In this work, a multi-wavelength model (MWM) is developed. It uses fluorescence bands in the fulvic acid (FA) spectrum that quench upon binding of inorganic Cu<sup>2+</sup> to FA. Quenching data at pH values of 5, 6, and 7 are placed in sets, containing fluorescence measures at select wavelengths versus added copper (<i>C</i><sub>M</sub>). Intensity data of wavelength set 1 are obtained from 25 nm constant offset synchronous fluorescence spectra (SyF), in which are observed distinct peaks (λ<sub>ex</sub> = 415 nm, λ<sub>em</sub> = 440 nm; and λ<sub>ex</sub> = 471 nm, λ<sub>em</sub> = 496 nm). Wavelength set 2 intensity data are obtained from the FA fluorescence excitation and emission maxima (λ<sub>ex</sub> = 335 nm, λ<sub>em</sub> = 450 nm; and λ<sub>ex</sub> = 471 nm, λ<sub>em</sub> = 496 nm). Application of MWM shows that the multi-wavelength data sets characterize ligands of different binding strength (log <i>K</i><sub>x</sub>) and concentration (<i>C</i><sub>Lx</sub>). Corresponding to pH values of 5, 6, and 7, mean and standard deviation values for wavelength set 1 are log <i>K</i><sub>415/440</sub> = 4.66 (0.12), 5.03 (0.12), and 5.05 (0.08), log <i>K</i><sub>471/496</sub> = 4.93 (0.06), 5.27 (0.11), and 5.39 (0.09), <i>C</i><sub>415/440</sub> = 3.1 (1.5), 10.9 (4.5), and 7.9 (3.9) μM, <i>C</i><sub>471/496</sub> = 14.3 (3.0), 1.7 (0.6), and 1.4 (0.5) μM. And for wavelength set 2, log <i>K</i><sub>335/450</sub> = 4.50 (0.03), 4.96 (0.27), and 5.22 (0.08), log <i>K</i><sub>471/496</sub> = 5.02 (0.04), 5.42 (0.32), and 5.71 (0.09), <i>C</i><sub>335/450</sub> = 8.8 (0.5), 21.9 (7.9), and 18.7 (0.3) μM, <i>C</i><sub>471/496</sub> = 21.0 (2.5), 7.17 (1.2), and 7.09 (0.3) μM. The ability of the 415/440 nm SyF transect to characterize the main excitation and emission maximum of FA at 335/440 nm is evaluated. Relatively low concentration values returned by the model for this transect (415/440 nm) suggest that it is not entirely illustrative of the maximum. The model predictive capability is verified at pH 6 with two fluorescing Cu<sup>2+</sup> chelating organic compounds, L-tyrosine and salicylic acid. This test confirms that the model is capable of providing good estimates of equilibrium binding parameters from multi-wavelength measurements of a mixed ligand system.
Michael D. Hays, David K. Ryan, and Stephen Pennell, "Multi-Wavelength Fluorescence-Quenching Model for Determination of Cu2+ Conditional Stability Constants and Ligand Concentrations of Fulvic Acid," Appl. Spectrosc. 57, 454-460 (2003)