Comparison of the Properties of Humic Acids Extracted from Soils by Alkali in the Presence and Absence of Oxygen

Humic acids (HAs) make up to 30–50% of the soil organic matter, which is the main reservoir of organic carbon in the biosphere. The common isolation protocol for HAs implies alkaline extraction from soils followed by acidification of the extract with HCl to pH 2. International Humic Substances Society (IHSS) recommends isolation of HAs in oxygen-free atmosphere (e.g., under nitrogen or inert gas purging) to prevent oxidative transformations of HAs during the extraction process. In the Russian school of soil science, extraction is usually conducted without the use of nitrogen. In the present work, we compared the physicochemical properties of HAs isolated from A1 horizons of soddy-podzolic soil (Retisol) and chernozem (Chernozem) by 0.1 M NaOH in the presence and absence of oxygen. The soils used in this study represented zonal types of southern taiga and steppe, respectively, and differed markedly with respect to humus formation conditions. The yield of humic substances (C_org content in the extracts), their elemental composition, functional groups content, molecular-weight distributions (gel filtration on Sephadex G-75), paramagnetic properties, and absorption spectra in the visible, UV, and IR regions were studied. For both soils, no statistically significant differences were found in the quantitative yield, molecular weight distribution, absorption spectra in the visible, UV and IR regions between HAs isolated by alkaline extraction in the presence and absence of oxygen. At the same time, for the HAs extracted from the Retisol soil in the presence of oxygen, higher O : C ratios, higher contents of quinone and carboxyl groups, and significantly higher content of free radicals were observed. This was revealed with the use of elemental analysis, potentiometric titration, ¹H and ¹³C NMR spectroscopy, and electron paramagnetic resonance spectroscopy. For the Chernozem HAs, these differences were not observed. The obtained results suggest that partial oxidation of the soil organic matter components takes place during alkaline extraction from the Retisol in the presence of oxygen. In the Chernozem, humification process is apparently accompanied by significant oxidative transformation of organic residues, so the presence of molecular oxygen does not cause further oxidation of HAs under alkaline conditions. Our results indicate that, for the isolation of HAs from the mineral horizons of Chernozems, the use of oxygen-free atmosphere is optional. In the case of Retisols, the use of oxygen-free atmosphere is desirable, especially if it is intended to study the reactions of HA oxidation upon, for example, enzymatic catalysis.