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 pK_a 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.