Splitting of Soil Humic Acid Fluorescence on Different Fluorophores

Polyacrylamide gel electrophoresis of chernozem soil humic acids (HA) followed by observation under UV light reveal new low molecular weight (MW) fluorescent fractions. Ultrafiltration of HA sample in 7 M urea on a membrane of low nominal MW retention (NMWR, 5 kDa) was repetitively used for separation of fluorescent and nonfluorescent species. Retentate R>5 kDa demonstrated very weak fluorescence under 270 nm excitation, while fluorescence intensity of ultrafiltrate UF<5 kDa was about six times higher than of the bulk HA. Fraction UF<5 kDa was further ultrafiltrated on membranes of MNWR 3 and 1 kDa, yielding subfractions UF3–5 kDa, UF 1–3 kDa with fluorescence maxima at 505and 488 nm, and UF<1 kDa with fluorescence maximum 465 nm and shoulder at 430nm. Fraction UF<1 kDa was purified by multiple ultrafiltration in H2O on 1-kDa membrane and demonstrated absorption spectrum with characteristic maximum at 268 nm and fluorescence maximum at 430nm. The results demonstrate that more than one fluorophore is present in chernozem soil HA complex. It is the first experimental verification of the assumption about the existence of different fluorophore in terrestrial HS.