[Show abstract][Hide abstract] ABSTRACT: Polyacrylamide gel electrophoresis of chernozem soil humic acids (HAs) followed by observation under UV (312 nm) excitation light reveals new low molecular weight (MW) fluorescent fractions. Ultrafiltration of HAs sample in 7 M urea on a membrane of low nominal MW retention (NMWR, 5 kDa) was repetitively used for separation of fluorescent and non-fluorescent species. Thirty ultrafiltrates and the final retentate R were obtained. Fluorescence maxima of separate ultrafiltrates were different and non-monotonously changed in the range of 475-505 nm. Fluorescence maxima of less than 490 nm were detected only in the four first utrafiltrates. For further physical-chemical analyses all utrafiltrates were combined into a fraction called UF<5 (NMW<5 kDa). Retentate R demonstrated very weak fluorescence under 270 nm excitation, while fluorescence intensity of UF<5 was about six times higher than of the bulk HAs. Fraction UF<5 was further ultrafiltrated on membranes of MNWR 3 kDa and 1 kDa, yielding three subfractions UF3-5, UF1-3 and UF<1 with NMW 3-5 kDa, 1-3 kDa and <1 kDa, respectively. The validation of the UF procedure was performed by size exclusion chromatography on Sephadex G-25 column. The fluorescence maxima were found to be at 505, 488 and 465 nm for UF3-5, UF1-3 and UF<1, respectively, with increasing of fluorescence intensity from UF3-5 to UF1-3 to UF<1 fraction. EPR analysis showed that the amount of free radicals was the largest in retentate R and drastically decreased in fluorescent ultrafiltrates. The results demonstrate that more than one fluorophore is present in chernozem soil HAs complex.
[Show abstract][Hide abstract] ABSTRACT: Soil humic acids are complex mixtures displaying several still unexplained spectral properties. In this work, we aimed to investigate the fluorescent properties of soil humic acid macromolecules of molecular sizes less than 5 kDa. To recover the fraction < 5 kDa, we employed a protocol based on repeated ultrafiltration of a soil humic acid HA with a 5 kDa membrane. The obtained retentate did not show any fluorescence. In contrast, the ultrafiltrate, which accounted for 20% by mass of the starting HA, contained a significant amount of the fluorescent components. This ultrafiltrate was in turn ultrafiltered through 3 kDa and 1 kDa membranes, and three subfractions were obtained. Their emission spectra, measured at an absorbance of 0.1 at 300 nm, were different in shape and intensity. In particular, the contribution of the long-wavelength emissions decreased and the intensity of emission at the maximum increased as the molecular weight decreased. These results show that a large portion of the fluorescent moieties of the soil HA are contained in the fraction < 5 kDa and that the repartitioning of fluorophores depends on their molecular size. This finding represents an interesting starting point for further studies.