Reversed-phase chromatography fractionation tailored to mass spectral characterization of humic substances

Large-scale structural characterization of humic substances via mass spectrometry requires reduction of complexity within nominal mass and separation of isomers, i.e., prefractionation. Humic substances (here loosely defined to encompass all humic, humic-like, and humic-containing material) are notoriously difficult to fractionate. Equally challenging is deriving information on whether and how fractionation has occurred. Here, reversed-phase high-performance liquid chromatography was used to induce tailored fractionation of Suwannee River fulvic acid (SRFA) within nominal mass. The fractionation was optimized on synthetic standards that differed in polarity and had elemental formulas similar to SRFA. Fractions were analyzed via electrospray ionization ion-cyclotron resonance mass spectrometry. Kendrick and Van Krevelen comparisons showed that fractionation occurred as predicted based on known molecular formula patterns.