1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 12966-2:2011Animal and vegetable fats and oils Gas chromatography of fatty acid methyl estersPart 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2011)BS EN
2、 ISO 12966-2:2011 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 12966-2:2011. It supersedes BS EN ISO 5509:2001,which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oil seeds, animal and vegetable fats and
3、 oils and their by products.A list of organizations represented 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. BSI 2011 ISBN 978 0 580 64653 9 IC
4、S 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 31 March 2011.Amendments issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME
5、EUROPENNE EUROPISCHE NORM EN ISO 12966-2 February 2011 ICS 67.200.10 Supersedes EN ISO 5509:2000English Version Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 2: Preparation of methyl esters of fatty acids (ISO 12966-2:2011) Corps gras dorigines animale et
6、 vgtale - Chromatographie en phase gazeuse des esters mthyliques dacides gras - Partie 2: Prparation des esters mthyliques dacides gras (ISO 12966-2:2011) Tierische und pflanzliche Fette und le - Gaschromatographie von Fettsuremethylestern - Teil 2: Herstellung von Fettsuremethylestern (ISO 12966-2:
7、2011)This European Standard was approved by CEN on 14 February 2011. 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 bibliographic
8、al 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 under the responsibility
9、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, France, Germany, Greece, Hung
10、ary, Iceland, Ireland, 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 Centr
11、e: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 12966-2:2011: EBS EN ISO 12966-2:2011 EN ISO 12966-2:2011 (E) 3 Foreword This document (EN ISO 12966-2:2011) has been prepared by Technic
12、al Committee ISO/TC 34 “Food products“ in collaboration with 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 AFNOR. This European Standard shall be given the status of a nationa
13、l standard, either by publication of an identical text or by endorsement, at the latest by August 2011, and conflicting national standards shall be withdrawn at the latest by August 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent ri
14、ghts. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 5509:2000. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Sta
15、ndard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the Unite
16、d Kingdom. Endorsement notice The text of ISO 12966-2:2011 has been approved by CEN as a EN ISO 12966-2:2011 without any modification. BS EN ISO 12966-2:2011ISO 12966-2:2011(E) ISO 2011 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scope1 2 Normative references1 3 Reactions .1 4
17、Methodology 2 4.1 Preparation of test sample .2 4.2 Rapid method.2 4.3 General method .4 4.4 Transmethylation using boron trifluoride (BF3) catalyst 6 4.5 Acid-catalysed transmethylation of glycerides9 Annex A (informative) Thin-layer chromatography method for testing the completeness of derivatizat
18、ion .11 Bibliography15 BS EN ISO 12966-2:2011ISO 12966-2:2011(E) iv ISO 2011 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carr
19、ied 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 the work.
20、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 Directives, Part 2. The main task of technical committees is to prepare Internat
21、ional 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 bodies casting a vote. Attention is drawn to the possibility that some of the e
22、lements 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 12966-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. This first edition of ISO 1
23、2966-2 cancels and replaces ISO 5509:2000, of which it constitutes a technical revision. 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 2: Preparation of methyl esters of fatty acids Part 3: P
24、reparation of methyl esters using trimethylsulfonium hydroxide (TMSH) The following part is under 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 c
25、hromatography BS EN ISO 12966-2:2011ISO 12966-2:2011(E) ISO 2011 All rights reserved vIntroduction General Oils and fats (i.e. liquid and solid lipids) are predominantly composed of fatty acid esters of glycerol (triacylglycerols, TAGs), with smaller amounts of fatty acid esters of sterols and long
26、chain aliphatic alcohols. Due to the high molecular mass of the TAGs and their consequent low volatility, they are difficult to analyse directly by gas chromatography (GC), especially if a detailed analysis of unsaturated fatty acids is required. Fatty acids themselves do not chromatograph well (exc
27、ept for short-chain-length fatty acids, e.g. butanoic and pentanoic acids). It is therefore better practice to form fatty acid esters, usually the fatty acid methyl esters (FAMEs), prior to GC. The analysis of oils and fats has been extensively reviewed in Reference 9. The formation of FAMEs is a cr
28、itical stage in the analysis of fatty acids. Non-quantitative conversion of fatty acids to FAMEs, modification of the structure of fatty acids (e.g. changes in positional and geometric isomers present) and formation of non-FAME artefacts may all affect the quantitative determination of fatty acid co
29、mposition. Transesterification is one mechanism which can be employed to form FAMEs from fatty acid esters in fats (i.e. triacylglycerol). Alkali- or acid-catalysed transesterification procedures can be used to form FAMEs in a methanolic medium; the procedure can be termed transmethylation. Transmet
30、hylation is a reversible process and a large excess of methanol is required to maintain an equilibrium position which favours formation of the FAMEs. Water can prevent the reaction going to completion, and its presence should therefore be minimized. Alkali-catalysed procedures do not produce FAMEs f
