资源与生态环境(文献资料)天然有机质的构造结构(The conformational structure of natural organic matter)

The conformational structure of naturalorganic matterAlessandroPiccolo,Riccardo SpacciniPellegrino Conte,Danca Smeikalova,DonatoSannino,Antonio Nebbioso,Barbara FontaineDipartimento di Scienze del Suolo, della Pianta.dell Ambiente e delle Produzioni AnimaliUniversita di Napoli Federico ll
The conformational structure of natural organic matter Alessandro Piccolo, Riccardo Spaccini, Pellegrino Conte, Dan ča Smejkalova, Donato Sannino, Antonio Nebbioso, Barbara Fontaine Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente, e delle Produzioni Animali Università di Napoli Federico II

WHAT IS HUMIC MATTER?
WHAT IS HUMIC MATTER?

ISOLATIONANDPURIEICATIONPROCEDURESTOOBTAINHUMUCANDFULVIC ACIDSFROMSOILPreparation ofhumic acidandfuhvicacid samples (iHss method)SoilHAfractionFAfractionHCl-extractinO.1NKOHtreatwrithO.1NHCaddKcItoO.3NKcentrifugeOn XAD-8SoilHCl-extractSolutionppton XAD-8(add 0.1 M HCI and0.1N NaOHacidity (pH 1.0)centrifugewashing to neutral pH)centrifugeHA pptExtractSoil residueonXAD-8<in O.1N HCIbackeluterepeataocidity to pH 1.0+0.3NHFwith 0.1N NaOH2timescentrifugeHAFApptpH5with0.1HCIfractionfractiondialysisfreeze-dryfreeze-dryHumin fractionHAsampleFAsample
ISOLATION AND PURIFICATION PROCEDURES TO OBTAIN HUMIC AND FULVIC ACIDS FROM SOIL Humin fraction pH 5 with 0.1 HCl freeze-dry (add 0.1 M HCl and washing to neutral pH)

Humic substances (humic andfulvicacids)havebeen(10-500KDa),traditionallydescribedasmacropolymerssimilarly to biopolymers, and their synthesis was attributed toan extracellularenzymatic catalysis.The assumed monomer of such a humic macropolymer has beenfrom time to time described with a hypothetical structureaccording to the updating of new experimental results obtainedwith progressively more advanced analytical techniques.HC:OSugarHC-OHCOOHHC=OCOOHCOOHOH-0R-CHHOCOOHCH-CH2HOCOOHOHOHYNHOH1R-CH/C=O-NHStevenson,1982+
Humic substances (humic and fulvic acids) have been traditionally described as macropolymers (10-500 KDa), similarly to biopolymers, and their synthesis was attributed to an extracellular enzymatic catalysis. The assumed monomer of such a humic macropolymer has been from time to time described with a hypothetical structure according to the updating of new experimental results obtained with progressively more advanced analytical techniques. Stevenson, 1982

Such macropolymericCHalo-(CHa)theory of humicsubstances became anaccepted paradigm.(CHyOlo-3CHnOHA number of structures,[CHa]o-(CHa)either linear or(CHa/0-2branched, but allCH2OFstabilized by covalent(CHa)0(CHglo-bonds, have beenproposed and becamecommon in textbooksand scientific reports50nm
Such macropolymeric theory of humic substances became an accepted paradigm. A number of structures, either linear or branched, but all stabilized by covalent bonds, have been proposed and became common in textbooks and scientific reports

By applying viscosity and and surface measurements to the Floryand Fox eguation derived to describe macropolymers,Goshand Schnitzer(1980)depictedhumicmatter aslinearpolymers having size and shapes depending on ionic strengthandpH of solution and concentration of sample.GOSHANDSCHNITZERMODELlonic strength(M)plSampleoncentratior54.05x10-1x1025x1021x101X1036.58国的DOLow Conc中High Conecf4
By applying viscosity and and surface measurements to the Flory and Fox equation derived to describe macropolymers, Gosh and Schnitzer (1980) depicted humic matter as linear polymers having size and shapes depending on ionic strength and pH of solution and concentration of sample

Assuming humic matter to be a polydisperse macropolymericmixture,Cameron etal.(1972)employedultracentifugation bysedimentation velocity technigue,to determine the MW of a numberof fractions ofHA separated by Gel Permeation ChromatographyThe MWvalues werereported torange from102 to 10Dalton3·0,C2B6870B52·0B40-30Mffminfmin083B2oB1A2C3A11-010*105104107Molecularweight
Assuming humic matter to be a polydisperse macropolymeric mixture, Cameron et al. (1972) employed ultracentifugation by sedimentation velocity technique, to determine the MW of a number of fractions of HA separated by Gel Permeation Chromatography. The MW values were reported to range from 102 to 106 Dalton

WERSHAW'SMODEITo explain the evident surfactant properties of humicsubstances, and their capacity to solvate hydrophobiccontaminants,Wershaw (1994)proposed that humic substancesbe aggregates or micelles of amphiphilic molecules with polarand apolar tails as much as in cell membrane arrangements.However, he did not directly approched the molecular size ofthe aggregating moleculesUHydrophilic headHydrophobic part
Hydrophilic head Hydrophobic part WERSHAW’s MODEL To explain the evident surfactant properties of humic substances, and their capacity to solvate hydrophobic contaminants, Wershaw (1994) proposed that humic substances be aggregates or micelles of amphiphilic molecules with polar and apolar tails as much as in cell membrane arrangements. However, he did not directly approched the molecular size of the aggregating molecules

THESUPRAMOLECOLARSTRUCTUREExperimental results (HPSEC, NMR, ESI-MS) indicate thathumic substances,rather than being macropolymers, aresupramolecular self-associations of heterogeneous and relatively“"small" molecules stabilized by weak bonds (H-bonds, van derWaals,元-π, CH-元), which may be disrupted by small amounts oforganic acids.CH,COOH00O50502525Void VolumeVoidVolumeRetentionVolumeRetentionVolumeTotalVolumeTotal VolumeSIZE-EXCLUSIONCHROMATOGRAPHY
CH3 COOH Retention Volume Void Volume Total Volume 100 50 25 I n t e n s i t y ( m V ) Void Volume Total Volume Retention Volume 100 50 25 I n t e n s i t y ( m V ) THE SUPRAMOLECOLAR STRUCTURE Experimental results (HPSEC, NMR, ESI-MS) indicate that humic substances, rather than being macropolymers, are supramolecular self-associations of heterogeneous and relatively “small” molecules stabilized by weak bonds (H-bonds, van der bonds, van der Waals, π-π, CH-π), which may be disrupted by small amounts of organic acids. SIZE-EXCLUSION CHROMATOGRAPHY

PRINCIPLESOESE-EXCLUSIONCHROMATOGRAPHYMobilePhaseB.Fig.2.1.Schematic of an SEC separation showing theseparation of low ()andhighMw()polymers:Astartofseparation; B smaller molecules diffuseintoporousparticles,whilelargermoleDcules eluteinthe interstitial regions ofthe packec bed; C size separation iscompleted; D large molecules,whichsamplelesscolumnvolume,elutefirst:EE small molecules having access to bothinterstitial and pore volumes eiute later
PRINCIPLES OF SIZE-EXCLUSION CHROMATOGRAPHY
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