1、BRITISH STANDARD BS EN 14106:2003 Fat and oil derivatives Fatty Acid Methyl Esters (FAME) Determination of free glycerol content The European Standard EN 14106:2003 has the status of a British Standard ICS 67.200.10 BS EN 14106:2003 This British Standard was published under the authority of the Stan
2、dards Policy and Strategy Committee on 14 May 2003 BSI 14 May 2003 ISBN 0 580 41839 1 National foreword This British Standard is the official English language version of EN 14106:2003. The UK participation in its preparation was entrusted to Technical Committee AW/307, Oil seeds animal and vegetable
3、 fats and oils, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catal
4、ogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its cor
5、rect application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests in
6、formed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 10, an inside back cover and a back cover. The BSI copyright notice displayed in this document i
7、ndicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14106 April2003 ICS67.200.10 Englishversion FatandoilderivativesFattyAcidMethylEsters(FAME) Determinationoffreeglycerolcontent Produitsdrivsdescorpsg
8、rasEstersmthyliques dacidesgras(EMAG)Dterminationdelateneuren glycrollibre ErzeugnisseauspflanzlichenundtierischenFettenund lenFettsureMethylester(FAME)Bestimmungdes GehaltesanfreiemGlycerin ThisEuropeanStandardwasapprovedbyCENon2January2003. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegul
9、ationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversion
10、s(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Gre
11、ece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitat
12、ioninanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14106:2003EEN14106:2003(E) 2 Foreword Thisdocument(EN14106:2003)hasbeenpreparedbyTechnicalCommitteeCEN/TC307“Oilseeds,vegetable andanimalfatsandoilsandtheirbyproductsMethodsofsamplingandanalysis“,thesecretariatofwhichisheld b
13、yAFNOR. This EuropeanStandard shallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltext orbyendorsement,atthelatestbyOctober2003andconflictingnationalstandardsshallbewithdrawnatthelatest byOctober2003. ThisdocumenthasbeenpreparedunderMandateM/245onFattyAcidMethylester(FAME)givent
14、oCENbythe EuropeanCommissionandtheEuropeanFreeTradeAssociation. AnnexAisinformative. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark,Finland, France,Germany,Greece,Hung
15、ary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal, Slovakia,Spain,Sweden,SwitzerlandandtheUnitedKingdom.EN14106:2003(E) 3 1Scope ThisEuropeanStandardspecifiesagaschromatographicmethodforthedeterminationoffreeglycerolcontentin FattyAcidMethylEsters(FAME)intherangeof0,005%to0,070%
16、,hereinafterreferredasFAME. ThismethodaimstoevaluatetheFAMEquality,intermsoftransesterificationbyproductscontentsuchas glycerol,whoseconcentrationmayaffectthefuelbehaviour. WARNINGTheuseofthismethodmayinvolvehazardousequipment,materialsandoperations.This methoddoesnotpurporttoaddresstoallofthesafety
17、problemsassociatedwithitsuse,butitisthe responsibilityoftheusertosearchandestablishappropriatesafetyandhealthpracticesanddetermine theapplicabilityofregulatorylimitationspriortouse. NOTE Inthecontextofthismethod( m/m)and( v/v)canbeused. 2 Normativereferences ThisEuropeanStandardincorporatesbydatedor
18、undatedreference,provisionsfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetext,andthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbyamendmentorrevisio
19、n.Forundatedreferencesthelatesteditionofthe publicationreferredtoapplies(includingamendments). ENISO661, AnimalandvegetablefatsandoilsPreparationoftestsample(ISO661:1989). 3 Termsanddefinitions ForthepurposesofthisEuropeanStandard,thefollowingtermanddefinitionapply. 3.1 freeglycerolcontent istheresi
