Moment Equations for Kinetic Study of Intermolecular Interaction by Size Exclusion Chromatography

Abstract

Abstract New moment equations were developed for size exclusion chromatography (SEC), in which chemical reactions simultaneously take place. It is expected that accurate information about affinity kinetics of intermolecular interactions with no immobilization and no chemical modification of solute and ligand molecules can be obtained from elution peak profiles measured by SEC. Moment equations for the first absolute (μ1A) and second central (μ2C) moment of elution peaks were developed on the basis of the Einstein equation for diffusion and the random walk model in order to analytically determine association (ka) and dissociation (kd) rate constant of intermolecular interactions. They were used to predict chromatographic behavior in hypothetical SEC systems to demonstrate their effectiveness. The influence of some parameters on μ1A and μ2C of elution peaks measured in the SEC systems was quantitatively evaluated. It was indicated that the contributions of axial dispersion and reaction kinetics to μ2C depend on some SEC conditions. It is important to control the SEC conditions so that the reaction kinetics has a predominant contribution to μ2C to determine accurate values of ka and kd. The moment equations are effective for optimizing experimental conditions to accurately analyze intermolecular interactions from kinetic points of view.