Loading, stationary phase, and salt effects during hydrophobic interaction chromatography: α-Lactalbumin is stabilized at high loadings

Amide hydrogen-deuterium exchange labeling has been used to study the effects of salt and protein loading on α-lactalbumin (BLA) stability during hydrophobic interaction chromatography (HIC). Stability in the adsorbed phase increased dramatically with increasing loading, and unfolding was nearly undetectable close to the resin saturation capacity. We also found that a butyl surface destabilized BLA more than a phenyl surface, despite the fact that BLA was bound more strongly on the phenyl surface. These observations have important implications for HIC process design and indicate that in some cases column capacity does not have to be sacrificed to preserve protein stability.