1、BSISOICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISHSTANDARDVegetablefatsandoilsI somericdiacylglycerolsDeterminationofrelativeamountsof1,2-and1,3-diacylglycerolsBS EN ISO 29822:2014BS EN ISO 29822:2014ISBN 978 0 580 83972 6Amendments/corrigenda issued sinc
2、e publicationDate Comments30 April 2014 This corrigendum renumbers BS ISO 29822:2009 as BS EN ISO 29822:2014This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 April 2009 The British Standards Institution 2014. Published by BSI Standards Limit
3、ed 2014National forewordThis British Standard is the UK implementation of EN ISO 29822:2014. It is identical ISO 29822:2009. It supersedes BS ISO 29822:2009, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats an
4、d oils and their by-products.A list of organizations represented on this committee can be obtained on request to its secretary.The publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard
5、cannot confer immunity from legal obligations.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 29822 April 2014 ICS 67.200.10 English Version Vegetable fats and oils - Isomeric diacylglycerols - Determination of relative amounts of 1,2- and 1,3-diacylglycerols (ISO 29822:2009) Corps gras dor
6、igine vgtale - Diacylglycrols isomriques - Dtermination des teneurs relatives en 1,2- et 1,3-diacylglycrols (ISO 29822:2009) Tierische und pflanzliche Fette und le - Isomere Diacylglyceride - Bestimmung der relativen Menge von 1,2-und 1,3- Diacylglyceride (ISO 29822:2009) This European Standard was
7、approved by CEN on 11 April 2014. 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 bibliographical references concerning such natio
8、nal 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 of a CEN member into its own langua
9、ge 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 Yugoslav Republic of Macedonia, France, Germany, Greece,
10、Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-
11、CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 29822:2014 EBS ISO 29822:2009ISO 29822:2009(E)PDF disclaimerThisPDF file may contain embedded typefaces. In accor
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14、y care has been taken to ensure that thefile is suitable for use by ISOmember bodies. Inthe unlikely eventthat a problem relating to it isfound, please inform the Central Secretariat at the address given below.COPYRIGHT PROTECTED DOCUMENT ISO 2009 All rights reserved. Unless otherwisespecified, no p
15、art of this publicationmay be reproduced or utilized in any form or by any means,electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below orISOs member body in the country ofthe requester.ISO copyright officeCase postale 56 CH
16、-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published inSwitzerland ii ISO 2009 All rights reservedBS EN ISO 29822:2014EN ISO 29822:2014 (E)EN ISO 29822:2014 (E) 3 Foreword The text of ISO 29822:2009 has been prepared by Technical Committee IS
17、O/TC 34 “Food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 29822:2014 by 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
18、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 October 2014, and conflicting national standards shall be withdrawn at the latest by October 2014. Attention is drawn to the possibility t
19、hat 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. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound
20、to implement this European Standard: 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,
21、Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 29822:2009 has been approved by CEN as EN ISO 29822:2014 without any modification. BS ISO 29822:2009INTERNATIONAL STANDARD ISO 29822:2009(E) ISO 2009 All rights reserved 1Vegeta
22、ble fats and oils Isomeric diacylglycerols Determination of relative amounts of 1,2- and 1,3-diacylglycerols1 Scope This International Standard specifies the determination of the degree of isomerization of diacylglycerols invegetable fats and oils. 1,2-Diacylglycerols are transformed to the more sta
