Comparing the Heterogeneity of Copper-Binding Characteristics for Two Different-Sized Soil Humic Acid Fractions Using Fluorescence Quenching Combined with 2D-COS

Heterogeneous distributions of copper-binding characteristics were compared for two ultrafiltered size fractions of a soil HA using fluorescence quenching combined with two-dimensional correlation spectroscopy (2D-COS). The apparent shapes of the original synchronous fluorescence spectra and the extent of the fluorescence quenching upon the addition of copper were similar for the two fractions. The stability constants calculated at their highest peaks were not significantly different. However, the 2D-COS results revealed that the fluorescence quenching behaviors were strongly affected by the associated wavelengths and the fraction's size. The spectral change preferentially occurred in the wavelength order of 467 nm → 451 nm → 357 nm for the 1–10 K fraction and of 376 nm → 464 nm for the >100 K fraction. The extent of the binding affinities exactly followed the sequential orders interpreted from the 2D-COS, and they exhibited the distinctive ranges of the logarithmic values from 5.86 to 4.91 and from 6.48 to 5.95 for the 1–10 K and the >100 K fractions, respectively. Our studies demonstrated that fluorescence quenching combined with 2D-COS could be successfully utilized to give insight into the chemical heterogeneity associated with metal-binding sites within the relatively homogeneous HA size fractions.