20、dualglycerolremaininginFAMEafterthevegetableoiltransesterificationreactionandtheseparationof soobtainedglycerol 4 Principleofmethod Ethylalcohol,waterandhexaneandaknownamountofinternalstandardareaddedtoaknownquantityof sample.Theadditionofthesesolventscausestheformationoftwophasesandfreeglycerolisqu
21、antitatively transferredintothelowerone. Thegaschromatographicanalysisoflowerphaseallowstoquantifytheconcentrationoffreeglycerol. 5Reagents Useonlyreagentsofrecognizedanalyticalgrade,unlessotherwisespecified. 5.1 Hexane. 5.2 1,4Butanediol, 99%minimum. 5.3 Ethylalcohol ,95%minimum. 5.4 Formicacid, 99
22、%minimum. 5.5 Glycerol,99%minimum.EN14106:2003(E) 4 5.6 Carriergas :helium,GLCgrade. 5.7 Auxiliarygases : hydrogen,purity99%minimum,freefrommoistureandorganiccompounds; air,freefromorganiccompounds. 5.8 InternalStandardSolution:weighapproximately80mg(accuracy 0,0001g)of1,4Butanediolina volumetricfla
23、sk,100mlcapacity.Dissolveinsomemlofdistilledwater,add1mlofformicacidandfilluptothe markusingdistilledwater.Thesopreparedsolutionisstablefor24hifstoredatambienttemperature. 6Apparatus 6.1 Gaschromatograph equippedbythefollowingdevices: 6.1.1 Thermostaticovenforcolumn,abletomaintainthesettemperaturewi
24、thanaccuracyof 1C. 6.1.2 Thermostaticinjectionport,equippedforcapillarycolumninsplitsplitlessmodeor,alternatively,forpacked column. 6.1.3 Flameionisationdetector(FID)andconverter/amplifierdevice. 6.1.4 Recorder/integrator,capabletorunwiththeconverter/amplifierwitharesponsetimeof1smaximumand variable
25、chartspeed. 6.1.5 Capillarycolumn,typePoraPLOTQ,length=10m,diameter0,32mm,filmthickness=10m(Note)or, alternativelypackedcolumnfilledwithCHROMOSORB101,diameter4mm,length=1m. NOTE Someproblemsanddiscrepanciesoncapillarycolumnbehaviourfromdifferentsuppliershavebeennoticed.Some Authorsreferalsothepossib
26、ilitytousecapillarycolumnscoatedwithFFAP(FreeFattyAcidPhase)orpolyethyleneglycol phasesinsteadofthesuggestedstationaryphase.Neverthelessthechoiceofcolumnshouldbebasedonthefollowingcriteria: theseparationbetweenglycerolandinternalstandardpeaksshouldbecomplete(baselinenearlytozerobetweentwo peaks); th
27、eanalysistimeshouldnotexceed15min; columnswithresponsefactorforglycerol,calculatedsuchas9.3,higherthan2,5arenotsuitable. 6.1.6 Flowcontrollers,forcarrierandauxiliarygases. 6.2 Microsyringe forgaschromatography ,5lor10l. 6.3 Volumetricflasks ,50mland100mlcapacity. 6.4 Twomarkprecisionpipettes ,1mlcap
28、acity. 6.5 Volumetricpipettes ,5mlcapacity. 6.6 Analyticalbalance ,accuracy 0,0001g. 6.7 Glassconicaltesttubes ,10mlcapacity. 6.8 Centrifugalmachine ,abletorunatthespeedof2000revolutionsperminute.EN14106:2003(E) 5 7Sampling SamplingisnotpartofthemethodspecifiedinthisEuropeanStandard.Arecommendedsamp
29、lingmethodisgiven inENISO55551. SamplingproceduresshalltakeintoaccountthatfreeglycerolcontainedinFAMEhasastrongaffinityforglass. Thereforesamplesshallbecollectedandstoredavoidingtheuseofglass. 8 Preparationoftestsample PreparethetestsampleinaccordancewithENISO661.Thetestsampleshallnotbeheatedand/orf
30、iltered. 9Procedure 9.1 Gaschromatographicconditions Thefollowinggeneraloperatingconditionshavebeenshowntobesatisfactory. Oventemperature:210C; injectortemperature:230C; detectortemperature:250C; splitratio:approximately50:1; carriergasflow:1ml/minto2ml/min. Conditionsforpackedcolumn: oventemperatur
31、e:200C; injectortemperature:230C; detectortemperature:250C; carriergasflow:20ml/minto30ml/min. 9.2Peaksidentification Theidentificationofpeaksbelongingtointernalstandardandglycerolcanbeachievedbyretentiontime comparisonofsuitablestandardsanalysedunderthesameanalyticalconditions.Anillustrativechromat
