To observe one-to-one binding between a single host (H) and guest (G) using UV/Vis absorbance, the Benesi–Hildebrand method can be employed. The basis behind this method is that the acquired absorbance should be a mixture of the host, guest, and the host–guest complex.
With the assumption that the initial concentration of the guest (G) is much larger than the initial concentration of the host (H), then the absorbance from H should be negligible.
The absorbance can be collected before and following the formation of the HG complex. This change in absorbance (ΔA) is what is experimentally acquired, with A being the initial absorbance before the interaction of HG and A being the absorbance taken at any point of the reaction.
Using the Beer–Lambert law, the equation can be rewritten with the absorption coefficients and concentrations of each component.
Due to the previous assumption that , one can expect that [G] = [G]. Δε represents the change in value between ε and ε.
A binding isotherm can be described as "the theoretical change in the concentration of one component as a function of the concentration of another component at constant temperature." This can be described by the following equation:
By substituting the binding isotherm equation into the previous equation, the equilibrium constant K can now be correlated to the change in absorbance due to the formation of the HG complex.
Further modifications results in an equation where a double reciprocal plot can be made with 1/ΔA as a function of 1/[G]. Δε can be derived from the intercept while K can be calculated from the slope.