Mechanism of protein retention in reversed-phase high-performance liquid chromatography

The retention of a number of proteins byalkylsilicas with phosphate buffer—isopropanol, buffer—ethanol and buffer—detergent mobile phases (all pH 2.1) was investigated. In each case the percentage of organic component was varied over a wide range without causing appreciable retention. However, a solution composition was reached where very small changes, less than 1% for the alcohols, caused dramatic increases in protein retention. Retention of proteins decreased with increased temperature. The mobile-phase composition was found to be characteristic of each protein at a given temperature so that separations could be achieved by careful selection of temperature and of buffer—organic component ratios. Calculations of the energy of interaction from surface tension and contact angel measurements supported the concept tht protein retention by alkylsilicas may be explained by Van der Waals interactions, and that the interactions may be attractive or repulsive depending on the surface tension of the mobile phase.