Reduced Heterogeneity of a Lignite Humic Acid by Preparative HPSEC Following Interaction with an Size-Separates by Pyr-GC-MS and 1H-NMR Spectroscopy

Preparative high performance size exclusion chromatography (HPSEC) was used to size-fractionate a humic acid (HA) solution with 0.05M ionic strength before and after having made the humic solution 0.0005M in acetic acid (AcOH). Size fractions were characterized by Pyrolysis-gas-chromatography-mass spectrometry (Pyr-GC-MS) and 1H NMR spectroscopy. Pyr-GC-MS showed that the AcOH treatment altered the distribution of humic molecolar components in the size fractions. The unsaturated alkyl chains were moved from size-fractions of larger molecular-size into those of lower molecular-sizes.Most of the aromatic moieties, which were found in larger molecular-size fractions for the untreated HA, were spread into fractions of lower molecular size after AcOH addition to HA. Carbohydrates, which were undetectable in any fraction of the untreated HA, appeared instead in the pyrogram of the lowest molecular-size and most hydrophilic fraction after treatment with AcOH. Our results suggested that AcOH disrupted the weakly bound association of humic supramolecular structures and HPSEC elution separated size-fractions of different composition without losing humic matter by adsorption on the HPSEC column. The fractions with the largest apparent molecular size were the richest in alkyl chains, thereby suggesting that humic molecules were stabilized into supramolecular associations by multiple weak interactions among apolar groups such as alkyl chains and aromatic moieties. 1H nmr spectra of size-fractions were greatly simplified and more resolved after AcOH treatment. This was attibuted to a less complex molecular association in the size separates which provided a large solubility in the NMR solvent and more favorable relaxation times. Combination of the procedure usedhere for size-fractionation with NMR and Pyr-GC-MS methods appear to be promising to advance knowledge on the molecular composition of humic substances.