A stopped-flow technique and three-dimensional excitation emission (Ex/Em) fluorescence spectrophotometer were used to detect the full fluorescence spectral kinetic changes that occurred during the complexation between fulvic acid (FA) and several metals [Cu(II), Ni(II), Co(II), Cd(II) and Ca(11)]. The study was carried outwith a fulvic acid isolated from Cavan Bog, Canada. At pH 7, the FA reacted rapidly with all metals studied. Two major kinetically distinguishable binding sites on FA (fast and slow), having reaction half-lives of 1.3-3.9 and 34.7-69.3 s, respectively, were identified using pseudo-first-order kinetic plots. Kinetic changes of Ex and Em wavelengths of the fluorescence maximums also indicate two major binding sites. For the fast-reacting binding site, the rate constant and the site relative contribution were as follows, Cu2+ > Ni2+ > Co2+ > Cd2+ > Ca2+, which agrees with the Irving-Williams series, indicating that complexation kinetics are affinity dependent. Within each kinetic phase, both Ex and Em wavelengths of fluorescence maximums increased with time, indicating the occurrence of structural changes during the binding process. Based on the results obtained, the use of full fluorescence spectra appears to be a promising tool for further understanding metal-FA complexation mechanisms.
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