DIN EN ISO 12966-4-2015 Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 4 Determination by capillary gas chromatography (ISO 12966-4 2015.pdf

1、November 2015 English price group 14No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.200.10!%GF4“2363517www.din.d

2、eDIN EN ISO 12966-4Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 4: Determination by capillary gas chromatography (ISO 12966-4:2015);English version EN ISO 12966-4:2015,English translation of DIN EN ISO 12966-4:2015-11Tierische und pflanzliche Fette und le Ga

3、schromatographie von Fettsuremethylestern Teil 4: Bestimmung mittels Kapillargaschromatographie (ISO 12966-4:2015);Englische Fassung EN ISO 12966-4:2015,Englische bersetzung von DIN EN ISO 12966-4:2015-11Corps gras dorigines animale et vgtale Chromatographie en phase gazeuse des esters mthyliques da

4、cides gras Partie 4: Dtermination par chromatographie capillaire en phase gazeuse (ISO 12966-4:2015);Version anglaise EN ISO 12966-4:2015,Traduction anglaise de DIN EN ISO 12966-4:2015-11Together with DIN EN ISO 12966-1:2015-10,supersedesDIN EN ISO 5508:1995-07,DIN EN ISO 15304:2002-07 andDIN EN ISO

5、 15304Corrigendum 1:2006-09www.beuth.deDocument comprises 27 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.10.15 DIN EN ISO 12966-4:2015-11 2 National foreword This document (EN ISO 12966-4:2015) has been prepared by Technica

6、l Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with Technical Committee CEN/TC 307 “Oil seeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). Based on the results of parallel voting, Techni

7、cal Committee CEN/TC 307 decided to adopt ISO 12966-4:2015 as a European Standard. The responsible German bodies involved in its preparation were the DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products) and the Deutsche Gesellscha

8、ft fr Fettwissenschaft (German Fat Research Society), Joint Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. This document is part of the ISO 12966 standards series on the gas chromatographic determination of fatty ac

9、id methyl esters. ISO 12966 consists of the following parts, under the general title Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters: Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters Part 2: Preparation of methyl esters of fatty acids Part

10、 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) Part 4: Capillary gas chromatographic method The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 661 DIN EN ISO 661 ISO 3696 DIN ISO 3696 ISO 12966-2 DIN EN ISO 1296

11、6-2 ISO 12966-3 DIN EN ISO 12966-3 Amendments This standard differs from DIN EN ISO 5508:1995-07, DIN EN ISO 15304:2002-07 and DIN EN ISO 15304 Corrigendum 1:2006-09 as follows: a) DIN EN ISO 5508:1995-07 has been editorially and technically revised on the basis of International Standards ISO 12966-

12、1:2015 and ISO 12966-4:2015; b) DIN EN ISO 15304:2002-07 and DIN EN ISO 15304 Corrigendum 1:2006-09, have been editorially and technically revised on the basis of International Standard ISO 12966-4:2015; c) the standards and the Corrigendum, now published together as DIN EN ISO 12966-4, specify a me

13、thod for the determination of fatty acid methylesters (FAMEs) derived by transesterification or esterification from fats, oils and fatty acids by capillary gas chromatography (GLC). Previous editions DIN EN ISO 5508: 1995-07 DIN EN ISO 15304: 2002-07 DIN EN ISO 15304 Corrigendum 1: 2006-09 A comma i

14、s used as the decimal marker. DIN EN ISO 12966-4:2015-11 3 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN EN ISO 12966-2, Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 2: Preparat

15、ion of methyl esters of fatty acids DIN EN ISO 12966-3, Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) DIN ISO 3696, Water for analytical laboratory use Specification and test methods DI

16、N EN ISO 12966-4:2015-11 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 12966-4 June 2015 ICS 67.200.10 Supersedes EN ISO 15304:2002, EN ISO 5508:1995English Version Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Par

17、t 4: Determination by capillary gas chromatography (ISO 12966-4:2015) Corps gras dorigines animale et vgtale - Chromatographie en phase gazeuse des esters mthyliques dacides gras - Partie 4: Dtermination par chromatographie capillaire en phase gazeuse(ISO 12966-4:2015) Tierische und pflanzliche Fett

18、e und le - Gaschromatographie von Fettsuremethylestern - Teil 4: Bestimmung mittels Kapillargaschromatographie(ISO 12966-4:2015) This European Standard was approved by CEN on 16 April 2015. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for g

19、iving 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 o

20、fficial 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

21、 of 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, Spai

22、n, 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 2015 CEN All rights of exploitation in any form and by any means reserved worldwide

23、 for CEN national Members. Ref. No. EN ISO 12966-4:2015 EForeword .31 Scope . 42 Normative references 43 Principle 44 Reagents and materials . 44.1 Reference fatty acid methyl esters (FAMEs) 54.2 Internal standards 55 Apparatus . 66 Sampling 77 Preparation of test sample . 78 Preparation of methyl e

24、sters from fats, oils, and fatty acids 79 Procedure. 79.1 General . 79.2 GC conditions 79.3 Performance check 810 Calculations 810.1 Qualitative analysis and peak identification 810.2 Quantitative analysis 810.2.1 Calculation of the composition of fatty acid methyl esters . 810.2.2 Calculation of th

25、e composition of fatty acid methyl esters using correction factors 910.2.3 Calculation of the composition of fatty acid methyl esters using an internal standard . 911 Precision . 1011.1 Results of interlaboratory test . 1011.2 Repeatability . 1011.3 Reproducibility . 1012 Test report . 10Annex A (in

