Hydrophobic interaction chromatography of proteins: Thermodynamic analysis of conformational changes

For BSA and β-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (ΔHads), entropy (ΔSads), free energy (ΔGads) and heat capacity (Δcp,ads) changes associated with adsorption we evaluated chromatographic data by the non-linear van’t Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van’t Hoff analysis revealed that a temperature raise from 278 to 308 K increasingly favoured adsorption seen by a decrease of ΔGads from −12.9 to −20.5 kJ/mol for BSA and from −6.6 to −13.2 kJ/mol for β-lactoglobulin. Δcp,ads values were positive at 1.2 m (NH4)2SO4 and negative at 0.7 m (NH4)2SO4. Positive Δcp,ads values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH4)2SO4 concentration. ITC measurements showed that ΔHads is dependent on surface coverage already at very low loadings. Discrepancies between ΔHads determined by van’t Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van’t Hoff analysis.