Abstract
Molecular mass distribution and fluorescence characteristics of organic ligands for copper(II) in freshwater were, for the first time, investigated using ultrafiltration, immobilized metal ion affinity chromatography (IMAC) and three-dimensional excitation-emission matrix spectroscopy (3DEEM). Two types of humic-like fluorescence (Peaks A and B) were found in all molecular ligand fractions, while an additional type of protein-like fluorescence (Peak C) was only observed in the 0.1 μm-GF/F molecular ligand fractions. The Ex/Em maxima of Peak A in the <5 kDa and 0.1 μm-GF/F molecular ligand fractions were blue-shifted (shift towards the shorter wavelength) relative to that in the 5 kDa–0.1 μm fractions. Contributions of total organic ligands were 1.6–4.0% of the bulk dissolved organic matter (DOM) in the original water, as determined by UV absorbance. In the molecular mass distribution of organic ligands, the relative contribution of the fraction with the <5 kDa molecular masses was dominant (67–79%), while 17–30% of the total organic ligands were in the 5 kDa–0.1 μm fraction, leaving 3–6% in the 0.1 μm–GF/F fraction. Fluorescence at the longer excitation wavelength was more heavily weighted in the lower molecular organic ligands relative to that at the shorter excitation wavelength. The binding affinity of organic ligands increased with their molecular weight. These results have significance for further understanding the nature of organic ligands and their biogeochemical role in the aquatic environment.
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