EN ISO 12966-3-2016 en Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 3 Preparation of methyl esters using trimethylsulfonium hydroxide .pdf

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1、BS EN ISO 12966-3:2016Animal and vegetable fats andoils Gas chromatography offatty acid methyl estersPart 3: Preparation of methyl esters usingtrimethylsulfonium hydroxide (TMSH) (ISO12966-3:2016)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 12966-3:2016

2、BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO12966-3:2016. It supersedes BS EN ISO 12966-3:2009 which iswithdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oilseeds, animal and vegetable fats and oils andtheir by-p

3、roducts.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016. Published by BSI

4、StandardsLimited 2016ISBN 978 0 580 92663 1ICS 67.200.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 June 2016.Amendments issued since publicationDate Text a

5、ffectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 12966-3 June 2016 ICS 67.200.10 Supersedes EN ISO 12966-3:2009English Version Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide

6、(TMSH) (ISO 12966-3:2016) Corps gras dorigines animale et vgtale - Chromatographie en phase gazeuse des esters mthyliques dacides gras - Partie 3: Prparation des esters mthyliques laide dhydroxyde de trimthylsulfonium (TMSH) (ISO 12966-3:2016) Tierische und pflanzliche Fette und le - Gaschromatograp

7、hie von Fettsuremethylestern - Teil 3: Herstellung von Methylestern mittels Trimethylsulfoniumhydroxid (TMSH) (ISO 12966-3:2016) This European Standard was approved by CEN on 12 May 2016. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giv

8、ing this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three off

9、icial versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies o

10、f Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,

11、 Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide f

12、or CEN national Members. Ref. No. EN ISO 12966-3:2016 EBS EN ISO 12966-3:2016EN ISO 12966-3:2016 (E) 3 European foreword This document (EN ISO 12966-3:2016) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable an

13、d animal fats and oils and their by-products - Methods of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by December 2016, and confl

14、icting national standards shall be withdrawn at the latest by December 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This docu

15、ment supersedes EN ISO 12966-3:2009. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugosl

16、av Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 12966-3:2016 ha

17、s been approved by CEN as EN ISO 12966-3:2016 without any modification. BS EN ISO 12966-3:2016ISO 12966-3:2016(E)Foreword iv1 Scope . 12 Normative references 13 Principle 14 Reagents 15 Apparatus . 26 Sampling 27 Preparation of the test sample . 28 Procedure. 29 Test report . 3Bibliography 4 ISO 201

18、6 All rights reserved iiiContents PageBS EN ISO 12966-3:2016ISO 12966-3:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through IS

19、O technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates c

20、losely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteri

21、a needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of pa

22、tent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents). Any trade name us

23、ed in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Ba

24、rriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils.This second edition cancels and replaces the first edition (ISO 12966-3:2009), of which it

25、constitutes a minor revision. The scope has been revised to state that the document is not applicable to milk and milk fat products.ISO 12966 consists of the following part, under the general title Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters: Part 1: Guidelines

26、on modern gas chromatography of fatty acid methyl esters Part 2: Preparation of methyl esters of fatty acids Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) Part 4: Determination by capillary gas chromatographyiv ISO 2016 All rights reservedBS EN ISO 12966-3:2016INTERN

27、ATIONAL STANDARD ISO 12966-3:2016(E)Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)1 ScopeThis part of ISO 12966 specifies a rapid base-catalysed transesterification method for fats and o

28、ils with trimethylsulfonium hydroxide (TMSH) to prepare fatty acid methyl esters. The method is exclusively applicable to the preparation of methyl esters of fats and oils for gas liquid chromatographic (GLC) analysis. It is applicable to all fats and oils, but excluding those coming from milk and m

29、ilk products. Isomerization of unsaturated fatty acids only occurs to a minor extent and isomerized fatty acids are only present at the determination limit. As isomerization takes place, the procedure is not recommended for conjugated linoleic acid (CLA).Only about 70 % to 80 % of the free fatty aci

30、ds are esterified. In the case of conjugated cyclopropyl and cyclopropenyl fatty acids, side reactions may occur, but these do not interfere with the determination of the fatty acids.NOTE This part of ISO 12966 is based upon German Standard Method C-VI 11e (98) (see Reference 8).2 Normative referenc

31、esThe following documents, in whole or in part, are normatively referenced in this document and indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 661, A

32、nimal and vegetable fats and oils Preparation of test sample3 PrincipleThe sample is dissolved in tert-butyl methyl ether (TBME) and mixed with a methanolic solution of trimethylsulfonium hydroxide. Glycerides are base-catalysed transesterified and fatty acid methyl esters are formed (see References

33、 4 to 8). Free fatty acids are converted to salts which are pyrolysed to methyl esters and dimethylsulfide in the injector. Excess reagent is also pyrolysed into methanol and dimethylsulfide. To obtain a complete pyrolytic reaction, a hot injector (split injection) of at least 250 C is necessary.For

