ph示差法原理

UNITF1.2CharacterizationandMeasurementofAnthocyaninsbyUV-VisibleSpectroscopyAnthocyaninpigmentcontenthasacriticalroleinthecolorqualityofmanyfreshandprocessedfruitsandvegetables.Thus,accuratemeasurementofanthocyanins,alongwiththeirdegradationindices,isveryusefultofoodtechnologistsandhorticulturistsinassessingthequalityofrawandprocessedfoods.Sincemanynaturalfoodcolorantsareanthocyaninderived(e.g.,grape-skinextract,red-cabbageextract,purple-carrotextract),thesamemeasurementscanbeusedtoassessthecolorqualityofthesefoodingredients.Inaddition,thereisintenseinterestintheanthocyanincontentoffoodsandnutraceuticalsbecauseofpossiblehealthbenefitssuchasreductionofcoronaryheartdisease(BridleandTimberlake,1996),improvedvisualacuity(TimberlakeandHenry,1988),antioxidantactivities(TakamuraandYamagami,1994;Wangetal.,1997),andanticanceractivities(Karaivanovaetal.,1990;Kameietal.,1995).Substantialquantitativeandqualitativeinformationcanbeobtainedfromthespectralcharacteristicsofanthocyanins.TheprotocolsdescribedinthisunitrelyonthestructuraltransformationoftheanthocyaninchromophoreasafunctionofpH,whichcanbemeasuredusingopticalspectroscopy.ThepH-differentialmethod,arapidandeasyprocedureforthequantitationofmonomericanthocyanins,isfirstdescribed(seeBasicProtocol1).Inaddition,otherauxiliaryspectrophotometrictechniquesareusedtomeasuretheextentofanthocyaninpolymeri-zationandbrowning(seeBasicProtocol2).BASICPROTOCOL1TOTALMONOMERICANTHOCYANINBYTHEpH-DIFFERENTIALMETHODAnthocyaninpigmentsundergoreversiblestructuraltransformationswithachangeinpHmanifestedbystrikinglydifferentabsorbancespectra(Fig.F1.2.1).ThecoloredoxoniumformpredominatesatpH1.0andthecolorlesshemiketalformatpH4.5(Fig.F1.2.2).ThepH-differentialmethodisbasedonthisreaction,andpermitsaccurateandrapidmeasurementofthetotalanthocyanins,eveninthepresenceofpolymerizeddegradedpigmentsandotherinterferingcompounds.ContributedbyM.MónicaGiustiandRonaldE.WrolstadCurrentProtocolsinFoodAnalyticalChemistry(2001)F1.2.1-F1.2.13Copyright©2001byJohnWiley&Sons,Inc.2.01.81.61.41.21.00.80.60.40.20.0260360460Wavelength(nm)AbsorbancepH1.0pH4.5560660760FigureF1.2.1Spectralcharacteristicsofpurifiedradishanthocyanins(acylatedpelargonidin-3-sophoroside-5-glucosidederivatives)inpH1.0andpH4.5buffers.F1.2.1AnthocyaninsMaterials0.025Mpotassiumchloridebuffer,pH1.0(seerecipe)0.4Msodiumacetatebuffer,pH4.5(seerecipe)1.Turnonthespectrophotometer.Allowtheinstrumenttowarmupatleast30minbeforetakingmeasurements.2.Determinetheappropriatedilutionfactorforthesamplebydilutingwithpotassiumchloridebuffer,pH1.0,untiltheabsorbanceofthesampleattheλvis-max(TableF1.2.1)iswithinthelinearrangeofthespectrophotometer(i.e.,formostspectrophotometerstheabsorbanceshouldbelessthan1.2).Dividethefinalvolumeofthesamplebytheinitialvolumetoobtainthedilutionfactor(DF;forexampleseestep7).IMPORTANTNOTE:Inordertonotexceedthebuffer’scapacity,thesampleshouldnotexceed20%ofthetotalvolume.3.Zerothespectrophotometerwithdistilledwateratallwavelengthsthatwillbeused(λvis-maxand700nm).Manyspectrophotometerswillallowforarapidbaselinecorrectiontozerobyusingbaselineadjust.4.Preparetwodilutionsofthesample,onewithpotassiumchloridebuffer,pH1.0,andtheotherwithsodiumacetatebuffer,pH4.5,dilutingeachbythepreviouslydetermineddilutionfactor(step2).Letthesedilutionsequilibratefor15min.R1R2OHquinonoidalbase:bluepH=7chalcone:colorlesspH=4.5carbinolpseudo-base(hemiketalform):colorlesspH=4.5flavyliumcation(oxoniumform):orangetopurplepH=1HOO-glyO-glyOO-glyO-glyOHOOHOHR1R2OHHOO-glyO-glyO+R1R2R1R2OHHO+H2O−H+−H+HOO-glyO-glyOFigureF1.2.2PredominantstructuralformsofanthocyaninspresentatdifferentpHlevels.CurrentProtocolsinFoodAnalyticalChemistryF1.2.2CharacterizationandMeasurementofAnthocyaninsbyUV-VisibleSpectroscopyTableF1.2.1ReportedMolarAbsorptivityofAnthocyaninsAnthocyaninaSolventsystemλvis-max(nm)Molarabsorptivity(ε)ReferenceCyanidin(Cyd)Cyd0.1%HClinethanol510.524600Schou,19270.1%HClinethanol54734700Ribereau-Gayon,1959Cyd-3-ara15:850.1NHCl/ethanol53844400ZapsalisandFrancis,196515:850.1NHCl/ethanol53544460FulekiandFrancis,1968aCyd-3,5-diglu0.1NHCl52030175Niketic-AleksicandHrazdina,1972MethanolicHCl508.535000BrouillardandElHacheChahine,1980Cyd-3-gal0.1%HClinmethanol53034300SiegelmanandHendricks,195815:850.1NHCl/ethanol53544900SakamuraandFrancis,196115:850.1NHCl/ethanol53546200ZapsalisandFrancis,196515:850.1NHCl/ethanol53546230FulekiandFrancis,1968aHClinmethanol53030200Swain,1965Cyd-3-gluAqueousbuffer,pH151026900JurdandAsen,19660.1NHCl52025740McClure,19671%HClinmethanol53034300SiegelmanandHendricks,195810%ethanol,pH1.551218800Herediaetal.,1998Cyd-3-rutAqueousbuffer,pH0.95107000Figueiredoetal.,19961%HCl52328840Swain,1965Cyd-3-sam-5-gluAqueousbuffer,pH0.95223600Figueiredoetal.,1996Cyd-3-sam-5-glu+sinapic+caffeic+malonicAqueousbuffer,pH0.953821200Figueiredoetal.,1996Cyd-3-sam-5-glu+sinapic+ferulicAqueousbuffer,pH0.952815100Figueiredoetal.,1996Cyd-3-sam-5-glu+sinapic+ferulic+malonicAqueousbuffer,pH0.953820100Figueiredoetal.,1996Cyd-3-sam-5-glu+sinapic+p-coum+malonicAqueousbuffer,p