Characterization of humic acids fractionated by ultrafiltration

Abstract

Humic acid (HA) is a mixture of natural organic macromolecules having a range of physicochemical properties and exhibiting different reactivities in environmental systems. The objective of this study was to characterize chemical and molecular heterogeneity of HA by fractionating a bulk HA (BHA) into a series of subsamples, each having relatively homogeneous properties. The BHA was base extracted from Pahokee peat and was fractionated into eight fractions using an ultrafiltration apparatus with membranes having seven molecular cut-offs. The eight HA fractions obtained have apparent molecular sizes of <1, 1–3, 3–5, 5–10, 10–30, 30–100, 100–300 and >300 kDa, respectively, and their molecular size distributions were further calibrated using high performance size exclusion chromatography (HPSEC). The chemical and structural properties of the eight HA fractions were characterized systematically using elemental analysis, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), Fourier transform infrared (FTIR), ultraviolet–visible (UV–vis), and solid state 13C-nuclear magnetic resonance ( 13C-NMR) spectroscopy. The results show that each HA fraction has a relatively narrow distribution of molecular sizes on HPLC chromatographs, suggesting that ultrafiltration technique is effective for fractionating broadly heterogeneous humic macromolecules into relatively homogeneous fractions. UV–vis spectroscopy and Py-GC-MS analyses indicate that the fractions with lower molecular weights have more heterogeneous functional groups, greater O/C atomic ratios, and higher contents of oxygen and lignin-derived aromatic structural units. Conversely, the HA fractions with higher molecular weights have lower contents of oxygen and aromatic structural units that correspond to greater H/C and lower O/C atomic ratios. This study suggests that HAs formed under the same biogeochemical conditions may consist of macromolecules with a range of chemical, structural and molecular properties.