Spectral Characterization And Proton Binding Behavior Of Soil Fulvic Acid

Soil fulvic acid?FA?is one of important components of soil dissolved organic matter,which is rich in different functional groups,such as carboxylic and phenolic groups,and has strong abilities of migration,transformation,complexation,adsorption and reduction-oxidation.The complexity and heterogeneity of the molecular structure for soil FA have become the main influencing factors for the exploration of FA compositions and environmental behaviors.Meanwhile,the proton binding behavior of soil FA significantly affects its functional group morphology,molecular structures,conformations,intramolecular reaction and other properties,and promotes or restricts the migration,transformation and bioavailability of organic pollutants and heavy metals in the soil.In this study,the extracted and purified FA from forest soil was selected to separate FA into five sub-fractions(marked as FA₃,FA₅,FA₇,FA₉ and FA₁₃)using XAD-8 adsorption resin coupled with stepwise elution with pyrophosphate buffers.Based on the spectroscopic and potentiometric titration experiments,spectroscopic methods combined with expanding model and techniques were used to characterize and study the properties of composition and acidic functional groups of the soil FA and its sub-fractions.The proton quenching mechanism,binding site heterogeneity and binding dissociation constants?pK_a?of fluorescence components in FA sub-fractions were also systematically explored.The spectroscopic methods mainly included ultraviolet-visible absorption spectroscopy,synchronous fluorescence spectroscopy and excitation-emission matrix spectroscopy.The expanding model and techniques mainly included two-dimensional correlation spectroscopy,parallel factor analysis?PARAFAC?,differential absorption and fluorescence spectroscopy,Gaussian peak fitting model and modified Stern-Volmer model.The major conclusions are as follows:1)Forest soil FA was successfully fractionated to FA sub-fractions.The fluorescence components,acidic functional groups and physicochemical properties of FA sub-fractions were significantly different.FA₃-FA₁₃ contained fulvic-like,humic-like and protein-like?including tryptophan-like and tyrosine-like?components.FA₇-FA₁₃ contained more protein-like components and easily degradable monocyclic aromatic compounds.During the potentiometric titration experiments,the charge density of FA sub-fractions increased with the increase of pH,and the contents of carboxylic and phenolic functional groups ranged 5.75-10.4 meq·g·C^-1 and 1.85-3.41 meq·g·C^-1 respectively.For the humic-like and fulvic-like components in FA sub-fractions,FA₃ and FA₁₃ contained more carboxylic and phenolic functional groups,respectively.For the protein-like components,FA₉ contained more carboxylic and phenolic functional groups than FA₇ and FA₁₃.Among five FA sub-fractions,FA3 and FA5 had higher or greater degree of molecular polymerization,molecular weight,aromaticity,and humification degree.Additionally,FA5 and FA9possessed higher fluorescence quantum yields.2)Forest soil FA and its sub-fractions showed different proton binding behaviors and different changing degrees of spectral characteristics due to the differences in molecular structure,acidic functional groups and other properties.The differential absorption spectroscopy combined with the Gaussian peak-fitting model had the advantages to highlight the protonation/deprotonation characteristics of trace FA,and the characteristics of fitted Gaussian peak were related not only to phenolic hydroxyl and carboxyl chromophores,but also to the effects of molecular charge transfers and chromophore interactions.The fluorescence spectra of FA sub-fractions showed the splitting,merging,disappearance red-shift/blue shift for the different fluorescence peaks.Based on the spectral characteristics and acid-base characteristics of FA sub-fractions,the fulvic-like and protein-like components in the FA sub-fractions were identified using the three-dimensional fluorescence spectroscopy combined with PARAFAC.In addition,the different PARAFAC components contained different contents of carboxyl-like,phenol-like,and protein-like components.3)The heterogeneous distributions of proton binding sites existed in both different fluorescence components and single fluorescence components of forest soil FA sub-fractions,reflecting in the three main aspects to the differences in fluorescence loading,the complex distributions of characteristic peaks in two-dimensional correlation spectra,and the diversity of acid dissociation constants(pK_a1).Based on the properties of characteristic peak in the synchronous and asynchronous correlation spectra with pH as the disturbance,the pK_a1 values of the fluorescence components in FA sub-fractions were consistent with their order of sequential changes.The sequential changes of fluorescence components were in the order of humic-like components>fulvic-like components with samechangingdirectionforFA3,tryptophan-likecomponents>humic-like components>fulvic-likecomponents>tyrosine-likecomponentsforFA₅,and tryptophan-like components>humic-like components>fulvic-like components for FA₇.The pK_a1 values and the order of sequential changes of tryptophan-like components in FA₉ and FA13 were greater or prior than those of tyrosine-like,fulvic-like and humic-like components.The spectral change from 374 nm to 350 nm was shown in the single fulvic-like components in FA₃,and the spectral change from 274 nm to 290 nm existed in the single tryptophan-like components in FA₉ and FA₁₃.4)The proton quenching mechanism of forest soil FA sub-fractions was dominated by static quenching,and different fluorescence components had different pKa1 values and alkaline dissociation constants(pK_a2).The pK_a1 and pK_a2 values of fluorescence components in FA sub-fractions rangd 2.43-4.76 and 9.33-31.27,respectively.The pK_a1values of humic-like and fulvic-like components?2.43-4.13?were similar to those of di-carboxylate molecules?2.5-4.3?,and pK_a1 values of tryptophan-like and tyrosine-like components?4.14-4.76?were about 2 units greater than that of amino acid?1.9-2.4?.The pKa2 values of fluorescence components in FA sub-fractions were similar to those of hydroxybenzene?8.0-10.0?and amino acids?9.61-12.02?.Under acidic pH conditions,the proton binding affinities of humic-like and fulvic-like components were weaker than those of tryptophan-like and tyrosine-like components.Moreover,the di-carboxylate groups might be the main proton binding site in humic-like and fulvic-like components.There are fewer contents of aggregated carboxyl functional groups in the aliphatic or alicyclic structures of tryptophan-like and tyrosine-like components.Under basic pH conditions,the phenolic functional groups and the amino acid compositions played a key role in the interactions between proton and fluorescent components in FA sub-fractions.