23、ble 1,3-isomers during storage ordue to acidic catalysed reaction. The massfraction of 1,2-diacylglycerols can be used as a quality criterion for vegetable fats and oils. 2 NormativereferencesThe following referenced documents are indispensable for the application of this document. For datedreferenc
24、es, only the edition cited applies. For undated references, the latest edition of the referenceddocument (including anyamendments) applies.ISO 661, Animal and vegetable fats and oils Preparation of test sample3 Terms and definitions For the purposes of this International Standard, the following term
25、s and definitions apply.3.1 degree of isomerization massfraction of the peak areas of all 1,2-diacylglycerols (C32, C34, C36) relative to the sum of the peaks of alldiacylglycerols (C32, C34, C36). NOTE The mass fraction is expressed as a percentage to one decimal place. 4 Principle A miniaturized c
26、olumn chromatographyon a silica gel column is used to separate the isomeric diacylglycerols as the more polar fraction from the major part of other lipids. The peak areas of 1,2- and 1,3-isomers are determined by gas chromatography after silylation. Only C32-, C34- and C36-diacylglycerols are taken
27、intoaccount. 5 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardoussubstances. Technical, organizational and personal safety measures shall be followed.During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distille
28、d ordemineralizedwater orwater of equivalent purity. BS EN ISO 29822:2014BS ISO 29822:2009ISO 29822:2009(E)2 ISO 2009 All rights reserved5.1 Silica gel 60 1), for column chromatography, (0,063 to 0,100 mm).5.1.1 Silica gel 60,with a moisture content, wH2O= 5 % mass fraction. Activate the silica gel
29、byheating overnight at (160 5) C. After heating, place the silica gel in a desiccator for cooling and then transfer the silica gel to a stoppered flask. Add 5 % mass fraction of water and shake until nolumps can be seen and the powder flows freely (1h in an automatic shaking machine). Store the cond
30、itionedsilica gel overnight before use. 5.2 Cotton wool, defatted. 5.3 Reference substances2). 5.3.1 Dilaurin (1,3-dilauroyl-sn-glycerol).5.3.2 Dipalmitin (1,2- and 1,3-dipalmitoyl-sn-glycerol, mixed isomers, w 99 % mass fraction). 5.3.3 Distearin (1,2- and 1,3-distearoyl-sn-glycerol, mixed isomers,
31、 w 99 % massfraction). 5.3.4 Diolein (1,2- and 1,3-dioleoyl-sn-glycerol, mixed isomers, w 99 % massfraction). 5.4 Reference solutions: For the reference substances dipalmitin and distearin, prepare a solution in toluene (mass concentration, = 1 mg/ml). 5.5 Isooctane (2,2,4-trimethylpentane).5.6 Diet
32、hyl ether, eluent.5.7 Diisopropyl ether. 5.8 Solvent mixture: isooctane/diisopropyl ether volume fraction (isooctane) = 85 ml/100 mland(diisopropyl ether) = 15 ml/100 ml. 5.9 1-Methylimidazole.5.10 N-Methyl-N-(trimethyl-silyl)heptafluorobutyramide (MSHFBA). 5.11 Silylatingreagent: 50 l 1-methylimida
33、zole in 1 ml of MSHFBA. 5.12 Toluene. 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1 Analyticalbalance, capable of weighing to the nearest 0,001 g and displaying 0,000 1 g. 1) Merck Art.-No612-1484 or 613-2623 (www.merck.de)are examples of suitable products available c
34、ommercially. Thisinformation is given for the convenience of users of this International Standard and does not constitute an endorsement byISO of these products. Equivalent products may be used if they can be shown to lead to the same results. 2) A suitable supplier is Sigma-Aldrich (). This informa
35、tion is given for the convenience of usersof this International Standard and does not constitute an endorsement by ISO of this supplier. BS EN ISO 29822:2014ISO 29822:2009(E)BS ISO 29822:2009ISO 29822:2009(E) ISO 2009 All rights reserved 36.2 Desiccator, for storing adsorbents after heating.6.3 Pipe
36、tte tip 3), capacity5 ml, length approx. 150 mm. 6.4 Pointed flask, capacity 10ml, with ground-glass neck and rounded bottom.6.5 Beaker, capacity 10 ml. 6.6 Rotary evaporator. 6.7 Gas chromatograph for capillary columns, split injector, flame ionization detector (FID) and suitableintegration system.
