Protonation-dependent heterogeneity in fluorescent binding sites in sub-fractions of fulvic acid using principle component analysis and two-dimensional correlation spectroscopy.

Heterogeneous distributions of proton binding sites within sub-fractions of fulvic acid (FA3-FA13) were investigated by use of synchronous fluorescence spectra (SFS), combined with principle component analysis (PCA) and two-dimensional correlation spectroscopy (2D-COS). Tryptophan-like, fulvic-like and humic-like materials were observed in SFS. Tyrosine-like materials were identified by use of SFS-PCA analysis. Combined information from synchronous-asynchronous maps and dissociation constants (pKa) was used to describe heterogeneity of binding sites for protons within each sub-fraction. Heterogeneous distributions of proton binding sites were observed in fulvic-like, humic-like, tryptophan-like, and tyrosine-like materials of five sub-fractions and even in the single fulvic-like materials in FA3 and tryptophan-like materials in FA9 and FA13. Values of pKa of sub-fractions ranged from 2.20 to 5.29, depending on associated wavelengths in synchronous-asynchronous maps and use of the modified Stern-Volmer equation. The larger values of pKa (4.17-5.29) were established for protein-like materials (including tryptophan-like and tyrosine-like materials) in comparison to those (2.20-3.38) for humic-like and fulvic-like materials in sub-fractions. Sequential variations of 274nm (pKa 4.15-5.29)→360-460nm (pKa 2.78-2.39) for FA5-FA13 revealed that binding of protons to tryptophan-like materials appeared prior to humic-like/fulvic-like materials. In FA9, protons were preferentially binding to tryptophan-like materials than tyrosine-like materials. In FA3, protons were preferentially binding to humic-like materials than fulvic-like materials. Relative differences of values of pKa for fluorescent materials within each sub-fraction were consistent with sequential orders derived from asynchronous maps. Such an integrated approach, SFS-PCA/2D-COS, has superior potential for further applications in exploring complex interactions between dissolved organic matter and contaminants in engineered and natural environments.