Interactions between stepwise-eluted sub-fractions of fulvic acids and protons revealed by fluorescence titration combined with EEM-PARAFAC

In aquatic environments, pH can control environmental behaviors of fulvic acid (FA) via regulating hydrolysis of functional groups. Sub-fractions of FA, eluted using pyrophosphate buffers with initial pHs of 3.0 (FA₃), 5.0 (FA₅), 7.0 (FA₇), 9.0 (FA₉) and 13.0 (FA₁₃), were used to explore interactions between the various, operationally defined, FA fractions and protons, by use of EEM-PARAFAC analysis. Splitting of peaks (FA₃ and FA₁₃), merging of peaks (FA₇), disappearance of peaks (FA₉ and FA₁₃), and red/blue-shifting of peaks were observed during fluorescence titration. Fulvic-like components were identified from FA₃-FA₁₃, and protein-like components were observed in fractions FA₉ and FA₁₃. There primary compounds (carboxylic-like, phenolic-like, and protein-like chromophores) in PARAFAC components were distinguished based on acid-base properties. Dissociation constants (pK_a) for fulvic-like components with proton ranged from 2.43 to 4.13 in an acidic pH and from 9.95 to 11.27 at basic pH. These results might be due to protonation of di-carboxylate and phenolic functional groups. At basic pH, pK_a values of protein-like components (9.77–10.13) were similar to those of amino acids. However, at acidic pH, pK_a values of protein-like components, which ranged from 3.33 to 4.22, were 1–2 units greater than those of amino acids. Results presented here, will benefit understanding of environmental behaviors of FA, as well as interactions of FA with environmental contaminants.