Fluorescence regional integration and differential fluorescence spectroscopy for analysis of structural characteristics and proton binding properties of fulvic acid sub-fractions.

Structural characteristics and proton binding properties of sub-fractions (FA3 -FA13 ) of fulvic acid (FA), eluted stepwise by pyrophosphate buffer were examined by use of fluorescence titration combined with fluorescence regional integration (FRI) and differential fluorescence spectroscopy (DFS). Humic-like (H-L) and fulvic-like (F-L) materials, which accounted for more than 80% of fluorescence response, were dominant in five sub-fractions of FA. Based on FRI analysis, except the response of F-L materials in FA9 and FA13 , maximum changes in percent fluorescence response were less than 10% as pH was increased from 2.5 to 11.5. Contents of carboxylic and phenolic groups were compared for fluorescence peaks of FA sub-fractions based on pH-dependent fluorescence derived from DFS. Static quenching was the dominant mechanism for binding of protons by FA sub-fractions. Dissociation constants (pKa) were calculated by use of results of DFS and the modified Stern-Volmer relationship. The pKa of H-L, F-L, tryptophan-like and tyrosine-like materials of FA sub-fractions exhibited ranges of 3.17-4.06, 3.12-3.97, 4.14-4.45 and 4.25-4.76, respectively, for acidic pHs. At basic pHs, values of pKa for corresponding materials were in ranges of 9.71-10.24, 9.62-10.99, 9.67-10.31 and 9.33-10.28, respectively. At acidic pH, protein-like (P-L) materials had greater affinities for protons than did either H-L or F-L materials. The di-carboxylic and phenolic groups were likely predominant sites of protonation for both H-L and F-L materials at both acidic and basic pHs. Amino acid groups were significant factors during proton binding to protein-like materials of FA sub-fractions at basic pH.