DIN EN ISO 29822-2014 Vegetable fats and oils - Isomeric diacylglycerols - Determination of relative amounts of 1 2- and 1 3-diacylglycerols (ISO 29822 2009) German version EN ISO .pdf

1、July 2014 Translation by DIN-Sprachendienst.English price group 9No 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!%2x$“2158501www.din.deDDIN EN ISO 29822Vegetable fats and oils Isomeric diacylglycerols Determination of relative amounts of 1,2- and 1,3-diacylglycerols(ISO 29822:2009);English version EN ISO 29822:2014,English translation of DIN EN ISO 29822:2014-07Tierische und pflanzliche Fette und le Is

3、omere Diacylglycerole Bestimmung der relativen Menge von 1,2- und 1,3-Diacylglycerolen (ISO 29822:2009);Englische Fassung EN ISO 29822:2014,Englische bersetzung von DIN EN ISO 29822:2014-07Corps gras dorigine vgtale Diacylglycrols isomriques Dtermination des teneurs relatives en 1,2- et 1,3-diacylgl

4、ycrols (ISO 29822:2009);Version anglaise EN ISO 29822:2014,Traduction anglaise de DIN EN ISO 29822:2014-07www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 13 pages06.14 DIN EN ISO 29822:2014-07 2 A comma is used as the decimal marker. Na

5、tional foreword The text of ISO 29822:2009 has been prepared by Technical Committee ISO/TC 34 “Food products” (Secre-tariat: AFNOR, France), Subcommittee SC 11 “Animal and vegetable fats and oils” (Secretariat: BSI, United Kingdom) and has been taken over as EN ISO 29822:2014 by Technical Committee

6、CEN/TC 307 “Oil seeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The document has been submitted to the Unique Acceptance Procedure (UAP). The responsible German bodies involved in its preparation were the Normenausschuss

7、 Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee) and the Deutsche 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 Sto

8、ffen und Rohstoffen. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 661 DIN EN ISO 661 ISO 5555 DIN EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and

9、 vegetable fats and oils Preparation of test sample DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measur

10、ement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 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-diac

11、ylglycero ls(ISO 29822:2009) Corps gras dorigine vgtale - Diacylglycrols isomriques - Dtermination des teneurs relatives en 1,2- et 1,3-diacylglycrols (ISO 29822:2009) Tierische und pflanzliche Fette und le - IsomereDiacylglycerole - Bestimmung der relativen Menge von 1,2-und 1,3-Diacylglycerolen (I

12、SO 29822:2009)This European Standard was 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 bibliog

13、raphical 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 responsib

14、ility 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 Yugoslav Republi

15、c 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. CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

16、 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 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMContents Page Fo

17、reword . 3 1 Scope . 4 2 Normative references. 4 3 Terms and definitions 4 4 Principle 4 5 Reagents . 4 6 Apparatus 5 7 Sample . 6 7.1 Sampling . 6 7.2 Preparation of test sample 6 8 Procedure 6 8.1 Preparation of the silica gel chromatography column . 6 8.2 Separation of the fraction containing non

18、 polar lipids 6 8.3 Preparation of trimethylsilyl ethers (silylation) . 7 8.4 Gas chromatography . 7 9 Result of the determination . 7 9.1 Identification of diacylglycerols . 7 9.2 Determination of the peak areas of 1,2- and 1,3- diacylglycerols in the oil . 8 10 Precision of the method 8 10.1 Inter

19、laboratory test . 8 10.2 Repeatability . 8 10.3 Reproducibility . 8 11 Test report . 8 Annex A (informative) Example of a typical chromatogram 9 Annex B (informative) Results of an interlaboratory test 10 Bibliography 11 2DIN EN ISO 29822:2014-07 EN ISO 29822:2014 (E) Foreword The text of ISO 29822:

20、2009 has been prepared by Technical Committee ISO/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

21、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 October 2014, and conflicting national standards shall be withdrawn at the latest by Octobe

22、r 2014. 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. According to the CEN-CENELEC Internal Regulations, the national standards orga

23、nizations of the following countries are bound 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,

24、 Malta, Netherlands, Norway, Poland, Portugal, 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. 3DIN EN ISO 29822:2014-07EN ISO 29822:2014 (E)1

25、 Scope This International Standard specifies the determination of the degree of isomerization of diacylglycerols in vegetable fats and oils. 1,2-Diacylglycerols are transformed to the more stable 1,3-isomers during storage or due to acidic catalysed reaction. The mass fraction of 1,2-diacylglycerols

26、 can be used as a quality criterion for vegetable fats and oils. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced docume

27、nt (including any amendments) applies. ISO 661, Animal and vegetable fats and oils Preparation of test sample 3 Terms and definitions For the purposes of this International Standard, the following terms and definitions apply. 3.1 degree of isomerization mass fraction of the peak areas of all 1,2-dia

28、cylglycerols (C32, C34, C36) relative to the sum of the peaks of all diacylglycerols (C32, C34, C36). NOTE The mass fraction is expressed as a percentage to one decimal place. 4 Principle A miniaturized column chromatography on a silica gel column is used to separate the isomeric diacylglycerols as

