Analysis of electrophoretic soil humic acids fractions by reversed-phase high performance liquid chromatography with on-line absorbance and fluorescence detection

A combination of reversed-phase high performance liquid chromatography (RP HPLC) with on-line absorbance and fluorescence detection was used for analysis of chernozem soil humic acids (HAs) and their fractions A, B and C + D with different electrophoretic mobility (EM) and molecular size (MS). Samples were injected onto the column at the identical volume and absorbance. All chromatograms exhibit the resolution of seven peaks. The estimation of relative recovery of HAs and fractions from the reverse-phase column has been done. High MS fraction A, which possesses the low EM, is essentially more hydrophobic (73% of the fraction amount remained adsorbed on the column) and aliphatic than medium MS and EM fraction B (33% of the fraction amount remained adsorbed on the column). The most hydrophilic and aromatic properties belong to low MS fraction C + D, which possess the highest EM and practically was not adsorbed on the column. The hydrophobicity of the bulk HAs lies within the range of fractions hydrophobicity. The absorption spectra of bulk HAs, electrophoretic fractions A, B, C + D and corresponding RP HPLC peaks were featureless but had differences in the values of absorbance ratio at 300 and 400 nm (A3/A4). For fractions A and B this ratio gradually decreased from peak 1 to 7 (from 3.05 to 2.80 and 3.00 to 2.40, respectively). This trend was less pronounced in HAs and practically absent in fraction C + D, where ratio A3/A4 varied within a small range. The strong relationship between fluorescence properties, EM, MS, polarity and aliphaticity/aromaticity of HAs fractions was found. Humic and protein-like fluorescence had different polarity nature. The protein-like fluorescence is located in humic material which irreversibly adsorbed on the reverse-phase column and not subjected to RP HPLC characterization. The humic-like fluorescence at Ex/Em 270/450 nm is mostly located in the hydrophilic peak of low MS fraction C + D. Taking into account that high MS fraction A consisted mainly of aliphatic components it is reasonable to suggest that these associations are capable of organizing into micellar structures. These data could be of great environmental importance, because the different fractions might reflect different soil physical–chemical properties.

► RP-UPLC with on-line absorbance and fluorescence of soil humic acids and fractions.
► Relative recovery of humic acid and fractions from the reverse-phase column.
► Relationship between fluorescence, absorbance, polarity and aliphaticity in fractions.