Structural features of aquatic fulvic acids by analytical and preparative HPLC followed by spectroscopic characterization

Analytical and preparative reversed-phase liquid chromatography were used to separate constituents of Suwannee River aquatic fulvic acid. Photodiode array UV-Vis, and fluorescence detectors were used to monitor the HPLC separation. Preparative FA fractions were subjected to 1H and 13C solid state NMR, FT-IR, and ESR. The preparative and analytical RP-HPLC separations have shown that the FA macromolecule consists of at least two polymeric fractions, each of which can further be separated into individual constituents by analytical RP-HPLC. Vanillic acid structures were identified in the analytical chromatograms of the total FA sample, but not in the fractions. The first fraction included the hydrophilic constituents of FA and represented about 40 percent of the total sample. It could be resolved into six constituents by analytical RP-HPLC. Their tR and UV-Vis scans were characteristic of carboxylic acids in rigid structures which exhibit strong hydrogen bonding. The second fraction included the hydrophobic constituents and represented about 30 percent of the total sample. It could be resolved into twelve constituents. Repeated structurally related units were separated and their UV-Vis scans were characteristic of conjugated ketone and phenolic structures. Free radical structures were present in both hydrophilic and hydrophobic fractions.