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OntheOriginoftheOpticalPropertiesofHumicSubstancesROSSANADELVECCHIO†ANDNEILV.BLOUGH*DepartmentofChemistryandBiochemistry,UniversityofMaryland,CollegePark,Maryland20742Absorptionandfluorescencespectroscopyandlaserphotobleachingexperimentswereemployedtoprobetheoriginsoftheopticalpropertiesofhumicsubstances(HS).LuminescencequantumyieldsandthewavelengthsofmaximumemissionwereacquiredforSuwanneeRiverhumicacid(SRHA)andfulvicacid(SRFA)atanextensiveseriesofexcitationwavelengthsacrosstheultravioletandvisible.Laserirradiationataserieswavelengthacrosstheultravioletandvisiblewasfurtheremployedtodestroyselectivelychromophoresabsorbingatspecificwavelengths,usingabsorptionspectroscopytofollowtheabsorptionlosseswithirradiationtime.Theresultsprovideunequivocalevidencethattheabsorptionandemissionspectraofthesematerialscannotresultsolelyfromasimplelinearsuperpositionofthespectraofnumerousindependentchromophores.Instead,thelongwavelengthabsorptiontailofHS(350nm)appearstoarisefromacontinuumofcoupledstates.Weproposethatthisbehaviorresultsfromintramolecularcharge-transferinteractionsbetweenhydroxy-aromaticdonorsandquinoidacceptorsformedbythepartialoxidationofligninprecursors.Wefurtherproposethatthesedonor-acceptorinteractionsmaybeacommonphenomenon,occurringwithinallnaturalhydroxy-orpolyhydroxy-aromaticpolymersthatformappropriateacceptorsuponpartialoxidation.Examplesofsuchspeciesincludelignin,polyphenols,tannins,andmelanins.IntroductionHumicsubstancesarecomplexorganicmaterialsfoundubiquitouslyinnaturewheretheyplayanessentialroleinnumerousenvironmentallyimportantprocesses(1-4).Be-causelightabsorptionbythesesubstancesincreasesexpo-nentiallywithdecreasingwavelengthacrossthevisible(VIS)andultraviolet(UV)spectrum(5-9),theycanprovideaquaticorganismsprotectionfromdamagingultravioletradiation(7,9-11).Atthehigherlevelsoftenfoundinfreshandcoastalwaters,absorptionbyHScanalsodominatetotallightabsorptionintheblueportionofthevisiblespectrum,thusalteringthequantityandqualityoflightavailableforphotosynthesis(4,7,12,13).Throughtheireffectontheaquaticlightfield,aswellasthroughtheirphotochemicalreactions(14-16),HScanhaveasubstantialimpactonthebiogeochemistryofnaturalwaters(7,14).Exhibitingnodistinctbands,theabsorptionspectraofHSdecreasewithincreasingwavelengthinanapproximatelyexponentialfashion,whereastheemissionspectra,whicharebroadandfeatureless,decreaseinintensityandshiftcontinuouslytotheredwithincreasingexcitationwavelength(17).AlthoughthesepropertiesofHShavebeenknownandstudiedextensivelyforover50years(1,2,4,7,17-19),anadequateexplanationofthesespectraldependencieshasyettobeachieved.Inparticular,nosatisfactoryexplanationhasyetbeenprovidedthatcanaccountforthelongwavelengthabsorptionandemissionpropertiesofthesematerials.Moreover,littleisknownatthemolecularlevelaboutthenatureoftheconstituentsorinteractionsthatproducetheseratheruniquespectralfeatures.Recently,workershavebeguntotestwhethertheopticalpropertiesofHScanbeattributedtoasimplesumofthespectraofaseriesofindependentchromophoresorwhetheramorecomplexsituationisoccurring(20-27).TheobservedopticalpropertiesofHScouldariseintwodistinctfashions.Thefirstpostulatesthattheabsorption(andemission)spectrumresultsfromasimplelinearsuperpositionoftheabsorption(emission)spectraofanensembleofindependent(noninteractingorelectronicallyisolated)chromophores(superpositionmodel)(20,22,23).Incontrast,thesecondpostulatesthepresenceofonlyafewdistinctchromophoresorcloselyrelatedclassesofchromophores,whichthroughelectronicinteractionproducenewopticaltransitionsthatareresponsiblefortheexponentiallydecreasing,longwavelengthabsorptiontail(interactionmodel)(17,20,21,25,28).Opticalcharge-transferbands,producedviaaground-stateinteractionbetweenanelectrondonor,D(e.g.,polyhydroxylatedaromatics,phenols,orindoles),andanelectronacceptor,A(e.g.,quinones),incloseproximityrepresentapossibleexplanationforthislongwavelengthabsorption(17,21,25,27-32).Thesedonor-acceptorcomplexesareknowntoexhibitnewbroadabsorptionbands(atlowerenergies)thatarenotshownbyeitherdonororacceptormoleculesindependently(29,33).Inearlierwork(21),weprovidedpreliminaryevidencethattheabsorptionspectraofHScannotarisefromasuperpositionofthespectraofnumerousindependentchromophores,basedonananalysisofthephotobleachingkineticsofHSandnaturalwatersfromtheDelawareandChesapeakeBays.Further,anattempttoaccountforthephotobleachingbehaviorofthesesamplesunderpolychro-maticlightusingasuperpositionmodelwasunsuccessful(20).Here,weemployroom-temperatureabsorptionandfluorescencespectroscopyandlaserphotobleachingexperi-mentstotestthisideaexplicitly.LuminescencequantumyieldsandthewavelengthsofmaximumemissionforSuwanneeRiverfulvicacid(SRFA)andhumicacid(SRHA)wereacquiredatexcitationwavelengthsacrosstheultravioletandvisiblewavelengths.Alaserwasfurtheremployedtodestroyselectivelyspeciesabsorbingatspecificwavelengthsacrosstheultravioletandthevisible,usingabsorptionspectroscopytofollowthephotobleaching.BecauseHScouldsensitizethephotobleachingthroughtheproductionofreactiveoxygenspecies(5,16,17,2