Influence of charge regulation in electrostatic interaction chromatography of proteins

Recently, the "slab model" was proposed to describe the interaction between a protein and the charged stationary phase surface in electrostatic interaction chromatography. The model is based on the solution of the linearized Poisson-Boltzmann equation for a system consisting of two charged planar surfaces in contact with an electrolyte solution. In the model it is assumed that the charge densities of both the protein and the stationary phase are constant during the adsorption process. However, as the protein comes close to the oppositely charged stationary phase surface, the protein net charge will change due to the electrical field from the stationary phase. In this paper, the theory for charge regulation is applied to the original slab model, and simple algebraic equations are developed in order to include the effect of charge regulation on the capacity factor. A large body of retention data are reanalyzed with the new model, and it is found that there is good agreement between the chromatographically and titrimetrically obtained protein net charge. An interesting consequence of charge regulation is that it gives a contribution to the retention of proteins with zero net charge and even to proteins with the same sign of charge as the stationary phase.