Moment Analysis Method for Measurement of Reaction Equilibrium and Rate Constants by Using High-Performance Liquid Chromatography

Abstract

The moment analysis method was developed for the determination of association equilibrium constant (K A ) and association (k a ) and dissociation (k d ) rate constants of intermolecular interactions between solute and ligand molecules. They are accurately determined by using moment equations from elution peak profiles because they are measured by using high-performance liquid chromatography (HPLC) under preferable conditions that neither immobilization nor chemical modification (i.e., fluorescence labeling) of solute and ligand molecules is required. To demonstrate the effectiveness of the method, it was applied to the inclusion complex formation system between dibenzo-18-crown-6 (DB18C6) and alkaline earth metal cations, i.e., Mg 2+ , Ca 2+ , and Sr 2+ , as a concrete example. Because the diameter of the three metal cations is smaller than that of the inner cavity of DB18C6, the values of K A , k a , and k d were analytically determined by assuming the stoichiometry of 1:1 between DB18C6 and the metal cation. They reflected the influence of the difference in the size between the inner cavity of DB18C6 and the metal cations on the inclusion complex formation. It seems that the moment analysis method based on HPLC separation is effective for the multifaceted analysis of chemical reactions because some characteristics of the method are different from those of other conventional methods. It must contribute to the dissemination of an opportunity for the study of chemical reactions to many researchers because of the versatility of HPLC.