DIN EN ISO 18395-2007 Animal and vegetable fats and oils - Determination of monoacylglycerols diacylglycerols triacylglycerols and glycerol by high-performance size-exclusion chrom.pdf

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1、October 2007DEUTSCHE NORM English price group 10No part of this standard 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 67.200.10!$IN“1389043w

2、ww.din.deDDIN EN ISO 18395Animal and vegetable fats and oils Determination of monoacylglycerols, diacylglycerols, triacylglycerolsand glycerol by high-performance size-exclusion chromatography(HPSEC) (ISO 18395:2005)English version of DIN EN ISO 18395:2007-10Tierische und pflanzliche Fette und le Be

3、stimmung von Monoacylglycerinen, Diacylglycerinen, Triacylglycerinen und Glycerindurch Hochleistungs-Grenausschluss-Chromatographie (HPSEC) (ISO 18395:2005)Englische Fassung DIN EN ISO 18395:2007-10www.beuth.deDocument comprises 14 pages DIN EN ISO 18395:2007-10 2 National foreword This standard has

4、 been prepared by Subcommittee SC 11 “Animal and vegetable fats and oils” (Secretariat: BSI, United Kingdom) of Technical Committee ISO/TC 34 “Agricultural food products” (Secretariat: MSZT, Hungary) in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils a

5、nd 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 (Foodstuffs and Agricultural Products Standards Committee) and the Deutsche Gesellscha

6、ft fr Fettforschung (German Fat Research Society), Joint Committee Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standard corresponding to the International Standard referred to in clause 2 and in the bibliography of the EN is as follows: ISO 661 DIN EN ISO 661

7、National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 18395August 2007ICS 67.200.10English VersionAnimal and vegetable fats and oils - Determination ofmonoacylglycerols, diacylg

8、lycerols, triacylglycerols and glycerolby high-performance size-exclusion chromatography (HPSEC)(ISO 18395:2005)Corps gras dorigines animale et vgtale - Dterminationde la teneur en monoacylglycrides, en diacylglycrides, entriacylglycrides et en glycrol par chromatographie18395:2005)Tierische und pfl

9、anzliche Fette und le - Bestimmung vonMonoacylglycerinen, Diacylglycerinen, Triacylglycerinenund Glycerin durch Hochleistungs-Grenausschluss-Chromatographie (HPSEC) (ISO 18395:2005)This European Standard was approved by CEN on 26 July 2007.CEN members are bound to comply with the CEN/CENELEC Interna

10、l Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.T

11、his European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are t

12、he national standards bodies of Austria, Belgium, Bulgaria, 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

13、 and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 183

14、95:2007: Edexclusion haute performance (CLHP dexclusion) (ISOForeword The text of ISO 18395:2005 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 18395:2007 by Technical Commi

15、ttee 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 national standard, either by publication of an identical text or by endorsement, at

16、the latest by February 2008, and conflicting national standards shall be withdrawn at the latest by February 2008. “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 a

17、ny or all such patent rights.” 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hun

18、gary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 18395:2005 has been approved by CEN as EN ISO 18395:2007 without any modification

19、s. 2 EN ISO 18395:2007 (E)1 Scope This International Standard specifies a method for the determination of monoacylglycerols, diacylglycerols and triacylglycerols and also free glycerol by high-performance size-exclusion chromatography. It is applicable to products (e.g. emulsifiers) comprising monoa

20、cylglycerols and diacylglycerols as main constituents in concentrations 10 %, and to triacylglycerols in a proportion of 3 %. No response factors at all are employed in the European Pharmacopoeia for acylglycerols. This shall be indicated in the test report. 6 Apparatus Usual laboratory equipment an

21、d, in particular, the following. 6.1 Analytical balance, with a readability of 0,1 mg. 6.2 Volumetric flask, of 10 ml capacity. 6.3 Pipette, of 5 ml capacity. 6.4 Ultrasonic bath. 6.5 HPLC/GPC pump. 6.6 Injector, equipped with a sample loop of 20 l capacity. 6.7 Detector, differential refractometer

