DIN EN ISO 12966-1-2015 Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 1 Guidelines on modern gas chromatography of fatty acid methyl es.pdf

1、October 2015 ICS 67.200.10!%FG partially supersedesDIN EN ISO 15304:2002-07 andDIN EN ISO 15304 Corrigendum 1:2006-09Tierische und pflanzliche Fette und le Gaschromatographie von Fettsuremethylestern Teil 1: Leitfaden fr die moderne Gaschromatographie von Fettsuremethylestern (ISO 12966-1:2014); Eng

2、lische Fassung EN ISO 12966-1:2014 + AC:2015,Englische bersetzung von DIN EN ISO 12966-1:2015-10Corps gras dorigines animale et vgtale Chromatographie en phase gazeuse des esters mthyliques dacides gras Partie 1: Lignes directrices relatives la chromatographie en phase gazeuse moderne des mthyliques

3、 dacides gras (ISO 12966-1:2014); Version anglaise EN ISO 12966-1:2014 + AC:2015, Traduction anglaise de DIN EN ISO 12966-1:2015-10Document comprises pages 15Translation by DIN-Sprachendienst. In case of doubt, the German-language original shall be considered authoritative. y be reproduced without p

4、rior permission of DIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany, No part of this translation maEnglish price group 10www.din.dewww.beuth.deesters (ISO 12966-1:2014);esters has the exclusive right of sale for German Standards (DIN-Normen). 09.15DIN EN ISO

5、12966-1:2015-10 2 National foreword This document (EN ISO 12966-1:2014 + AC:2015) has been prepared by Technical 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

6、Methods of sampling and analysis” (Secretariat: AFNOR, France). 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 Gesellschaft fr Fettwissensch

7、aft (German Fat Research Society), Joint Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. ISO 12966-1 is part of the ISO 12966 standards series on the gas chromatographic determination of fatty acid methyl esters. ISO

8、 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 3: Preparation of me

9、thyl 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 12966-2 DIN EN ISO 12966-2 ISO 12966-3 DIN EN ISO 12966-3 ISO 12966-4 DIN EN ISO 12966-4

10、Amendments This standard differs from DIN EN ISO 5508:1995-07 as follows: a) the standard has been developed on the basis of ISO 12966-1:2014 and ISO/FDIS 12966-4:2014; the text has been revised in form and substance; b) the standard, which has been renamed DIN EN ISO 12966-1, provides guidelines on

11、 modern gas chromatography of fatty acid methyl esters; c) to meet the various requirements, Part 2 and Part 3 of the DIN EN ISO 12966 series cover the preparation of fatty acid methyl esters by different methods. Compared with DIN EN ISO 12966-1:2015-03, DIN EN ISO 15304:2002-07 and DIN EN ISO 1530

12、4 Corrigen-dum 1:2006-09 the following corrections have been made: a) Corrigendum EN ISO 12966-1:2014/AC:2015 has been incorporated; b) the superseding note on the national and European title pages and in the European foreword has been corrected; A comma is used as the decimal marker.DIN EN ISO 1296

13、6-1:2015-10 3 c) DIN EN ISO 15304:2002-07 and DIN EN ISO 15304 Corrigendum 1:2006-09 have been replaced by DIN EN ISO 12966-1, which has been technically and editorially revised. The standard gives an overview of the gas chromatographic determination of fatty acids, free and bound, in animal and veg

14、etable fats and oils following their conversion to fatty acid methyl esters (FAMEs). Previous editions DIN EN ISO 5508: 1995-07 DIN EN ISO 15304: 2002-07 DIN EN ISO 15304 Corrigendum 1: 2006-09 DIN EN ISO 12966-1: 2015-03 DIN EN ISO 12966-1:2015-10 4 National Annex NA (informative) Bibliography DIN

15、EN ISO 12966-2, Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 2: Preparation 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 trime

16、thylsulfonium hydroxide (TMSH) DIN EN ISO 12966-4, Animal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 4: Capillary gas chromatographic method EN ISO 12966-1 December 2014 + AC May 2015 ICS 67.200.10 Supersedes EN ISO 5508:1995and EN ISO 15304:2002English Version A

17、nimal and vegetable fats and oils Gas chromatography of fatty acid methyl esters Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters (ISO 12966-1:2014) Corps gras dorigines animale et vgtale Chromatographie en phase gazeuse des esters mthyliques dacides gras Partie 1: Lignes

18、directrices relatives la chromatographie en phase gazeuse moderne des esters mthyliques dacides gras (ISO 12966-1:2014) Tierische und pflanzliche Fette und le Gaschromatographie von Fettsuremethylestern Teil 1: Leitfaden fr die moderne Gaschromatographie von Fettsuremethylestern (ISO 12966-1:2014) E

19、N ISO 12966-1:2014 was approved by CEN on 2014-10-20 and Corrigendum AC:2015 on 2015-05-06. 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

20、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 any other language made by translation un

21、der 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 Republic, Denmark, Estonia, Finland, Former

22、 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. Management Centre: Avenue Marnix 17, B-1000 Bru

23、ssels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 12966-1:2014 + AC:2015 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NOR

24、MContents EN ISO 12966-1:2014 + AC:2015 (E) Page Foreword 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Principle . 5 4 Preparation of FAME 6 5 Columns 7 6 GLC of FAMEs . 8 7 Evaluation of the chromatograms . 8 7.1 Peak area and area per cent 8 7.2 Evaluation by means of an internal standard

25、 or correction factors . 9 8 Test report . 9 Bibliography . 10 DIN EN ISO 12966-1:2015-10 2Foreword to EN ISO 12966-1:2014 + AC:2015 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 2015, and

26、conflicting national standards shall be withdrawn at the latest by June 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 doc

