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 7M urea treatment (HAU) were examined using both solid 13C and 1H in D2O and DMSO-d6 NMR. The ratio C ar (165-108ppm)/Calk (108-0ppm) 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-d6 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.