Abstract
For BSA and β-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (Δ H ads), entropy (Δ S ads), free energy (Δ G ads) and heat capacity (Δ c p,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 Δ G ads from −12.9 to −20.5 kJ/mol for BSA and from −6.6 to −13.2 kJ/mol for β-lactoglobulin. Δ c p,ads values were positive at 1.2 m (NH 4) 2SO 4 and negative at 0.7 m (NH 4) 2SO 4. Positive Δ c p,ads values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH 4) 2SO 4 concentration. ITC measurements showed that Δ H ads is dependent on surface coverage already at very low loadings. Discrepancies between Δ H ads 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.
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