STUDY OF THE RETENTION MECHANISM OF PROTEINS IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY

A stoichiometric displacement retention model for proteins based on hydrophobic interaction chromatography (HIC) is presented. Several methods were used to demonstrate that water is the displacing agent in this process. Salt not only affects the molar concentration of water, but also changes the confomation of proteins, hydrophobic interaction forces and the number of water molecules in a series of hydrated protein molecules. An equation that relates the capacity factor of proteins, k′, to the water concentration and the stoichiometric displacement factor, Z (the number of water molecules required to displace a protein from ligands) was derived. The intercept of this equation, log I, contains a number of constants that relate to the affinity of protein to the ligands. There is a good linear relationship between log k′ and log [H2O] under different chromatographic conditions. Although Z and log I varied with pH, salt temperature, the plot of log I vs. Z was always an excellent straight line, with a slope j ≈ 1.74, the logarithm of the molar concentration of pure water. Hydrophobic interations dominate the retention of proteins in HIC at high salt concentrations. © 1990.