26、formative) Theoretical flame ionization detector correction factor (TCF) for fatty acid methyl esters (FAMEs) . 11Annex B (informative) Examples of chromatograms 12Annex C (informative) Comparison of FAME composition with two different GC columns.14Annex D (informative) Results of an interlaboratory

27、 trial .16Bibliography .22Contents Page2DIN EN ISO 12966-4:2015-11 EN ISO 12966-4:2015 (E) Foreword This document (EN ISO 12966-4:2015) 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 oil

28、s 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 2015, and conflicting national standar

29、ds shall be withdrawn at the latest by December 2015. 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 document supersedes EN ISO

30、5508:1995, EN ISO 15304:2002. 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 Yugoslav Repu

31、blic 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 12966-4:2015 has been

32、approved by CEN as EN ISO 12966-4:2015 without any modification. 3DIN EN ISO 12966-4:2015-11 EN ISO 12966-4:2015 (E) 1 ScopeThis part of ISO 12966 specifies a method for the determination of fatty acid methyl esters (FAMEs) derived by transesterification or esterification from fats, oils, and fatty

33、acids by capillary gas chromatography (GLC). Fatty acid methyl esters from C8 to C24 can be separated using this part of ISO 12966 including saturated fatty acid methyl esters, cis- and trans-monounsaturated fatty acid methyl esters, and cis- and trans-polyunsaturated fatty acid methyl esters.The me

34、thod is applicable to crude, refined, partially hydrogenated, or fully hydrogenated fats, oils, and fatty acids derived from animal and vegetable sources.This method is not suitable for the analysis of dairy, ruminant fats and oils, or products supplemented with conjugated linoleic acid (CLA). Milk

35、and milk products (or fat coming from milk and milk products) are excluded from the scope of this part of ISO 12966.This part of ISO 12966 is not applicable to di-, tri-, polymerized and oxidized fatty acids, and fats and oils.2 Normative referencesThe following documents, in whole or in part, are n

36、ormatively referenced in this document and are 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, Animal and vegetable fats and oils Preparation of

37、test sampleISO 3696, Water for analytical laboratory use Specification and test methodsISO 6353, Reagents for chemical analysisISO 12966-2, Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 2: Preparation of methyl esters of fatty acidsISO 12966-3, Animal and veg

38、etable fats and oils Gas chromatography of fatty acid methyl esters Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)3 PrincipleUsing capillary gas chromatography, FAMEs are separated on a highly polar stationary phase with respect to their chain length, degree of (un)sa

39、turation, and geometry and position of the double bonds.4 Reagents and materialsUnless otherwise stated, use only reagents as specified in ISO 6353-2 and ISO 6353-3 (if listed there). If not, then use reagents of recognized analytical grade and water of at least grade 3, as defined in ISO 3696.4DIN

40、EN ISO 12966-4:2015-11 EN ISO 12966-4:2015 (E) WARNING Attention is drawn to the regulations which specify the handling of dangerous matter. Technical, organizational, and personal safety measures shall be followed.4.1 Reference fatty acid methyl esters (FAMEs)4.1.1 Reference mixtures of pure FAMEs

41、and/or oils with known fatty acid composition should be used for the identification of fatty acids analysed under the test conditions of this method.4.1.2 Fats and oils with certified fatty acid composition, e.g. certified reference material BCR 162.4.1.3 Reference fatty acid methyl esters (FAMEs) -

42、 Methyl esters of pure fatty acids, in particular, cis- and trans-isomers of octadecenoic (oleic), trans-isomers of octadecadienoic (linoleic), and octadecatrienoic (-linolenic) acids. Wide ranges of cis- and trans-octadecenoic methyl ester isomers are available on the market. Trans-geometrical isom

43、ers of linoleic and -linolenic acids can be prepared in the laboratory with the aid of p-toluenesulfonic acid. In addition to pure compounds, convenient mixtures of FAMEs are also commercially available.4.2 Internal standardsFor the quantification of the fatty acids, in grams per 100 g, the use of a

44、 FAME as an internal standard (IS) is necessary. An external calibration with mixtures of different fatty acids is also possible.NOTE If it is necessary to check the recovery and the effectiveness of the derivatization method, then either or both a TAG and a FAME internal standard should be used. Wh

45、ile the TAG-IS is added to the sample prior to the FAME preparation, the FAME-IS is added before or after the FAME preparation. The FAME-IS is used to calculate the recovery of the FAME from the TAG-IS and therefore, the efficiency of the derivatisation procedure. In this case, a different chain len

46、gth of the standards is required.Depending on the type of fat, different internal standards can be used (C11:0 FAME, C17:0 FAME, C19:0 FAME, C21:0 FAME, C23:0 FAME, etc.). An external calibration with mixtures of different fatty acids is also possible. It is recommended to carry out further analysis

47、 of the sample without the addition of the internal standard to check the natural content of the fatty acid which is used as the internal standard. The content shall be considered in the calculation.IMPORTANT If the TAG-IS (4.2.2) is hard to dissolve in the cold, a hot methylation procedure, as spec

48、ified in ISO 129662:2011, 4.3, 4.4, and 4.5, shall be used.The internal standard solutions are stable if precautions are taken to eliminate the loss of solvent and therefore, a change in the concentration of the IS. For example, store the solution in a refrigerator in a well-sealed amber bottle when

49、 not in use. Pure standards are available on the market. Purity of the IS shall be confirmed by thin-layer chromatography, high-performance liquid chromatography, gas chromatography analysis, or by any other appropriate technique.The following are examples of suitable standards (as FAME and TAG):4.2.1 Fatty acid methyl ester (FAME) as internal standard (IS) solution:C21:0 FAME heneicosanoic acid methyl ester (purity 99 %), mass