ABSTRACT
This chapter provides a brief examination of the effects of the more common hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) high-performance liquid chromatography (HPLC) solvents on protein conformation, together with a demonstration of protein denaturation by hydrophobic stationary phases. It attempts to demonstrate potential effects of HPLC solvents and hydrophobic matrices on the secondary, tertiary, and quaternary structures of proteins. The most commonly used solvent systems for RPC of peptides and proteins involve linear increasing gradients, starting with water and increasing concentrations of organic solvent. Several research groups have demonstrated an increase in protein retention times during HIC with increasing length and, thus, hydrophobicity of alkyl functional ligands, e.g., pentyl, butyl, propyl, and methyl. The overall hydrophobicity of a HIC or RPC stationary phase, and its subsequent effect on protein structure, is a combination of the hydrophobicity of the functional ligand and the ligand density.