22、(RI detector). 6.8 GPC column combination, with an effective molecular mass up to 4 000 Da (e.g. three columns of 300 mm 7,5 mm Plgel2), 5 m, 100 ). It is also possible to use other columns (or column combinations) provided that the separation of the monoacylglycerols and diacylglycerols and also of

23、 the diacylglycerols and triacylglycerols is ensured. The use of only two columns is also possible to achieve the required separation. 1) Available for example from Sigma-Aldrich (http:/ 2) Available for example from Polymer Laboratories (http:/ This information is given for the convenience of users

24、 of this International Standard and does not constitute an endorsement by ISO of these products. 4 EN ISO 18395:2007 (E)6.9 Column oven (column thermostat). 6.10 Data integration and evaluation system. 7 Sampling A representative sample should have been sent to the laboratory. It should not have bee

25、n damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 55555. 8 Preparation of test sample Prepare the test sample in accordance with ISO 661. Before taking the test portion from the

26、 sample, mix the sample thoroughly to ensure homogeneity. For the same reason, melt solid samples completely for proper mixing. 9 Procedure 9.1 Preparation of the sample solution Weigh, to the nearest 0,1 mg, about 100 mg of the sample into a 10 ml volumetric flask. The concentration is reported in

27、milligrams of sample per 10 ml. After diluting to the mark with THF, place the flask in the ultrasonic bath for 5 min to 10 min. The clear solution obtained is directly injected into the GPC system. 9.2 Preparation of reference solutions Weigh, to the nearest 0,1 mg, amounts of glycerol, monoacylgly

28、cerols, diacylglycerols and triacylglycerols above and below the expected concentration in the sample into 10 ml volumetric flasks. After diluting to the mark with THF, place the flasks in the ultrasonic bath for 5 min to 10 min. The clear solutions obtained in this way are directly injected into th

29、e GPC system. The concentration is reported in milligrams of component per 10 ml for each reference solution. If the expected content is not known, it is possible to make up a number of calibration solutions that cover a wider range. NOTE Refer to 5.2.2. 9.3 Gel permeation chromatography (GPC) Set u

30、p the GPC system as follows: Injection volume: 20 l Separation columns: Range: minimum 0 to 4 000 Da (6.8) Oven temperature: 35 C or 40 C 0,1 C Flow rate: 0,8 ml/min to 1,0 ml/min RI-detector: Temperature: 35 C or 40 C. Typical chromatograms obtained under these conditions are given in Annex A. 5 EN

31、 ISO 18395:2007 (E)Qualitative testing for the presence of monoacylglycerols, diacylglycerols and triacylglycerols and of glycerol is carried out by comparing the retention times with comparative substances (e.g. C18-monoacylglycerols/-C18-diacylglycerols/C18-triacylglycerols) or acylglycerol groups

32、. For the quantitative determination of the contents, prepare at least two calibration solutions. Choose the concentrations of the solutions so that they bracket the contents in the sample above and below. These solutions are injected in succession, twice in each case. The calibration curves produce

33、d make it possible to calculate the glycerol and acylglycerol concentrations in the sample. NOTE Refer to 5.2.2. 10 Calculation The glycerol content in the sample, wG, expressed as a mass fraction in percent, is calculated as follows: GGGS100 %mFwm= where wGis the glycerol content of the sample; mGi

34、s the mass of the glycerol in the sample, in milligrams, calculated from the calibration curve; mSis the mass, in milligrams, of the test portion; FGis the response factor for glycerol. The mono-, di- and tri-acylglycerol contents are calculated in the same way. In the evaluation of the peak areas,

35、a horizontal baseline is used as the integration boundary. The response factors for glycerol, monoacylglycerols, diacylglycerols and triacylglycerols are generally the same under the conditions indicated here, so that in most cases the use of quantitative reference solutions is unnecessary and the r

36、esponse factors are assumed to be F = 1. However it is necessary to determine a response factor for glycerol when the concentration in the sample is 3 %. In most cases, free fatty acids are not separated from the monoacylglycerol peak group. Therefore in samples with acid values 1 the amount of fatt