27、ument supersedes EN ISO 5508:1995 and 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, Fi

28、nland, 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 the United Kingdom. Endorsement notice The text of

29、 ISO 12966-1:2014 has been approved by CEN as EN ISO 12966-1:2014 + AC:2015 without any modification. EN ISO 12966-1:2014 + AC:2015 (E) DIN EN ISO 12966-1:2015-10 3AFNOR. This document (EN ISO 12966-1:2014 + AC:2015) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration

30、 Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” the secretariat of which is held by IntroductionThis part of ISO 12966 is one of a suite of four International Standards for the preparation and determination of fatt

31、y acid methyl esters by gas chromatography in animal and vegetable fats and oils. ISO 12966 (all parts) is applicable to crude, refined, partially hydrogenated or fully hydrogenated fats, oils and fatty acids derived from animal and vegetable sources.ISO 12966 (all parts) is not suitable for the ana

32、lysis of dairy, ruminant fats and oils (including milk and milk products or fat coming from milk and milk products), or products supplemented with conjugated linoleic acid (CLA). Furthermore it is not intended to be applied to polymerized and oxidized fats and oils.This part of ISO 12966 is a guidel

33、ine to the modern gas chromatography of fatty acid methyl esters, while ISO 12966-2 and ISO 12966-3 cover the preparation of fatty acid methyl esters by different methods. In ISO 12966-4, the conditions for the analysis of fatty acid methyl esters by capillary gas chromatography are given.This suite

34、 of International Standards replaces the following International Standards: ISO 5508:1990 is replaced by ISO 12966-1 and ISO 12966-4 ISO 15304:2002 is replaced by ISO 12966-1 and ISO 12966-4 ISO 5509:2000 is replaced by ISO 12966-2 and ISO 12966-3EN ISO 12966-1:2014 + AC:2015 (E) DIN EN ISO 12966-1:

35、2015-10 41 ScopeThis part of ISO 12966 gives an overview of the gas chromatographic determination of fatty acids, free and bound, in animal and vegetable fats and oils following their conversion to fatty acid methyl esters (FAMEs).The qualitative and quantitative determination of the composition of

36、fatty acids by gas liquid chromatography (GLC) is a widely used application in lipid analysis. It is used for the characterization of fats and oils, or fatty foodstuffs after the extraction of the oil from the matrix. The bound fatty acids of the triacylglycerols (TAGs) and, depending on the esterif

37、ication method, the free fatty acids (FFA) and other lipids, are converted to fatty acid methyl esters (FAMEs), which are determined by capillary gas chromatography. Depending on the number of different fatty acids (theoretically more than 50 different fatty acids can be present) capillary columns w

38、ith a length of 10 m to 100 m are used for a separation.The GLC of FAMEs is applicable to all natural and synthetic mixtures of tri-, di- and monoacylglycerols, to fatty acid esters, free fatty acids, soaps and other fatty compounds. With this suite of standards, FAMEs from C4 to C26 can be determin

39、ed, including saturated fatty acid methyl esters, cis- and trans-monounsaturated fatty acid methyl esters, and cis- and trans-polyunsaturated fatty acid methyl esters.For the determination of short chain fatty acids, isopropyl and butyl esters are often used so as to avoid interferences with the sol

40、vent peak and in order to reduce differences in detector responses.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references,

41、the latest edition of the referenced document (including any amendments) applies.ISO 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 chromatography of fat

42、ty acid methyl esters Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH)ISO 12966-4:1), Animal and vegetable fats and oils Determination of methyl esters of fatty acids Part 4: Capillary gas chromatographic method3 PrincipleGas liquid chromatography (GLC) is used for the

43、qualitative and quantitative analysis of FAMEs. The FAMEs are prepared in accordance with ISO 12966-2 or ISO 12966-3 and the dissolved FAMEs are then injected into and vaporized within the injector. The separation of FAMEs is achieved on analytical 1) Under preparation. To be published.EN ISO 12966-

44、1:2014 + AC:2015 (E) DIN EN ISO 12966-1:2015-10 5columns of different polarity and lengths. For the detection of the FAMEs, a flame ionization detector (FID) is used.In the gas chromatography of FAMEs with FID, hydrogen should be used as the carrier gas (mobile phase). For MSD (mass selective detect

45、or) applications helium shall be used. The separation can be done in a shorter time with sharper peaks by using hydrogen. The stationary phase is a microscopic layer of a thin liquid film on an inert solid surface, made of steel, glass or fused silica.The volatilized compounds being analysed interac

46、t, during their passage through the capillary tubing, with the stationary phase coating the inner surface of the column. Due to this different interaction of different compounds, they exit the column, or elute, at a different time, which is called the retention time of the compound at a given set of

47、 analysis parameters. The comparison of retention times is used for the identification of the different compounds.CAUTION When a mass selective detector is used, special care shall be taken in order to achieve quantitative results due to differences in fragmentation patterns of individual FAMEs. Als

48、o, for mass spectrometry, derivatives other than the FAMEs discussed in this part of ISO 12966 are used, as for example, picolinyl esters or dimethyl oxazolidines and others.4 Preparation of FAMEThe preparation of the fatty acid methyl esters shall be carried out in accordance with ISO 12966-2 and I

49、SO 12966-3. An overview is given in Table 1.Table 1 Overview on the different esterification methodsMethod Principle GC-Injector Note ISO 12966-2, Subclause 4.2Rapid transmethylation method under alkali-cata-lysed conditions with KOHFFA are not converted to FAMEDanger of soap formationOn-columnSplit/SplitlessApplicable to fats and oils containing fatty acids down to butanoic (C4:0)Transesterifi-cation at room temperature, no solvent evapora-tion necessaryAlso for medium chain triglyce