Fluorescence Quenching of Humic Substances and Natural Organic Matter by Nitroxide Free Radicals

Fluorescence spectroscopy is one of the most frequently used techniques for studying dissolved organic matter (DOM) in natural and engineered systems. However, the spatial distribution and fluorophores, including local and interacting states, within DOM's larger structure remains poorly understood. In this study, we used two nitroxide fluorescence quenchers to evaluate the chemical and spatial heterogeneity of DOM fluorophores. Several results from quenching experiments with cationic 4-aminoTEMPO (tempamine), including downward-curving Stern- Volmer plots and spectral dependent quenching, show that multiple emitting species contribute to the observed emission even at a single excitation wavelength. Furthermore, for DOM isolates of diverse geographic origins (soil vs aquatic) and isolation procedures (reverse osmosis vs humic substances), the maximum extent of quenching occurs on the red edge of the emission spectra. For soil humic substance isolates, the spectral dependent quenching was significant enough to affect a blue shift in the average emission wavelength. The same soil humic substance isolates whose emission spectra were blue shifted by tempamine quenching were also blue shifted by decreasing solution pH and decreasing solvent polarity, which suggests a role for anionic fluorophores (e.g., hydroxybenzoic acids) in long wavelength fluorescence. Finally, curvature in Stern-Volmer plots indicate that between 10 and 50% of emitting species detected by steady-state fluorescence are inaccessible to quenching by tempamine, suggesting that this fraction of fluorophores may be inaccessible to water solvent. Results from this study provide an assessment of the spatial distribution of fluorophores within DOM and help to reconcile prior studies on the role of solvent polarity and pH on DOM fluorescence.