Retention model for proteins in reversed-phase liquid chromatography.

This paper presents a retention model for proteins on an reversed-phase chromatography support in which retention is a function of the number (Z) of solvent molecules required to displace the solute from the surface. An equation is derived that relates the capacity factor of a protein to the displacing agent concentration and the stoichiometry of solvent-solute displacement. Experimental tests of the model indicate that each protein has a unique Z value and that Z is directly proportional to the molecular weight of a series of proteins when 60% formic acid is used as the mobile phase additive. This relationship is attributed to a direct relationship between Z and the contact surface area between polypeptide solutes and the support. Desorption curves for proteins also become more convex with increasingly molecular weight, as predicted by the retention model. In the solvent series of methanol, ethanol, propanol, the Z number decreases from the C1 to C3 alcohol. The Z number for any particular solvent is also related to other mobile phase additives, such as acids, and the concentration of additives.