COI-T.20-Doc.-no.20ECN甘油三酯

INTERNATIONALOLIVECOUNCILCOI/T.20/Doc.no.20/Rev.22008ENGLISHOriginal:ENGLISHPríncipedeVergara,154–28002Madrid–EspañaTelef.:[phone]Fax:[phone]-e-mail:[e-mail]-(TAGs)withanequivalentcarbonnumberof42(ECN42theoretical)calculatedfromthefattyacidcomposition,andtheanalyticalresults(ECN42HPLC)obtainedbydeterminationintheoilbyhighperformanceliquidchromatography.2.FieldofapplicationThestandardisapplicabletooliveoils.Themethodisapplicabletothedetectionofthepresenceofsmallamountsofseedoils(richinlinoleicacid)ineveryclassofoliveoils.3.PrincipleThecontentoftriacylglycerolswithECN42determinedbyHPLCanalysisandthetheoreticalcontentoftriacylglycerolswithECN42(calculatedonthebasisofGLCdeterminationoffattyacidcomposition)correspondwithinacertainlimitforgeniuneoliveoils.Adifferencelargerthanthevaluesadoptedforeachtypeofoilpointsoutthattheoilcontainsseedoils.4.MethodThemethodforthecalculationofthetheoreticalcontentoftriacylglycerolswithECN42andofthedifferencewithrespecttotheHPLCdataisessentiallybytheco-ordinationofanalyticaldataobtainedbymeansofothermethods.Itispossibletodistinguishthreephases:determinationoffattyacidcompositionbycapillarygaschromatography,calculationoftheoreticalcompositionoftriacylglycerolswithECN42,HPLCdeterminationofECN42triacylglycerols.COI/T.20/Doc.no.20/Rev.2page24.1.Apparatus4.1.1Round-bottomedflasks,250and500mL.4.1.2Beakers100mL.4.1.3Glasschromatographiccolumn,21mminternaldiameter,450mmlength,withcockandnormalisedcone(female)atthetop.4.1.4Separatingfunnels,250mL,withnormalisedcone(male)atthebottom,suitableforconnectiontothetopofthecolumn.4.1.5Glassrod,600mmlength.4.1.6Glassfunnel,80mmdiameter.4.1.7Volumetricflasks,50mL.4.1.8Volumetricflasks,20mL.4.1.9Rotaryevaporator.4.1.10Highperformanceliquidchromatograph,allowingthermostaticcontrolofcolumntemperature.4.1.11Injectionunitsfor10μldelivery.4.1.12Detector:differentialrefractometer.Thefullscalesensitivityshouldbeatleast10-4unitsofrefractiveindex.4.1.13Column:stainlesssteeltube250mmlengthx4.5mminternaldiameterpackedwith5μmdiameterparticlesofsilicawith22to23%carbonintheformofoctadecylsilane*.4.1.13Recorderand/orintegrator.*Examples:Lichrosorb(Merck)RP18Art50333Lichrosphere(Merck)100CH18Art50377orequivalent4.2.ReagentsThereagentsshouldbeofanalyticalpurity.Elutionsolventsshouldbede-gassed,andmayberecycledseveraltimeswithouteffectontheseparations.4.2.1Petroleumether40-60°Cchromatographicgrade.4.2.2Ethylether,peroxide-free,freshlydistilled.4.2.3Elutionsolventforpurifyingtheoilbycolumnchromatographymixturepetroleumether/ethylether87/13(v/v).4.2.4Silicagel,70-230mesh,typeMerck7734,withwatercontentstandardisedat5%(w/w/).COI/T.20/Doc.no.20/Rev.2page34.2.5Glasswool.4.2.6AcetoneforHPLC.4.2.7AcetonitrileforHPCL.4.2.8HPLCelutionsolvent:acetonitrile+acetone(proportionstobeadjustedtoobtainthedesiredseparation;beginwith50:50mixture)orpropionitrile.4.2.9Solubilisationsolvent:acetone.4.2.10Referencetriglycerides:commercialtriglycerides(tripalmitin,triolein,etc.)maybeusedandtheretentiontimesthenplottedinaccordancewiththeequivalentcarbonnumber,oralternativelyreferencechromatogramsobtainedfromsoyaoil,mixture30:70soyaoil-oliveoilandpureoliveoil(seenotes1and2andfigures1,2,3,4).4.3.SamplepreparationAsanumberofinterferingsubstancescangiverisetofalsepositiveresults,thesamplemustalwaysbepurifiedaccordingtoIUPACmethod2.507,usedforthedeterminationofpolarcompoundsinfryingfats.4.3.1ChromatographiccolumnpreparationFillthecolumn(4.1.3.)withabout30mLofelutionsolvent(4.2.3.),thenintroduceinsidethecolumnsomeglasswool(4.2.5.)pushingittothebottomofthecolumnbymeansoftheglassrod(4.1.5.)Ina100mLbeaker,suspend25gofsilicagel(4.2.4.)in80mLofelutionmixture(4.2.3.),thentransferittothecolumnbymeansofaglassfunnel(4.1.6.).Toensurethecompletetransferofthesilicageltothecolumn,washthebeakerwiththeelutionmixtureandtransferthewashingportionstothecolumntoo.Openthecockandletthesolventelutefromthecolumnuntilitslevelisabout1cmoverthesilicagel.COI/T.20/Doc.no.20/Rev.2page44.3.2ColumnchromatographyWeighwiththeaccuracyof0.001g,2.5±0.1gofoil,previouslyfiltered,homogenisedandanhydrified,ifnecessary,ina50mLvolumetricflask(4.1.7.).Dissolveitinabout20mLofelutionsolvent(4.2.3.).Ifnecessary,slightlyheatittomakethedissolutioneasily.Coolatroomtemperatureandadjustthevolumewithelutionsolvent.Bymeansofavolumetricpipette,introduce20mLofsolutioninsidethecolumnpreparedaccordingto4.3.1.,openthecockandletthesolventelutetothesilicagellayerlevel.Thenelutewith150mLofelutionsolvent(4.2.3.),adjustingthesolventrateatabout2mL/min(150mLwilltakeabout60-70minutestopassthroughthecolumn).Theeluateisrecoveredina250mLround-bottomedflask(4.1.1.)previouslytaredinanovenandexactlyweighed.Eliminatethesolventatreducedpressure(Rotavapor)andweightheresiduethatwillbeusedtopreparethesolutionforHPLCanalysisandf