Studies on the relationship between humic acid fluorescence, their physical properties, and the effects of metal complexation

Fluorescence spectroscopy is routinely applied to the study of humic substances (HS) and their reactions with metals, but little is known about the relationship between HS physical properties (e.g., size, conformation), ligand types, and their fluorescence.; Steady-state fluorescence, fluorescence lifetime, and fluorescence anisotropy experiments were performed to study these relationships in five humic acids (HA). Four purified and one commercial HA were used. Aluminum complexation was used both to investigate whether different ligands are represented in different spectral regions and to control the aggregation state of the HA. Differences in the effect of Al between the purified and commercial HA are explained by the presence of contaminating metals and fulvic acid in the commercial sample. Fluorescence quenching by Al is shown to be due to precipitation of HA. An additional mechanism involving self-quenching of HA in Al-induced aggregates is supported by the fluorescence anisotropy results.; High-performance size exclusion chromatography was employed to study the relationship between HA size, fluorescence, and metal binding properties. A method for estimating HA molecular weight from fluorescence spectra is proposed. Smaller HA fractions are over-represented in the fluorescence signal and have longer fluorescence lifetimes than larger fractions. These results can be explained by self-quenching and energy transfer in larger HA fractions. Titrations with Cu2+ and Pb2+ show that larger HA have larger metal binding constants.