DIN EN ISO 12966-2-2011 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) .pdf

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1、May 2011 Translation by DIN-Sprachendienst.English price group 13No 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

2、.200.10!$pWC“1775232www.din.deDDIN EN ISO 12966-2Animal 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)English translation of DIN EN ISO 12966-2:2011-05Tierische und pflanzliche Fette und le Gaschromato

3、graphie von Fettsuremethylestern Teil: 2: Herstellung von Fettsuremethylestern (ISO 12966-2:2011)Englische bersetzung von DIN EN ISO 12966-2:2011-05Corps gras dorigine animale et vgtale Chromatographie en phase gazeuse des esters mthyliques dacides gras Partie 2: Prparation des esters mthyliques dac

4、ides gras (ISO 12966-2:2011)Traduction anglaise de DIN EN ISO 12966-2:2011-05SupersedesDIN EN ISO 5509:2001-01www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.25s04.11 DIN EN ISO 12966-2:2011-05 2 A comma is used as the decimal mark

5、er. National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 11 “Animal and vegetable fats and oils” (Secretariat: BSI, United Kingdom) in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable an

6、d animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee) and the D

7、eutsche Gesell-schaft fr Fettwissenschaft (German Fat Research Society), Joint Committee NA 057-05-05 AA Gemein-schaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. ISO 12966 consists of the following parts, under the general title Animal and vegetable

8、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 trimethylsulfonium hydroxide (TMSH) Part 4: Determination of cis-, trans-, saturated, mono- and polyunsaturated fatty acids in vegetable or non

9、-ruminant oils and fats is under preparation Part 1: Guidelines on gas chromatography is planned. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 661 DIN EN ISO 661 ISO 835 DIN EN ISO 835 ISO 1042 DIN EN ISO 1042 ISO 3696 DIN ISO 3696 I

10、SO 4800 DIN ISO 4800 ISO 8655-2 DIN EN ISO 8655-2 Amendments This standard differs from DIN EN ISO 5509:2001-01 as follows: a) the standard has been revised in form and substance; b) the number of the standard has been changed; c) to meet the various needs, this standard specifies four different met

11、hylation methods. Previous editions DIN EN ISO 5509: 2001-01 DIN EN ISO 12966-2:2011-05 3 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN EN ISO 835, Laboratory glassware Graduated pipettes DIN EN ISO 1042, Laboratory gla

12、ssware One-mark volumetric flasks DIN EN ISO 8655-2, Piston-operated volumetric apparatus Part 2: Piston pipettes DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 4800, Laboratory glassware Separating funnels and dropping funnels DIN EN ISO 12966-2:2011-05 4 T

13、his page is intentionally blank EUROPEAN STANDARD NORME 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

14、acids (ISO 12966-2:2011) Corps gras dorigines animale et 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 - Te

15、il 2: Herstellung von Fettsuremethylestern (ISO 12966-2:2011)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 wi

16、thout 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 any othe

17、r 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 Republic,

18、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 United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMA

19、LISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: 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: EContents Page Foreword3 Introduction .4 1 Scope 7 2 Normative refe

20、rences 7 3 Reactions7 4 Methodology.8 4.1 Preparation of test sample8 4.2 Rapid method.8 4.3 General method10 4.4 Transmethylation using boron trifluoride (BF3) catalyst.12 4.5 Acid-catalysed transmethylation of glycerides 15 Annex A (informative) Thin-layer chromatography method for testing the com

21、pleteness of derivatization17 Bibliography 21 DIN EN ISO 12966-2:2011-05 EN ISO 12966-2:2011 (E) 2 Foreword This document (EN ISO 12966-2:2011) has been prepared by Technical Committee ISO/TC 34 Food products” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats

22、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 national standard, either by publication of an identical text or by endorsement, at the latest by August 2011, and conflicting national st

23、andards 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 rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN I

24、SO 5509:2000. 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, France, Germany, Greece, Hungary, Ic

25、eland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 12966-2:2011 has been approved by CEN as a EN ISO 12966-2:2011 without any modificati

26、on. “DIN EN ISO 12966-2:2011-05 EN ISO 12966-2:2011 (E) 3 Introduction 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 chain aliphatic alcohols. Du

27、e 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 (except for short-chain-length f

28、atty 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 critical stage in the analysis

29、 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 composition. Transesterificati

30、on 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. Transmethylation is a reversible pro

31、cess 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 from free fatty acids, due to

32、 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 termed methylation. Again, a

33、n 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 methyl esters are specified. T

34、hese 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 conditions This method is appl

35、icable 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 presence of anhydrous methanol (tr

36、ansmethylation) 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 acids). The most commonly used r

37、eagents are potassium and sodium hydroxide and sodium methoxide in the presence of anhydrous methanol. DIN EN ISO 12966-2:2011-05 EN ISO 12966-2:2011 (E) 4 “General” transmethylation/methylation under sequential alkaline and acid conditions This method under sequential alkali- and acid-catalysed con

38、ditions 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 recommended. During methylation, substances containi

39、ng 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 toxicity of BF3it is recommended that thismethod

40、 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 following configurations can be totally or partially dec

41、omposed: 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, otherwise the “rapid” or “general” transmethylation/met

42、hylation 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 trifluoride is a strong Lewis acid, and in the form of

43、 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 fatty acids. Methanolic boron trifluoride solutio

44、n 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 fraction) produce methoxy artefacts from unsaturated f

45、atty 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 its lifetime. d) Methanolic boron trifluoride i

46、s an acidic reagent and therefore may produce derivatives of fatty acids containing labile groups which may give rise to spurious peaks on FAME chromatograms. DIN EN ISO 12966-2:2011-05 EN ISO 12966-2:2011 (E) 5 Additional information Much attention has been given to the preparation and analysis of

47、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 described in the preceding paragraphs, but quantitative recovery from t

48、he 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 refluxing the esterification medium, they are more soluble in water than lon

49、ger-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 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