ИСТИНА

Humic substances (HS) are characterized with non-stoichiometric elemental compositions and extreme structural heterogeneity. This hinders greatly their structural analysis. One approach to solve this problem is to use Fourier Transform Ion Cyclotron Resonance Mass spectrometry (FTICR MS), which due to its high resolution and mass accuracy, enables observation of all ionizable molecules in the HS ensemble1. However, it’s a major limitation is tolerance to isomers, which makes it impossible to explain differences in properties of HS with similar molecular compositions. This problem is particular important in case of HS isolated from brown coal which is widely considered as a source of novel materials for agricultural and medicinal application. Recently, we have developed methods for H/D exchange (HDX) of mobile and back-bone protons2,3 and 18O/16O exchange4 in complex organic mixtures. FTICR MS enabled enumeration of reactive sites for all parent-ions in the mass-spectra. Our intention was to applied these approaches to reveal isomers and structural patterns of individual compartments in coal HS (CHS). For HDX of skeletal protons mixtures of 300 μl of 4 M NaOD or 16% DCl in D2O with 5 mg of CHS were heated at 120 °C for 40 hours in a sealed tube. 18O/16O exchange was performed by incubation of CHS samples in H218O with CF3COOH (1%) for 48 hours. HDX of mobile protons was conducted by a twofold dilution of the CHS solution with D2O. All samples were analyzed using 7T FTICR MS Bruker Apex Ultra with harmonized cell. To avoid back-exchange nebulizing gas flow and heating temperature were adjusted according to published recommendation5. Exchange series were identified by analyses of mass-differences between peaks of parent and labeled ions. Application of acid/base catalysed HDX allowed to distinguish isomers in common molecular composition of HS from different coals. We found that lignin components of HS differ by the number of OCH3- groups substituents in aromatic rings. This collaborated with oxygen exchange, wherein ether oxygen atoms remain intact. Moreover, we explored that in the single sample individual components possess different substitution patterns as it was shown using conventional Van Krevelen diagram6. The exchange character was in agreement with model structures proposed for major HS precursor. The maximum of HDX was characteristic for lignin region. Within this region the number of acid/base catalyzed HDX increased with a drop in H/C and an increase of O/C atomic ratios. This lies on the humification pathway with the evolutional transition of phenylpropanoid building blocks from sinapyl to coniferyl and p-cumaryl alcohols. Additional application of HDX of mobile protons enabled distinction of pyridine and pyrrole-like nitrogen heterocycles in aromatic N-containing species. Therefore, combination of isotopic labeling with FTICR MS can provide the necessary information on the structure of the individual components of HS.