EN ISO 12966-3-2009 0625 Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 3 Preparation of methyl esters using trimethylsulfonium hydroxid.pdf

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1、BS EN ISO12966-3:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal and vegetablefats and oils Gaschromatography offatty acid methyl estersPart 3: Preparation of methyl estersusing trimethylsulfonium hydroxide(TMSH) (ISO 12966-3:2009)Copyrig

2、ht European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 January2010 BSI 2010ISBN 978 0 5

3、80 65361 2Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 12966-3:2009National forewordThis British Standard is the UK implementation of EN ISO12966-3:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats an

4、d oils andtheir by products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard ca

5、nnot confer immunityfrom legal obligations.Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 12966-3 December 2009 ICS 67.2

6、00.10 English Version Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) (ISO 12966-3:2009)Corps gras dorigines animale et vgtale - Chromatographie en phase gazeuse des esters mthyliques

7、 dacides gras - Partie 3: Prparation des esters mthyliques laide dhydroxyde de trimthylsulfonium (TMSH) (ISO 12966-3:2009) Tierische und pflanzliche Fette und le - Gaschromatographie von Fettsuremethylestern - Teil 3: Herstellung von Methylestern mittels Trimethylsulfoniumhydroxid (TMSH) (ISO 12966-

8、3:2009) This European Standard was approved by CEN on 18 November 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliograp

9、hical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a

10、CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,

11、 Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B

12、-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 12966-3:2009: ECopyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted wit

13、hout license from IHS-,-,-BS EN ISO 12966-3:2009EN ISO 12966-3:2009 (E) 3 Foreword This document (EN ISO 12966-3:2009) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-pr

14、oducts - 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 June 2010, and conflicting national standards shall be withdra

15、wn at the latest by June 2010. 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. According to the CEN/CENELEC Internal Regulations, the

16、national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norw

17、ay, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 12966-3:2009 has been approved by CEN as a EN ISO 12966-3:2009 without any modification. Copyright European Committee for Standardization Provided by IHS under lic

18、ense with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 12966-3:2009ISO 12966-3:2009(E) ISO 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO memb

19、er bodies). The work of preparing International Standards is normally carried out through ISO 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, government

20、al and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Direct

21、ives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodie

22、s casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 12966-3 was prepared by Technical Committee ISO/TC 34, Food products, Subcommit

23、tee SC 11, Animal and vegatable fats and oils. 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 2: Preparation of methyl esters of fatty acids Part 3: Preparation of methyl esters using trimethyl

24、sulfonium hydroxide (TMSH) The following part is in preparation: Part 4: Determination of cis-, trans-, saturated, mono- and polyunsaturated fatty acids in vegetable or non-ruminant oils and fats The following part is planned: Part 1: Guidelines on gas chromatography Copyright European Committee for

25、 Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 12966-3:2009Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted

26、 without license from IHS-,-,-BS EN ISO 12966-3:2009INTERNATIONAL STANDARD ISO 12966-3:2009(E) ISO 2009 All rights reserved 1Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) 1 Scope This p

27、art of ISO 12966 specifies a rapid base-catalysed transesterification method for fats and oils 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)

28、analysis. It is applicable to all fats and oils including milk fat and blends containing milk fat. 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 recomme

29、nded for conjugated linoleic acid (CLA). As CLA is not correctly analysed, this method is not applicable to the determination of the complete fatty acid composition of milk fat samples. Only about 70 % to 80 % of the free fatty acids are esterified. In the case of conjugated cyclopropyl and cyclopro

30、penyl 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 references The following referenced documents are indispensable for the app

31、lication of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 661, Animal and vegetable fats and oils Preparation of test sample 3 Principle The sample is dissolved in ter

32、t-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 4 to 8). Free fatty acids are converted to salts which are pyrolysed to methyl esters and dimethylsu

33、lfide 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 the determination of short-chain fatty acids (C4to C8), valeric acid methyl ester is used as an int

34、ernal 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 (Reference 9). In the early part of the chromatogram (region of C4), peaks may occur, which are from the re

35、agent. These peaks are not taken into account. Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 12966-3:2009ISO 12966-3:2009(E) 2 ISO 2009 All rights reserved4 Reagents

36、WARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational, and personal safety measures shall be followed. During the analysis, unless otherwise stated, use only reagents and solvents of recognized analytical grade. 4.1 tert-Butyl met

37、hyl ether (TBME). 4.2 Trimethylsulfonium hydroxide (TMSH), methanolic solution, 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 aga

38、inst 0,1 mol/l HCl. The concentration should be at least 0,15 mol/l. NOTE The solution remains stable for at least two months when stored at 4 C in small quantities in closed tubes. 5 Apparatus Usual laboratory equipment, and in particular: 5.1 Test tubes, of capacity 2 ml with ground glass stopper

39、(autosampler vials). 5.2 Graduated pipettes, of capacities 250 l, 500 l, and 1 000 l, ISO 8351class A. 6 Sampling A 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 t

40、his part of ISO 12966. A recommended sampling method is given in ISO 55553. 7 Preparation of the test sample The 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

41、 maximum 10 C above their melting point and mixed. Avoid overheating. Samples containing water are dissolved in petroleum 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 carefully washed with

42、petroleum ether. The solvent is subsequently removed with the aid of a rotary evaporator. 8 Procedure 8.1 Weigh, 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 determina

43、tion of short-chain fatty acids (C4to C8), valeric acid methyl ester is used as an internal standard. The internal standard solution is used to dissolve the sample. Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking perm

44、itted without license from IHS-,-,-BS EN ISO 12966-3:2009ISO 12966-3:2009(E) ISO 2009 All rights reserved 38.3 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. As the methyl

45、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 methyl esters

46、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 vent valve sh

47、all be protected with an activated charcoal trap. 9 Test report The test report shall contain at least the following information: a) result of the determination; b) the method used, together with reference to this part of ISO 12966; c) all information necessary for the complete identification of the

48、 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). Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 12966-3:2009ISO 12966-3:2009(E) 4 ISO 2009 All rights reservedBibliography 1 ISO 835, Laboratory glassware Graduated pipettes 2 ISO 1042, Laboratory glassware One-mark volumetric flas