Oleg Trubetskoj

Polyacrylamide gel electrophoresis of soil humic acid fractionated by size-exclusion chromatography and ultrafiltration

Abstract A humic acid (HA) from chernozem soil has been fractionated either by size-exclusion chromatography (SEC) on Sephadex G-75 using water, Tris-HCl or 7 M urea as eluents, or by ultrafiltration (UF) in the presence of 7 M urea or water. UF-fractionated HA was classified as 100K (nominal retention size > 100 000); 30K (100 000–30 000); 10K (30 000–10 000); 5K (10 000–5000). Several pools from each chromatography and all ultrafiltration retentates were assayed by polyacrylamide gel electrophoresis (PAGE). The results indicate that SEC fractionation gave a better separation of HA on fractions differing in electrophoretic mobility and molecular size (MS), and that apparently SEC is a more feasible technique than UF for soil HA fractionation; nevertheless the UF in 7 M urea solution might be useful for MS evaluation of HA fractions obtained by association SEC-PAGE. PAGE in the presence of denaturing agents can be successfully used for checking the purity of HA fractions obtained by both SEC and UF.

Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis

Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500–510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions.

Compositional differences between soil humic acids extracted by various methods as evidenced by photosensitizing and electrophoretic properties.

Humic acids (HAs) were isolated from Elliott soil provided by the International Humic Substances Society (1BS102M) by three commonly used methods: (i) 0.1M NaOH (EI-HA), (ii) neutral 0.1M Na(4)P(2)O(7) (L-HA) and subsequent 0.1M NaOH (S-HA), and (iii) 0.1M NaOH+0.1M Na(4)P(2)O(7) (NP-HA). The objective was to evaluate the impact of these extractants on the photosensitizing properties of the isolated HAs. HAs were analyzed for their elemental composition, functional acid groups content, absorption and emission properties, electrophoretic characteristics and ability to produce singlet oxygen using furfuryl alcohol (FFA) as a scavenger. L-HA was slightly more aromatic and oxygenated than the other HAs and contained a higher portion of long-wavelength fluorophores and macromolecules showing low molecular size (MS) and high electrophoretic mobility. L-HA also gave a rate of FFA photooxygenation between 1.25- and 1.6-fold higher than the other HAs. This suggests that the free humic macromolecules ionized at pH 7 and/or weakly bounded on mineral surfaces via cation bridges are of relatively low MS and contribute significantly to the photosensitizing and long-wavelength emitting properties. Differences among the other HAs were more subtle, but the parallel evolution of the reactivity and electrophoretic characteristics was observed. Photochemical and electrophoretic measurements seem to be sensitive indicators to evaluate differences among the extraction procedures of HAs.

Photochemical activity and fluorescence of electrophoretic fractions of aquatic humic matter

A combination of sephadex gel-chromatography and polyacrylamide gel electrophoresis was used to obtain stable electrophoretic fractions with different molecular sizes from a preparation of humic substances isolated from river water by reverse osmosis. The photochemical activity of humic substances and their fractions was determined. For the first time it is shown that low-molecular fractions of aquatic humic substances, when irradiated by polychromatic light, can intensely fluoresce and initiate the formation of singlet O2, effectively destroying model compound furfural. The photochemical activity and intensity of fluorescence of high-molecular fractions is insignificant. The obtained data will help to extend the current knowledge in the field of natural water hydrochemistry about the ways of its natural self-purification from anthropogenic pollutants.

Weight and optical differences between soil humic acids fractions obtained by coupling SEC-PAGE

Humic acids (HAs) from four soils of different origin (podzoluvisol, phasozem, chernozem and ferralsol) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EM) have been obtained from all the samples. Fractions identically marked had similar EM, independently of HA sources. The weight content, extinction coefficients (ECs), E4/E6 ratios have been studied for all HAFs. EC values and E4/E6 ratios increased as MS decreased within an individual HA sample. The weight content of high MS fraction was the highest in podzoluvisol and ferralsol and the smallest in phasozem and chernozem. On the other hand, chernozem and phasozem HAs contained considerably more of the lower MS fraction than HAs from the podzoluvisol and ferralsol. The knowledge of weight and optical differences between soil humic acids fractionated by coupling SEC-PAGE could be valued for explanation of some physico-chemical properties of soils and influence of soil genesis on humic matter properties.

Photoinductive efficiency of soil extracted humic and fulvic acids

Humic and fulvic acids extracted from soils of different genesis were investigated for their ability to photoinduce the transformation of fenuron (2 x 10(-4) mol(-1)) at 365 nm. The ratio of the initial rate of fenuron consumption over the rate of light absorption by humic substances was found to be higher for fulvic acids (range 2.0 x 10(-3) to 9.0 x 10(-5)) than for humic acids (range 1.7 x 10(-4) to - 3.6 x 10(-5)). Within the FAs population, this ratio decreased as the specific absorption coefficient at 365 nm increased. It seems therefore that most of 365-nm absorbing components have no photoinductive activity and even reduce that of photoinductive chromophores.

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HAs130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.

1H-NMR and 13C-NMR spectroscopy of chernozem soil humic acid fractionated by combined size-exclusion chromatography and electrophoresis

Humic acid (HA) from chernozem soil was fractionated using combination size-exclusion chromatography–polyacrylamide gel electrophoresis, and three fractions showing distinct electrophoretic mobilities (EM) and molecular sizes (MS) were obtained. Unfractionated HA, all fractions and HA after 7 M urea treatment (HAU) were examined using both solid 13C and 1H in D2O and DMSO-d 6 NMR. The ratio C ar (165−108 ppm)/C alk (108−0 ppm) increased by a factor of 5 from the highest to the lowest MS fraction. This suggests that there is significant heterogeneity in terms of molecular structure and functionality among the various fractions, which differ in both EM and MS. A significant contrast in aromatic range was observed between 1H-NMR spectra of the original unfractionated HA and HAU in DMSO-d 6 solvent. Taking into account that the high MS fraction consists mainly of aliphatic components and the low MS fraction mainly of aromatic components, it is reasonable to suggest that these fractions have different chemical structures. These results are of great environmental importance because different MS fractions might be differently involved in chemical/biological processes in soils.

Surface-Enhanced Raman Spectroscopy of Chernozem Humic Acid and Their Fractions Obtained by Coupled Size Exclusion Chromatography—Polyacrylamide Gel Electrophoresis (SEC-PAGE):

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.

Humic acid characteristics in podzol soil chronosequence

Humic acid (HA) characteristics were studied in a natural revegetation chronosequence of 10-, 20-, 60-year-old and mature podzol soils under a pine forest, along the spoil heaps of a sand quarry. The elemental composition of HAs showed a trend towards carbon increase and hydrogen decrease with soil age. Essential differences in the atomic ratios between humic acids extracted from the different soils may be due to the intensity of the humification process as related to soil age. Compared with HAs extracted from organic horizons, those from mineral horizons showed lower C/N and H/C ratios, whereas O/C ratios were higher. Electrophoretic data showed that the proportion of the more-mobile fraction (L-MS) was higher in the mineral horizons than in the organic horizons and, among organic layers, in the Oe and Oa horizons compared with Oi. Intensive transformation of pine remains may have occurred and led to HAs with an increasing degree of humification in only 60 years. Moreover, progressive accumulation of the L-MS fraction in the E and Bs horizons with soil age might be the result of the ongoing migration of the most-polar organic compounds down through the soil profiles.