is one of the most widely used separation technology in industry due to its unique selectivity. However, there is minimal understanding of the binding mechanism, nature of selectivity in HIC systems, and how to develop efficient HIC processes. This dissertation investigates several experimental and theoretical aspects of preparative HIC systems.; We first demonstrate the utility of hydrophobic displacement chromatography to purify an industrial fusion protein. Then, a preferential interaction quadratic (PIQ) isotherm is developed to predict solutes' behaviors under both linear and nonlinear conditions at a wide range of salt concentrations. To provide significant insight into the solute transport in HIC systems, a simple method is presented for HIC resin characterization. The results indicate that both pore and surface diffusion are rate-limiting mechanisms. The general rate model combined with the PIQ isotherm is employed to predict the solutes' eluent profiles under both displacement and gradient conditions. To aid high efficiency displacer design, isocratic experiments were conducted to obtain 43 solutes' affinities on 4 HIC resins. Results indicate that a small solute interacts with both the resin backbone chemistry as well as ligands due to the low ligands density. The backbone chemistry plays a dominant role in determining the small molecules' affinities on HIC resins. Finally, the predictive QSRR models are developed to predict the solute's affinity. To quantitatively describe protein surface properties, a new set of physically interpretable molecular descriptors is generated from protein crystal structures. Further, the effect of water and salt types (kosmotropic, chaotropic and neutral) on protein binding is studied. The preferential interaction approach was used to calculate the total number of water molecules released during the protein adsorption/desorption process, which was then used to explain the salt selectivity to proteins in HIC systems. Finally, the pH effects on protein affinity in HIC systems are investigated. The numbers of released water molecules were computed as a function of pH, salt types, and resin hydrophobicity.