31、rom free fatty acids, due to the formation of soaps. Esterification is an acid-catalysed mechanism which can be employed to form FAMEs from fatty acids. It is possible that the fatty acids are naturally present in the sample of fat under examination. Formation of FAMEs by this mechanism is commonly
32、termed methylation. Again, an excess of methanol and the absence of water are preconditions for the quantitative formation of FAMEs. This part of ISO 12966 provides guidelines for the preparation of fatty acid methyl esters. In support of these guidelines, various procedures to prepare fatty acid me
33、thyl esters are specified. These include: a) “rapid” transmethylation under alkaline conditions; b) “general” transmethylation/methylation under sequential alkaline and acid conditions; c) boron trifluoride (BF3) transmethylation/methylation. “Rapid” transmethylation method under alkali-catalysed co
34、nditions This method is applicable to the routine analysis of edible fats and oils containing fatty acids down to butanoic acid (C4:0) and/or for the determination of butanoic acid or hexanoic acid (C6:0) by GC using an internal standard. Alkaline catalysts transesterify neutral lipids in the presen
35、ce of anhydrous methanol (transmethylation) more rapidly than acid catalysts. The disadvantages of such alkali-catalysed procedures are that free fatty acids are not esterified, and the presence of water may prevent the transmethylation going to completion (hydrolysis of the FAMEs to free fatty acid
36、s). The most commonly used reagents are potassium and sodium hydroxide and sodium methoxide in the presence of anhydrous methanol. BS EN ISO 12966-2:2011ISO 12966-2:2011(E) vi ISO 2011 All rights reserved“General” transmethylation/methylation under sequential alkaline and acid conditions This method
37、 under sequential alkali- and acid-catalysed conditions is applicable to all oils and fats including distillate and acid oils, but is not recommended for lauric oils. Short-chain fatty acid methyl esters are easily lost during reflux. For lauric acid oils, the “rapid” transmethylation method is reco
38、mmended. During methylation, substances containing the following configurations can be totally or partially decomposed: a) keto, epoxy, hydroxyl, hydroperoxy groups; b) cyclopropyl and cyclopropenyl groups; c) acetylenic fatty acids. Boron trifluoride (BF3) transmethylation/methylation Owing to the
39、toxicity of BF3it is recommended that thismethod only be used in extremis. The BF3method is applicable for most oils, fats and derivatives (fatty acids, soaps) with the exception of milk fats and fats containing fatty acids with specific groups. During methylation, substances containing the followin
40、g configurations can be totally or partially decomposed: a) keto, epoxy, hydroxyl, hydroperoxy groups; b) cyclopropyl and cyclopropenyl groups; c) acetylenic fatty acids. If the fatty matter contains such substances in only very small amounts (e.g. cottonseed oil), the method can be applied, otherwi
41、se the “rapid” or “general” transmethylation/methylation methods should be followed. For GC, the optimum recovery of the methyl esters from the reaction mixture is obtained by using isooctane (2,2,4-trimethylpentane). However, only about 75 % of the methyl caproate present is recovered. Boron triflu
42、oride is a strong Lewis acid, and in the form of its coordination complex with methanol, under reflux conditions, it can rapidly methylate fatty acids. Methanolic boron trifluoride does transmethylate fatty acid esters (e.g. triglyceride), but the rate of reaction is slower than the methylation of f
43、atty acids. Methanolic boron trifluoride solution is commercially available, which enhances the attractiveness of this acid catalyst, but there are potential disadvantages associated with the use of this reagent. a) It has been reported that high concentrations of boron trifluoride (50 % mass fracti
44、on) produce methoxy artefacts from unsaturated fatty acids. b) The reagent has a limited shelf-life at ambient temperature and should be kept refrigerated. c) Aged reagent may produce artefacts and therefore it is recommended that each new batch purchased be tested before use and periodically during
45、 its lifetime. d) Methanolic boron trifluoride is an acidic reagent and therefore may produce derivatives of fatty acids containing labile groups which may give rise to spurious peaks on FAME chromatograms. BS EN ISO 12966-2:2011ISO 12966-2:2011(E) ISO 2011 All rights reserved viiAdditional informat
46、ion Much attention has been given to the preparation and analysis of esters of short-chain fatty acids by GC, largely because of their occurrence in milk fats. Short-chain fatty acids, in the free state or esterified to glycerol, can be converted completely to methyl esters by any of the reagents de
47、scribed in the preceding paragraphs, but quantitative recovery from the reaction medium may not be achieved unless special precautions are taken. Losses can occur at several stages in any procedure. Short-chain fatty acid esters (methyl especially) are volatile and may be lost selectively on refluxi
48、ng the esterification medium, they are more soluble in water than longer-chain esters and can be lost in an aqueous extraction step or they may be distilled off when the extracting solvent is evaporated. Selective losses can also occur if non-saponifiable impurities have to be removed by sublimation
49、 or thin-layer chromatography (TLC) purification. The best esterification procedures for short-chain fatty acids are those in which heating of the reagents is avoided and in which stages involving aqueous extraction and solvent removal are absent. Injection of reaction media containing basic and acidic esterification catalysts directly on to GC columns shortens their working lives. The top few centimetres of packed columns can be replenished periodically, while lengths of deactivated tubing or “retention gaps” ahead of capillary columns
