Multi-wavelength fluorescence-quenching model for determination of Cu2+ conditional stability constants and ligand concentrations of fulvic acid.

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 Cu2+ 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 (CM). Intensity data of wavelength set 1 are obtained from 25 nm constant offset synchronous fluorescence spectra (SyF), in which are observed distinct peaks (lambda(ex) = 415 nm, lambda(em) = 440 nm; and lambda(ex) = 471 nm, lambda(em) = 496 nm). Wavelength set 2 intensity data are obtained from the FA fluorescence excitation and emission maxima (lambda(ex) = 335 nm, lambda(em) = 450 nm; and X(ex) = 471 nm, lambda(em) = 496 nm). Application of MWM shows that the multi-wavelength data sets characterize ligands of different binding strength (log K(x)) and concentration (C(Lx)). Corresponding to pH values of 5, 6, and 7, mean and standard deviation values for wavelength set 1 are log K(415/440) = 4.66 (0.12), 5.03 (0.12), and 5.05 (0.08), log K(471/496) = 4.93 (0.06), 5.27 (0.11), and 5.39 (0.09), C(415/440) = 3.1 (1.5), 10.9 (4.5), and 7.9 (3.9) microM, C(471/496) = 14.3 (3.0), 1.7 (0.6), and 1.4 (0.5) microM. And for wavelength set 2, log K(335/450) = 4.50 (0.03), 4.96 (0.27), and 5.22 (0.08), log K(471/496) = 5.02 (0.04), 5.42 (0.32), and 5.71 (0.09), C(335/450) = 8.8 (0.5), 21.9 (7.9), and 18.7 (0.3) microM, C(471/496) = 21.0 (2.5), 7.17 (1.2), and 7.09 (0.3) micrpM. 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 Cu2+ 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.