29、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 into account. 5 Reagents WARNING Attention is drawn to the regulations which specify the handli

30、ng of hazardous substances. Technical, organizational and personal safety measures shall be followed. During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. 4DIN EN ISO 29822:2014-07 EN ISO 29

31、822:2014 (E) 5.1 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 by heating overnight at (160 5) C. After heating, place the silica gel in a desiccator for cooling and then transfer the s

32、ilica gel to a stoppered flask. Add 5 % mass fraction of water and shake until no lumps can be seen and the powder flows freely (1 h in an automatic shaking machine). Store the conditioned silica gel overnight before use. 5.2 Cotton wool, defatted. 5.3 Reference substances 2). 5.3.1 Dilaurin (1,3-di

33、lauroyl-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, w 99 % mass fraction). 5.3.4 Diolein (1,2- and 1,3-dioleoyl-sn-glycerol, mixed isomers, w 99 % mass fraction). 5.4

34、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 Diethyl ether, eluent. 5.7 Diisopropyl ether. 5.8 Solvent mixture: isooctane/diisopropyl ether volume fraction (isooc

35、tane) = 85 ml/100 ml and (diisopropyl ether) = 15 ml/100 ml. 5.9 1-Methylimidazole. 5.10 N-Methyl-N-(trimethyl-silyl)heptafluorobutyramide (MSHFBA). 5.11 Silylating reagent: 50 l 1-methylimidazole in 1 ml of MSHFBA. 5.12 Toluene. 6 Apparatus Usual laboratory equipment and, in particular, the followi

36、ng. 6.1 Analytical balance, capable of weighing to the nearest 0,001 g and displaying 0,000 1 g. 1) Merck Art.-No 612-1484 or 613-2623 (www.merck.de) are examples of suitable products available commercially. This information is given for the convenience of users of this International Standard and do

37、es not constitute an endorsement by ISO 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 information is given for the convenience of users of this International Standard and does not constitute an en

38、dorsement by ISO of this supplier. 5DIN EN ISO 29822:2014-07EN ISO 29822:2014 (E)6.2 Desiccator, for storing adsorbents after heating. 6.3 Pipette tip 3), capacity 5 ml, length approx. 150 mm. 6.4 Pointed flask, capacity 10 ml, with ground-glass neck and rounded bottom. 6.5 Beaker, capacity 10 ml. 6

39、.6 Rotary evaporator. 6.7 Gas chromatograph for capillary columns, split injector, flame ionization detector (FID) and suitable integration system. 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 chromat

40、ography (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 thickness. Other columns of similar polarity and selectivity may also be used. 7 Sample 7.1 Sampling Sampling is not part of the method specified in this

41、International Standard. A recommended sampling method is given in ISO 5555 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 7.2 Preparation of test sample Prepare the test sample in accordance with

42、ISO 661. 8 Procedure 8.1 Preparation of the silica gel chromatography column Insert a small stopper of cotton wool (5.2) approx. 5 mm high, in the lower part of a pipette tip (6.3) followed by 1 g silica gel 60 (5.1). Cover the silica layer with a 5 mm high stopper of cotton wool. Compress the filli

43、ng by slight pressing with a flat ended rod. 8.2 Separation of the fraction containing non polar lipids 8.2.1 Weigh, to the nearest 0,1 mg, about 100,0 mg of the test sample into a beaker (6.5) and add 1 ml of toluene (5.12). 8.2.2 Transfer the test portion on to the column, carefully purging the fl

44、ask 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 with solvent mixture (5.8) and discard the solvent. 3) VWR/Merck Art.-No 1.15101.1000 (www.merck.de) is an example of a suitable product available commercially

45、. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. 6DIN EN ISO 29822:2014-07 EN ISO 29822:2014 (E) 8.2.3 Elute the dia

46、cylglycerols with two 3,5 ml portions of diethyl ether (5.6) and collect the eluate in a pointed flask (6.4). 8.2.4 Remove the solvent from the eluate to about 1 ml with a rotary evaporator (6.6) at 20 C. Transfer the remaining solution into a reaction vial. Blow off the solvent in the reaction vial

47、 with a stream of nitrogen. 8.3 Preparation of trimethylsilyl ethers (silylation) 8.3.1 Add 200 l of the silylation reagent (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

48、 1 l to 2 l of the solution for the gas chromatography. 8.4 Gas chromatography The conditions in Table 1 for the gas chromatograph have been found to give useful chromatograms. Table 1 Gas chromatographic conditions Function Condition Capillary GC column Restek RTX5 4), 60 m; internal diameter 0,25

49、mm, film thickness 0,1 m Injection volume 1 l (split 1:50) Carrier gas Hydrogen at 2 ml/min, constant flow Gas for FID Hydrogen at 33 ml/min and air at 420 ml/min Injector temperature 340 C Detector temperature 340 C Temperature oven 240 C; maintain for 1 min, heat at 10 C/min to 320 C, maintain for 10 min Optimize the temperature programme and the velocity of the carrier gas flow so that chromatograms simila