Differences in fluorescence properties between humic acid and its size fractions separated by preparative HPSEC

Abstract We applied three dimensional excitation–emission-matrix (3D-EEM) spectrofluorometry to investigate the fluorescence properties of a humic acid (HA) solution at different pH values and its size-fractions separated by preparative high performance size exclusion chromatography (HPSEC). 3D-EEM fluorescence spectra revealed peak maxima in the bulk HA which were assigned to protein-like, phenol-like and naphthol-like components. Moreover, changes of peak maxima with pH showed that phenol-like and naphthol-like molecular classes were composed of sub-components with different acidities, and, thus, different structures. The 3D-EEM spectra of three size-fractions separated by HPSEC showed similar peak maxima as for the bulk HA, with additional components, such as highly condensed aromatic systems, which had not been visible in the bulk HA. Furthermore, the fluorescence intensity of peak maxima in 3D-EEM spectra of size-fractions appeared invariably greater than that for the bulk HA. This phenomenon as well as the appearance of new maxima in size-fraction spectra is explained by the reduced self-quenching effect in the less complex and conformationally looser size-fractions. In fact, the close mutual proximity of fluorescent humic molecules in the tight supramolecular association of the bulk HA decreased or even inhibited their fluorescence quantum yield. This work shows that, due to self-quenching of humic molecules, fluorescence spectroscopy cannot be used to characterise the molecular complexity of humic matter and, thus, differentiate its environmental origin.