Quantifying Thiol Abundance and p <i>K</i> _a Values in Natural Organic Mixtures Using HERFD-XANES Spectroscopy

Reduced organic sulfur (S) functional groups play critical roles in diverse environmental and biological processes, but an enduring analytical challenge is resolving thiols (R-SH) and thioethers (R-S-R') in complex mixtures. Here, we demonstrate the use of S Kα₁ high-energy-resolution fluorescence detected (HERFD) X-ray absorption near-edge structure (XANES) spectroscopy to distinguish thiol groups from thioether moieties in complex environmental mixtures as a function of pH. Experiments with model S thiol compounds showed a quantitative decrease in the normalized amplitude of the reduced S peak in spectra with thiol deprotonation (R-SH ↔ R-S^- + H⁺), a phenomenon not observed for other S functionalities. Spectra were collected across a pH range relevant to thiol p<i>K</i>_as (3-11) for five well-characterized dissolved organic matter (DOM) samples from natural environments, containing different S content and various mixtures of S functionalities. Using a quantitative relationship between the decrease in the reduced S peak amplitude and thiol deprotonation in composite spectra of model S compounds, the proportions of reduced S as thiols (<i>f</i>_RS-) in the five DOM samples were quantified to range from 3% to 41%. For two DOM samples, we quantified thiol p<i>K</i>_a distributions and verified reversibility through stepwise, bidirectional pH adjustments. The increase in thiol abundance of one DOM sample following experimental abiotic sulfurization was also quantified to demonstrate method application. We discuss opportunities to optimize S Kα₁ HERFD-XANES spectroscopy for enhanced thiol sensitivity in various research applications.