Molecular characterization of an end-residue of humeomics applied to a soil humic acid

Humeomics encompasses step-wise chemical fractionation and instrumental determination to fully characterize the heterogeneous molecular composition of natural organic matter. Humeomics applied to a terrestrial humic acid produced a recalcitrant end-residue, that was solubilized in alkali and size-fractionated by preparative high performance size exclusion chromatography (HPSEC). The ten separated size-fractions were then subjected to analytical HPSEC hyphenated with a high-resolution electrospray mass spectrometer (ESI-MS). Total ion chromatograms of size-fractions showed two eluting peaks, the molecular masses of which were identified with empirical formulae by ESI-MS. Most empirical formulae were easily associated with linear alkanoic, unsaturated, hydroxylated and hydroxy-unsaturated acids, as well as cyclic acids, but some compound structures were unclear. Tandem MS fragmentation was applied to one linear and three cyclic compounds, to elucidate their structures as hydroxyunsaturated hexanoic acid, two furane rings and a norbornane-like ring, respectively. The latter three compounds were never reported for terrestrial humic acid, but they resembled the carboxyl-rich alicyclic molecules, which had been proposed previously for dissolved organic matter. Quantitative measurement of components indicated that long-chain saturated acids were present in large-sized fractions more than in short-chain homologues, whereas unsaturated, hydroxylated and most cyclic acids were more abundant in small-sized fractions. This suggests that long, saturated and unsubstituted linear acids allow formation of large suprastructures, probably as a result of favorable intermolecular packing, compared with the irregularly shaped cyclic, unsaturated or hydroxylated compounds. We showed that humeomics clarifies the molecular composition and conformational arrangement of natural organic matter, and may help to elucidate the relationship between humic structure and environmental activity.