Improved Accuracy and Precision in the Determination of Association Constants

Association constants for inclusion complexes are commonly determined by varying the analytical concentration of the “host” species while maintaining the analytical concentration of “guest” species constant. In implementing the experiment, commonly referred to as Benesi−Hildebrand analysis, conditions are controlled such that all necessary concentration requirements are fulfilled and the guest molecule in the unbound state does not contribute appreciably to the overall signal (<5%). In this paper, we demonstrate that this commonly accepted constraint, while clearly important, is insufficient for the accurate determination of association constants. Systematic error introduced by variation in the host species concentration, and therefore the equilibrium concentration of unbound guest molecules, results in overestimation of the complexation constant. These errors in the association constant determination appear to be the most troublesome when steady-state fluorescence or absorbance measurements, two of the most common techniques, are utilized. We demonstrate that correction of this systematic error can be accomplished by iterative correction for the equilibrium concentration of the unbound guest.