Limitations on the determination of the stoichiometry and equilibrium constants of weak complexes by computer fitting methods: experimental verification

We present experimental evidence showing that computer fitting methods are inadequate for the study of weak complexations, whenever a certain equation relates the reaction parameters. In such cases, one must resort to direct (non-computational) methods for the determination of the stoichiometry of the interaction and the equilibrium constants. Our data refer to complex formation between cyclodextrins and diphenylpolyenes, and we show that differentiation between the 1/1 and 2/1 stoichiometries, by computer fitting, is not always possible. The direct method employed here to determine the correct interaction parameters was primarily time-resolved fluorescence spectroscopy.