37、y acids, calculated from the acid value, may be subtracted from the monoacylglycerol content. This shall be indicated in the test report. Report the result of the determination, expressed as a mass fraction in percent, to one decimal place. 11 Precision 11.1 Interlaboratory test Details of an interl

38、aboratory test on the precision of the method are summarized in Annex B. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. 11.2 Repeatability The absolute difference between two independent single test results, obtain

39、ed using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases exceed the value of r given in Tables B.1 to B.5. 6 EN ISO 18395:2007 (E)11.3 Reproducibility The absolute diff

40、erence between two single test results, obtained using the same method on identical test material in different laboratories by different operators using different equipment, will in not more than 5 % of cases exceed the value of R given in Tables B.1 to B.5. 12 Test report The test report shall spec

41、ify: all information necessary for the complete identification of the sample; the sampling method used, if known; the test method used, with reference to this International Standard; all operating details not specified in this International Standard, or regarded as optional, together with details of

42、 any incidents which may have influenced the test result(s); the test result obtained; if the repeatability has been checked, the final quoted result obtained. 7 EN ISO 18395:2007 (E)Annex A (informative) Examples of chromatograms Key X retention time, min Y peak intensity, mV 1 triacylglycerols (39

43、,132) 2 diacylglycerols (40,407) 3 monoacylglycerols (42,552) 4 free glycerol (47,560) 5 diglycerol monostearate (41,719) aPartial separation of C16-/C18-monoacylglycerols. NOTE Five columns of 300 mm 7,5 mm Plgel, 5 m, 100 were used. Figure A.1 Chromatogram of a sample comprising mono-, di- and tri

44、-acylglycerols derived from tallow Table A.1 Sample comprising mono-, di- and tri-acylglycerols derived from tallow Substance Retention time min Area % Area Triacylglycerols 39,132 1 694 320 6,2 Diacylglycerols 40,407 10 574 930 38,5 Diglycerol-monoester 41,719 311 730 1,2 Monoacylglycerols 42,552 1

45、4 719 338 53,5 Free glycerol 47,560 155 732 0,57 8 EN ISO 18395:2007 (E)Key X retention time, min Y peak intensity, mAU (absorption units) 1 triacylglycerols 2 diacylglycerols 3 monoacylglycerols 4 glycerol NOTE Two columns of 300 mm 7,5 mm Plgel, 5 m, 100 were used. Figure A.2 Separation of a test

46、mixture 9 EN ISO 18395:2007 (E)Annex B (informativ) Results of interlaboratory test A collaborative study was organized by the Joint Committee for the Analysis of Fats, Oils, Fat Products, Related Products and Raw Materials in 2002 on an international basis. Five different samples were investigated.

47、 The results, evaluated in accordance with ISO 5725-26are given in Tables B.1 to B.5. Table B.1 Results of interlaboratory test (fatty acids) Sample identification: Mono-/di-acylglycerols of C8/C10 fatty acids Monoacyl-glycerols Di-/triacyl-glycerols Glycerol Number of participating laboratories 10

48、10 10 Number of laboratories retained after eliminating outliers 10 10 8 Number of test results in all laboratories 20 20 16 Mean 45,78 41,76 10,96 Repeatability standard deviation (sr) 0,24 0,27 0,14 Repeatability relative standard deviation 0,5 0,7 1,20 Repeatability limit (r) 0,67 0,77 0,38 Repro

49、ducibility standard deviation (sR) 1,38 1,08 0,61 Reproducibility relative standard deviation 3,0 2,6 5,5 Reproducibility limit (R) 3,85 3,03 1,70 Table B.2 Results of interlaboratory test (tallow) Sample identification: Mono-/di-acylglycerols based on tallow Monoacyl-glycerols Diacyl-glycerols Triacyl-glycerols Glycerol Number of participating laboratories 10 10 10 9 Number of laboratories retained after eliminating outliers 10 9 9 8 Number of test results