Novel Method for Characterizing Humic Substances Using Fluorescent Solvatochromism

Charge-transfer-type fluorochromes, which exhibit shifts in fluorescence intensity and emission wavelength in response to solvent polarity changes, have been widely employed to investigate solute-solvent interactions. Humic substances (HSs) are naturally occurring macromolecular organic acids derived from plant residues, with structural properties that vary depending on their origin and environmental conditions. The polarity of HSs is closely associated with the mobility and toxicity of environmental pollutants, making their chemical characterization essential. In this study, we developed a rapid and straightforward method to characterize HS polarity using fluorescent solvatochromism. The fluorescence peak shifts of four dyes-8-anilino-1-naphthalenesulfonic acid (ANS), acridine orange (AO), methylene blue (MB), and Rhodamine B (RhB)-were evaluated in the presence of humic acids (HAs), a major component of HSs. To assess environmental variability, a total of twelve HS samples were tested, including HSs derived from soils of different origins, compost, commercial reagents, and standard reference materials. Among these, AO and MB exhibited distinct spectral shifts without overlapping with the intrinsic fluorescence of HAs. Notably, MB displayed a consistent blue shift dependent on HA concentration, with the most stable response observed at 5 mg/L. The magnitude of this shift was significantly correlated with UV-Vis parameters associated with the aromaticity, humification degree, and polarity of HSs. Overall, this study demonstrates that MB-based fluorescent solvatochromism can function as an empirical and facile indicator for assessing the structural and microenvironmental characteristics of HSs, providing a rapid and complementary screening approach for HSs extracted and purified from environmental samples.