Fluorescence spectroscopy is one of the most utilized tools for studying DOM source and structure in natural and engineered systems, yet a molecular-level understanding of fluorophores and their location within the larger DOM three-dimensional structure remains poorly characterized. In this study, we employed fluorescence quenching experiments for studying the accessibility of DOM fluorophores utilizing cationic, hydrophilic, and hydrophobic nitroxide free radicals at two different pH values (4 and 9). Quantitative assessment of fluorescence quenching via Stern-Volmer constants indicates that cationic tempamine was a stronger quencher of DOM fluorescence than neutral tempol at both pH 4 and 9. Additionally, quenching by cationic tempamine at pH 9 resulted in an apparent blue shift in fluorescence spectra for all DOM isolates, with the greatest shifts observed for soil humic substances. Conversely, quenching at pH 4 showed blue shifts only for soil humic acids. Fluorescence quenching data were also analyzed via a modified Stern-Volmer equation, which indicated that a fraction of fluorophores was inaccessible to cationic tempamine. Overall, the results of this study suggest that a small fraction of fluorophores in DOM may be inaccessible to the solvent. Both the inaccessible fraction and blue shift in fluorescence emisssion during quenching are dependent upon DOM type and pH value.

Quenching of dissolved organic matter fluorescence by nitroxide free radicals

ACS Fall 2021

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