1、BSI Standards PublicationBS EN ISO 12966-4:2015Animal and vegetable fats andoils Gas chromatography offatty acid methyl estersPart 4: Determination by capillary gaschromatographyBS EN ISO 12966-4:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 12966-4:2
2、015. Together with BS EN ISO 12966-1:2014, it supersedes BS EN ISO 5508:1995 and BS EN ISO 15304:2002 which arewithdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and their by-products.A list of organizations rep
3、resented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0
4、580 83509 4 ICS 67.200.10 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e d
5、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 - Part 4: Determination by capillary gas chromatography (ISO 1296
6、6-4:2015) 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 Fette und le - Gaschromatographie von Fettsuremethylestern - Te
7、il 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 giving this European Standard the status of a national stan
8、dard 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 official versions (English, French, German). A version in a
9、ny 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 of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Rep
10、ublic, 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, Sweden, Switzerland, Turkey and United Kingdom. EUROPEA
11、N 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 for CEN national Members. Ref. No. EN ISO 12966-4:2015 EB
12、S EN ISO 12966-4:2015EN ISO 12966-4:2015 (E) 3 Foreword This document (EN ISO 12966-4:2015) has been prepared by Technical Committee ISO/TC 34 “Foodproducts“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats andoils and their by-products - Methods of sampling
13、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 identicaltext or by endorsement, at the latest by December 2015, and conflicting national standards shall be withdrawnat the latest by Decembe
14、r 2015. Attention is drawn to the possibility that some of the elements of this document may be the subject of patentrights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 5508:1995, EN ISO 15304:2002. According to the
15、CEN-CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, CzechRepublic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,Hungar
16、y, 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 approved by CEN as EN ISO 12966-4:2015 without any
17、modification. BS EN ISO 12966-4:2015ISO 12966-4:2015(E)Foreword iv1 Scope . 12 Normative references 13 Principle 14 Reagents and materials . 14.1 Reference fatty acid methyl esters (FAMEs) 24.2 Internal standards 25 Apparatus . 36 Sampling 47 Preparation of test sample . 48 Preparation of methyl est
18、ers from fats, oils, and fatty acids 49 Procedure. 49.1 General . 49.2 GC conditions 49.3 Performance check 510 Calculations 510.1 Qualitative analysis and peak identification 510.2 Quantitative analysis 510.2.1 Calculation of the composition of fatty acid methyl esters . 510.2.2 Calculation of the
19、composition of fatty acid methyl esters using correction factors 610.2.3 Calculation of the composition of fatty acid methyl esters using an internal standard . 611 Precision . 711.1 Results of interlaboratory test . 711.2 Repeatability . 711.3 Reproducibility . 712 Test report . 7Annex A (informati
20、ve) Theoretical flame ionization detector correction factor (TCF) for fatty acid methyl esters (FAMEs) . 9Annex B (informative) Examples of chromatograms 10Annex C (informative) Comparison of FAME composition with two different GC columns.12Annex D (informative) Results of an interlaboratory trial .
21、14Bibliography .20 ISO 2015 All rights reserved iiiContents PageBS EN ISO 12966-4:2015ISO 12966-4:2015(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 norma
22、lly 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, governmental and non-governmental, in liaison with ISO, also take part in th
23、e work. ISO collaborates closely 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
24、different approval criteria 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 documen
25、t may be the subject of patent 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/p
26、atents).Any trade name used 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 princ
27、iples in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary Information The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils.This first edition cancels and replaces ISO 5508:1990 and ISO 153
28、04:2002, which have been technically revised.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
29、 esters of fatty acids Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) Part 4: Determination by capillary gas chromatographyiv ISO 2015 All rights reservedBS EN ISO 12966-4:2015INTERNATIONAL STANDARD ISO 12966-4:2015(E)Animal and vegetable fats and oils Gas chromatogra
30、phy of fatty acid methyl esters Part 4: Determination by capillary gas chromatography1 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 acids by capillary gas chromatog
31、raphy (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 method is applicable to crude, ref
32、ined, 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 and milk products (or fat coming
33、 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 normatively referenced in this do
34、cument 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 test sampleISO 3696, Water for a
35、nalytical 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 vegetable fats and oils Gas chromat
36、ography 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)saturation, and geometry and posit
37、ion 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. ISO 2015 All rights reserved 1BS EN
38、ISO 12966-4:2015ISO 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 and/or oils
39、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) - Methyl este
40、rs 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 isomers of linol
41、eic 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 FAME as an
42、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. While the TAG-
43、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 length of the s
44、tandards 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 of the samp
45、le 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 specified in ISO
46、 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 not in use.
47、 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 aci
48、d methyl ester (FAME) as internal standard (IS) solution:C21:0 FAME heneicosanoic acid methyl ester (purity 99 %), mass concentration 5,0 mg/ml in iso-octane or MTBE should be used as the internal standard.4.2.2 Triacylglycerol (TAG) internal standard (IS) solution:C21:0 TAG - triheneicosanoin (puri
49、ty 99 %), mass concentration 5,0 mg/ml in chloroform. The TAG internal standard solution is 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 not in use. Pure triheneicosanoin is 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.2 I