37、 Do not use on-column or splitless injectors, as this results in isomerization of 1,2-diacylglycerols.6.8 Fused silica capillary column, for gas chromatography (internal diameter, 0,25 mm or 0,32 mm,length 12 m, 15 m, 30 m or 60 m) coated with 5 % diphenyl-,95 % dimethylpolysiloxane, 0,1 m film thic
38、kness.Other columns of similar polarity and selectivity mayalso be used. 7 Sample 7.1 Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling methodis given in ISO5555 1. It is important that the laboratory receive a sample which is truly represen
39、tative and has not been damaged orchanged during transport or storage. 7.2Preparation of test sample Prepare the test sample inaccordance with ISO 661. 8 Procedure 8.1Preparation of the silica gel chromatography column Insert a small stopper of cotton wool (5.2) approx. 5 mm high, in the lower part
40、ofa pipette tip (6.3) followed by1 g silica gel 60 (5.1). Cover the silica layer with a 5 mm high stopper of cotton wool. Compress the filling byslight pressingwith a flat ended rod. 8.2Separation of the fraction containing non polar lipids 8.2.1 Weigh, to the nearest 0,1 mg, about 100,0 mg of the t
41、est sample into a beaker (6.5) and add 1ml of toluene (5.12). 8.2.2 Transfer the test portion on to the column, carefullypurging the flask with 1 ml solvent mixture (5.8).Wash the column with two 3,5 ml portions of the solvent mixture (5.8). Rinse the end of the pipette tip withsolvent mixture (5.8)
42、 and discard the solvent. 3) VWR/Merck Art.-No 1.15101.1000 (www.merck.de) is an exampleof a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement byISO of this product. Equivalent product
43、s may be used if they can be shown to lead to the same results. BS EN ISO 29822:2014ISO 29822:2009(E)BS ISO 29822:2009ISO 29822:2009(E)4 ISO 2009 All rights reserved8.2.3 Elute the diacylglycerols with two 3,5 ml portions of diethylether (5.6) and collect the eluate in a pointed flask (6.4).8.2.4 Re
44、move the solvent from the eluate to about 1 ml with a rotaryevaporator (6.6) at 20 C. Transfer theremaining solution into a reaction vial. Blow off the solvent in the reaction vial with a stream of nitrogen. 8.3Preparation of trimethylsilyl ethers (silylation)8.3.1 Add 200l of the silylationreagent
45、(5.11) to the reaction vial containing the diacylglycerols, seal and allow the mixture to react for 20 min at room temperature. 8.3.2 After silylation, add 1 ml acetone and use 1 l to 2 l of the solution for the gas chromatography.8.4 Gas chromatographyThe conditions in Table 1 for the gas chromatog
46、raph have been found to give useful chromatograms.Table 1 Gas chromatographic conditionsFunction Condition Capillary GC column Restek RTX54), 60 m; internaldiameter 0,25 mm, film thickness 0,1 mInjection volume 1 l (split 1:50) Carrier gas Hydrogen at 2 ml/min, constant flowGas for FIDHydrogen at 33
47、 ml/min and air at 420 ml/min Injector temperature 340 CDetector temperature 340 CTemperature oven 240 C; maintain for 1 min, heat at 10 C/min to 320 C, maintain for 10 minOptimize the temperature programme and the velocityof the carrier gas flow so that chromatograms similar toFigure A.1 are obtain
48、ed. Test the separation with silylated diacylglycerol fractions. 8.5Identification of diacylglycerol isomers Prepare a solution of dipalmitin (1,2-C32,1,3-C32) (5.3.2), distearin (1,2-C36, 1,3-C36) (5.3.3),and diolein (1,2-C36, 1,3-C36) (5.3.4),each with a mass concentration, = 1 mg/ml toluene. Add
49、100 l of this solution toa derivatization vial. Blow off the solvent in the reaction vial with a stream of nitrogen and silylate. Do not add acetone to the reference solutions.9 Result of the determination 9.1Identification of diacylglycerols To identify the 1,2- and 1,3-diacylglycerols in the test portion, determine the retention times of the referencestandards. 4) Example of a suitable product available commercially. This information is given for the convenience of users of thisIntern
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