Characterization of Natural Organic Matter and Humic Substance Isolates by Size Exclusion Chromatography following Reduction with Sodium Borohydride

Chemical reduction with sodium borohydride has been used for over four decades to probe the presence and function of carbonyl-containing moieties in dissolved organic matter (DOM). One of these structure-property relationships is the attenuation of UV-visible absorbance after borohydride reduction, an effect that has been observed universally across DOM of different origins. We previously demonstrated that DOM with similar bulk physicochemical properties exhibits bifurcating reactivity with borohydride depending on the source (i.e., soil vs. aquatic), as judged by the kinetics of fractional absorbance removal during reduction at a fixed borohydride:DOM mass ratio. This result and data from other studies suggest that a portion of borohydride-reducible chromophores in DOM may be inaccessible to the water solvent, explaining the incomplete absorbance attenuation even at very high borohydride mass excesses. Here, we study the reactivity of five DOM isolates with sodium borohydride via size exclusion chromatography coupled to total organic carbon, absorbance, and fluorescence detectors. Reduction with sodium borohydride resulted in quantifiable yet exceedingly small decreases in DOM molecular weight, suggesting that the reduction of carbonyl groups to alcohols does not markedly impact the DOM secondary structure. Interestingly, higher molecular weight DOM exhibited the most prominent changes in optical properties after reduction, suggesting that larger molecules contain a high proportion of borohydride-reducible moieties. Optical surrogates were inversely correlated to molecular weight across a single isolate, both native and reduced. However, the correlation broke down at lower molecular weights, wherein optical surrogates remained constant with continued decreases in elution volume, suggesting that there is an intrinsic lower limit to the ability of optical surrogates to capture further decreases in molecular weight. Overall, these results provide insights into the DOM structure and help inform future applications of sodium borohydride for studying the DOM source and reactivity.