Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatography-on line Fourier transform ion cyclotron resonance (FTICR-) mass spectrometry

Size-exclusion chromatography was coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (SEC-FTICR-MS) to separate a fulvic acid isolate into three size fractions and to determine the elemental composition of fulvic acids in these fractions. Molecular formulas of about 3000 ions in the mass range of 200-700 Da were derived, many of which occur in all three fractions and follow the same system of elemental composition. Product ion spectra generated by SEC coupled to quadrupol-time-of-flight-MS (Q-TOF-MS) confirmed that the ions of all three fractions are basically polycarboxylates with hardly any other functional moiety. However, SEC-FTICR-MS revealed that the ions generated from the high molecular weight (HMW) fraction are enriched in carboxyl groups and are more aromatic as compared with the low molecular weight (LMW) fraction. These findings support the idea that the HMW fulvic acids are formed from LMW fulvic acids. The shift in the relative frequency of ions from the LMW to the HMW fraction is in line with different interaction mechanisms: HMW fulvic acids may be aggregates held together by electrostatic interaction of the carboxylate groups via hydrogen bonds or with polyvalent cations or by hydrophobic interaction of their carbon backbone, or consist of LMW fulvic acids covalently bound to each other or to (aliphatic) alcohols