Applications of isocratic hydrophobic interaction chromatography: Small molecules to proteins

Although hydrophobic interaction chromatography is useful for separating proteins, the retention mechanisms are not well understood. This study explores the effect of solute structure, and surfactant and salt concentration in the mobile phase on isocratic retention in HIC. A homologous series of acyl Coenzyme A derivatives are used to probe the effect of solute structure. Dansyl amino acids and water-soluble proteins are used to investigate the effect of ammonium sulfate concentration. The influence of the nonionic surfactant MEGA-8 (octanoyl-N-glucamide) in submicellar HIC mobile phases on water-soluble protein retention is examined. The use of MEGA-8 for the elution of the membrane protein D-lactate dehydrogenase in its active form is investigated.;From the study of the homologous acyl Coenzyme A derivatives, the free energy of transfer of a methylene group from water to a propyl HIC stationary phase is approximately ;Increasing salt concentration increases dansyl amino acid retention time, but affects each to varying degrees. The effect of ammonium sulfate concentration on their retention is correlated with the apparent "hydrophobicities" of the amino acids based on others' published tables of amino acid hydrophobic parameters.;The effect of increasing salt concentration on the retention, presumed to be related to the solute's nonpolar contact area with the stationary phase, is determined for the water-soluble proteins and compared to that of the small molecules studied. There is a rough correlation between molecular weight and the effect of increasing salt concentration on increasing solute retention.;As the amount of MEGA-8 in the mobile phase increases, retention is decreased to varying extents for all of the proteins. Protein retention is decreased as the amount of surfactant adsorbed to the stationary phase surface increases.;Post-column D-LDH activity is not significantly decreased using isocratic micellar HIC with MEGA-8. The type of nonionic surfactant with which D-LDH is solubilized changes its elution characteristics dramatically.