32、ogramis reportedbelow.EN14106:2003(E) 6 GLCAnalysis Key 1 1,4Butanediol 2G l y c e r o l Figure1ChromatogramofasyntheticsolutionusedforresponsefactordeterminationDetermination offreeglycerolcontent 9.3 Determinationofresponsefactor Weighapproximately100mg 0,1mgofglyceroland100mg 0,1mgof1,4butanediol
33、ina100mlvolumetric flask.Dissolvein50mlofethylalcohol(5.3)andfilluptothemarkwithwater.Considertheheatingandthevolume diminutionduetothewater/alcoholmixing.Injectatleastthreetimesinthechromatograph1 m lofthissolution usingtheexperimentalconditionslistedabove,inordertocalculatetheresponsefactor.Thesol
34、utionisstablefor someweeksevenifstoredatroomtemperature.Usingtheobtainedchromatogramstheresponsefactor F r canbe calculatedasfollows: 2 2 1 1 / / ) ( factor Response M A M A F r = (1)EN14106:2003(E) 7 where A 1 isthepeakareaofinternalstandard; A 2 isthepeakareaofglycerol; M 1 isthemassof1,4butanedio
35、linresponsefactorsolution,expressedinmg; M 2 isthemassofglycerolinresponsefactorsolution,expressedinmg. Theresponsefactor F r ,suchascalculatedaboveshallbe2,5orlower(seenotein6.1.5). 9.4 Quantitativeanalysis 9.4.1 Samplepreparation Weighapproximately3,5g 0,0001gofsample(correspondingtoabout4ml)intoa
36、10mltesttube.Add1mlof ethylalcohol(5.3)andgentlyshaketoensuremixing.Addexactly1mlofinternalstandardsolution(5.8)and4ml ofhexane.Tightlyplugandshakevigorouslythetubeforfiveminutes.Centrifugethesamplefor15min.Usethe lowerphaseforgaschromatographicanalysis. 9.4.2 Gaschromatographyanalysis Takeapproxima
37、tely1 m lofthelowerphaseusingtheGCmicrosyringeanddrawenoughairtoemptytheneedle (“hotneedle“technique).Carefullycleanbodyandneedleofsyringeusingapapertowel.Introducetheneedle throughthemembraneofinjectionport,waitfor5s,thenrapidlyinjectthesample.After5sdrawofftheneedle. Takenoteofinjectionmomentonchr
38、omatogram.Continueelutionsomeminutesafterthecompletedetectionof glycerolpeak. 10Results Calculate,bymeansofintegrator,theareasofpeaksbelongingtotheunknownsample.Thepercentcontentof freeglycerolinsampleisgivenbythefollowingformula: 100 ) / ( (m/m) % glycerol, Free 1 1 2 = m m F A A r where A 1 , A 2
39、, F r havethesamemeaningasinformula(1); m 1 isthemassofinternalstandardinsample(milligrams); m isthemassofsample(milligrams). Theresultshallbeexpressedasamassfractioninpercent,roundedtotwodigits 11Precision 11.1 Interlaboratorytest DetailsofinterlaboratorytestaregiveninannexA.Thevaluesderivedfromthe
40、setestsmaynotbeapplicableto concentrationrangesandmatricesotherthanthosegiven.EN14106:2003(E) 8 11.2Repeatability Thedifferencebetweentworesultsobtainedbythesameoperator,withthesameapparatusunderconstant operatingconditionsonthesamesample,willbehigherthantherepeatabilitylimit( r)ascalculatedfromthe
41、formulaeinfollowingtable,onlyinonecaseintwenty. 11.3Reproducibility Theabsolutedifferencebetweentwoindependentsingletestresults,obtainedbytwodifferentoperatorsworkingin differentlaboratoriesusingthesamemethodonidenticaltestmaterialwithinashortintervaloftimewillbehigher thanthereproducibilitylimit( R
42、)ascalculatedfromtheformulaeinfollowingtable,onlyinonceoutof20 determinations. Table1Repeatabilityandreproducibilitylimits( rand R) FreeglycerolinFAME,in%( m/m) r=0,4664 X0,0012 R=0,7812 X+0,0032 X correspondingtofreeglycerolconcentrationvalue. 12 Testreport Thetestreportshallspecify: allinformation
43、necessaryforthecompleteidentificationofthesample; thesamplingmethodusedifknown; thetestmethodused,withreferencetothisEuropeanstandard; alloperatingdetailsnotspecifiedinthisEuropeanStandard,orregardedasoptional,togetherwithdetailsof anyincidentswhichmayhaveinfluencedthetestresult(s); thetestresult(s)obtained,oriftherepeatabilityhasbeenchecked,thefinalquotedresultobtained.EN14106:2003(E) 9 Ann