34、 the determination of short-chain fatty acids (C4to C8), valeric acid methyl ester is used as an internal standard. Lipids containing hydroxy groups are partially converted to the corresponding O-methyl ether derivatives which may interfere with fatty acid methyl esters in the GLC separation (Refere

35、nce 9). In the early part of the chromatogram (region of C4), peaks may occur, which are from the reagent. These peaks are not taken into account.4 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational, and personal safety

36、 measures shall be followed. ISO 2016 All rights reserved 1BS EN ISO 12966-3:2016ISO 12966-3:2016(E)During the analysis, unless otherwise stated, use only reagents and solvents of recognized analytical grade.4.1 tert-Butyl methyl ether (TBME).4.2 Trimethylsulfonium hydroxide (TMSH), methanolic solut

37、ion, amount of substance concentration c(Me3SOH) = 0,2 mol/l.The content of the solution can be determined by acidimetry: dilute 5,0 ml of the solution with 10 ml of methanol, add two drops of phenolphthalein and titrate against 0,1 mol/l HCl. The concentration should be at least 0,15 mol/l.NOTE The

38、 solution remains stable for at least two months when stored at 4 C in small quantities in closed tubes.5 ApparatusUsual laboratory equipment, and in particular:5.1 Test tubes, of capacity 2 ml (autosampler vials).5.2 Graduated pipettes, of capacities 250 l, 500 l, and 1 000 l, ISO 8351class A.6 Sam

39、plingA representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage.Sampling is not part of the method specified in this part of ISO 12966. A recommended sampling method is given in ISO 55553.7 Preparation of the test sampleThe

40、 test sample shall be liquid, dry and clear. Proceed in accordance with ISO 661, but heat the sample to just above the melting point.Solid samples are carefully melted at a temperature of maximum 10 C above their melting point and mixed. Avoid overheating.Samples containing water are dissolved in pe

41、troleum ether and dried for 30 min by addition of anhydrous sodium sulfate. The drying agent is removed by filtration through a fluted filter paper and the residue is carefully washed with petroleum ether. The solvent is subsequently removed with the aid of a rotary evaporator.8 Procedure8.1 Weigh,

42、into a test tube (5.1), (10 2) mg of the test sample.8.2 Pipette (5.2) 500 l of TBME (4.1) into the test tube and dissolve the sample, warming gently if necessary.NOTE For the determination of short-chain fatty acids (C4to C8), valeric acid methyl ester is used as an internal standard. The internal

43、standard solution is used to dissolve the sample.2 ISO 2016 All rights reservedBS EN ISO 12966-3:2016ISO 12966-3:2016(E)8.3 Pipette (5.2) 250 l of TMSH solution (4.2) into the test tube and shake vigorously for about 30 s. After this, the solution is ready for injection into the gas chromatograph. A

44、s the methyl esters are formed during injection, an injector temperature of at least 250 C is required.If necessary, use a mixture of TBME and methanol (9 volumes + 1 volume) to dilute the solution.IMPORTANT Free fatty acids react with TMSH to form the corresponding salts, which are pyrolysed to met

45、hyl esters and dimethylsulfide in the injector. Therefore, an injector temperature of 250 C is necessary. To prevent blocking, the capillary of the split vent shall have an appropriate internal diameter (1 mm). It should be cleaned by regular heating or flushing with solvent. Moreover, the split ven

46、t valve shall be protected with an activated charcoal trap.9 Test reportThe test report shall include the following information:a) result of the determination;b) the method used, together with a reference to this part of ISO 12966, i.e. ISO 12966-3;c) all information necessary for the complete ident

47、ification of the sample;d) all operating details not specified in this part of ISO 12966, or regarded as optional, together with details of any incident that may have influenced the result(s). ISO 2016 All rights reserved 3BS EN ISO 12966-3:2016ISO 12966-3:2016(E)Bibliography1 ISO 835, Laboratory gl

48、assware Graduated pipettes2 ISO 1042, Laboratory glassware One-mark volumetric flasks3 ISO 5555, Animal and vegetable fats and oils Sampling4 Butte W.J. Rapid method for the determination of fatty acid profiles from fats and oils using trimethylsulfonium hydroxide for transesterification. J. Chromat

49、ogr. A. 1983, 261 pp. 1421455 Schulte E., & Weber K. Rapid preparation of fatty acid methyl esters from fats with trimethylsulfonium hydroxide or sodium methylate. Fat Sci. Technol. 1989, 91 pp. 1811836 Arens M., Schulte E., Weber K. Fettsuremethylester, Umesterung mit trimethylsulfoniumhydroxid Fatty acid methyl esters, transesterification with trimethylsulfonium hydroxide. Fat Sci. Technol. 1994, 96 pp. 67687 El-Hamdy A.H., & Christie W.W. Preparation of methyl esters of fatty acid