ИСТИНА

Introduction. Optical methods are routinely used to characterize dissolved organic matter (DOM) in a wide range of environmental research due to their simplicity, short acquisition time and sensitivity to structural alterations. The fundamental problem in DOM analysis is the extreme complexity of its molecular composition. The FTICR mass-spectrometry revealed that single DOM sample consists of thousands of individual molecules, which interaction leads to unique optical properties inherent to supramolecular systems rather than to sum of mixture components. Much attention is paid to identification of DOM components responsible for unique absorption and fluorescence properties. Here we present synthetic and acidity-based fractionation approaches coupled to FTICR MS on order to determine relations between gradients of molecular composition, structure and optical properties of DOM. Materials and Methods DOM samples were isolated from two organic-rich soils by solid-phase extraction. Fracitonation was performed by step-wise extraction on nonionic sorbent at steadily lowered pH values. Carboxylic groups in DOM fractions were determined by selective deuteromethylation using CD3OD/SOCl2 reaction. Synthetic complex mixture with a gradient in optical properties was obtained by continues ozonation of phenolic model compounds. Fluorescence spectra were recorded using the FluoroMax-4 fluorometer (Horiba Hobin Yvon). DOM samples were analyzed using 7T FT MS Bruker Apex Ultra with harmonized cell (Bruker Daltonics). Synthetic mixtures were analyzed using QExactive Orbitrap mass-spectrometer (Thermo). FTICR MS data were processed using the lab-made “Transhumus” software with sensible chemical constraints. Deuteromethylation data treatment and statistical analysis were perfomed using custom R-script. Preliminary Data For the first time formation of DOM optical properties was investigated by tailoring its molecular composition using novel fractionation technique and model retrosynthesis, reconstructed major aromatic constituents of DOM. Fractionation of DOM on hydrophobic resin resulted in a clear shift of molecular composition from relatively saturated molecules to condensed oxidized compounds as it was shown by FTICR MS. This shift was accompanied by a red shift in fluorescence spectra and increase of long-wave tail of absorbance spectra. The complete shift of molecular composition toward condensed oxidized molecules resulted in the atypical non-exponential absorbance. Therefore, application of FTICR MS enabled to show that optical properties depend on the proportion of oxygen-rich (electron acceptors) and reduced (electron donors) compounds. The depletion with one of the components results in the distortion of sample optical properties. Novel approach of deuteromethylation coupled to FTICR MS enabled enumeration of carboxylic group number in individual components of DOM and its fractions. We explored that DOM molecular shift corresponded to a deep separation of COOH-depleted molecules and polycarboxylic compounds. At the same time, DOM fractions possessed a number of common formulae. According to deuteromethylation results they varied by the number of carboxylic groups. This was a reliable proof of isomeric complexity of DOM. At the same time, components observed in most acidic fraction possessed less amounts of COOH-groups as compared to pH=3. This indicated the presence of keto-acids at pH=2 fraction, which are the most acidic organic carboxylic compounds. Dominating of electron-depleted compounds resulted in red-shift in fluorescence spectrum. Kinetic experiments of oxidative condensation of model compounds under ozone atmosphere also enabled tailoring optical properties and connect them to the molecular constituents appeared during reaction. We observed that increase of the total number of new molecules significantly affect the absorbance spectra reaching exponential DOM-like decay after 2 hours. Novel Aspect Fractionation and deuteromethlyation explored isomeric complexity of DOM. The developed approaches enable deeper insight on structure-optical properties relationships of DOM.