SPLIT-PEAK AFFINITY CHROMATOGRAPHIC STUDIES OF THE IMMOBILIZATION-DEPENDENT ADSORPTION-KINETICS OF PROTEIN-A

An equation was derived to describe the unusual chromatographic situation in which part of a sample of a pure solute eluted in the column void volume while the remainder was strongly retained. It was shown that this behavior was a natural consequence of slow diffusion or slow adsorption kinetics. Kinetic parameters were measured for an affinity chromatographic system consisting of immobilized protein A and immunoglobulin G. By use of different immobilization methods and supports, it was found that diffusion was rate-limiting in some cases while adsorption was rate-limiting in others. One immobilization method decreased the adsorption rate constant of protein A by an order of magnitude.