| Abstract
| - Association constants for inclusion complexes are commonly determined by varying the analytical concentrationof the “host” species while maintaining the analyticalconcentration of “guest” species constant. Inimplementing the experiment, commonly referred to as Benesi−Hildebrand analysis, conditions are controlled such thatall necessary concentration requirements are fulfilled andthe guest molecule in the unbound state does not contribute appreciably to the overall signal (<5%). Inthispaper, we demonstrate that this commonly acceptedconstraint, while clearly important, is insufficient fortheaccurate determination of association constants.Systematic error introduced by variation in the host speciesconcentration, and therefore the equilibrium concentration of unbound guest molecules, results in overestimation of the complexation constant. These errors in theassociation constant determination appear to be the mosttroublesome when steady-state fluorescence or absorbance measurements, two of the most common techniques, are utilized. We demonstrate that correctionofthis systematic error can be accomplished by iterativecorrection for the equilibrium concentration of the unbound guest.
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