BS EN ISO 16931-2009 Animal and vegetable fats and oils - Determination of polymerized triacylglycerols by high-performance size- exclusion chromatography (HPSEC)《动植物油脂 高效体积排阻色谱法(H.pdf

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1、BS EN ISO16931:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal and vegetablefats and oils Determinationof polymerizedtriacylglycerols byhigh-performancesize-exclusionchromatography(HPSEC) (ISO16931:2009)This British Standardwas published

2、under theauthority of the StandardsPolicy and StrategyCommittee on 31 May2009. BSI 2009ISBN 978 0 580 63529 8Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 16931:2009National forewordThis British Standard is the UK implementation of EN ISO 16931:2009.It supersedes BS EN ISO 169

3、31:2001 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats and oils andtheir by products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not

4、 purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 16931April 2009ICS 67.200.10 Supersedes EN ISO 16931:

5、2001 English VersionAnimal and vegetable fats and oils - Determination ofpolymerized triacylglycerols by high-performance size-exclusionchromatography (HPSEC) (ISO 16931:2009)Corps gras dorigines animale et vgtale - Dterminationde la teneur en triacylglycrols polymriss parchromatographie liquide dex

6、clusion haute performance(CLHP dexclusion) (ISO 16931:2009)Tierische und pflanzliche Fette und le - Bestimmung desGehaltes an polymerisierten Triglyceriden mitHochleistungs-Ausschlusschromatographie (HPSEC) (ISO16931:2009)This European Standard was approved by CEN on 16 March 2009.CEN members are bo

7、und to comply with the CEN/CENELEC Internal 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 CE

8、N 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 translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status

9、 as theofficial versions.CEN members are the 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, Slov

10、akia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for

11、CEN national Members.Ref. No. EN ISO 16931:2009: EBS EN ISO 16931:2009EN ISO 16931:2009 (E) 3 Foreword This document (EN ISO 16931:2009) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and anima

12、l 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 the latest by October 2009, and conflicting

13、national standards shall be withdrawn at the latest by October 2009. 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 sup

14、ersedes EN ISO 16931:2001. 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, Hungary

15、, Iceland, 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 16931:2009 has been approved by CEN as a EN ISO 16931:2009 without any modificati

16、on. BS EN ISO 16931:2009ISO 16931:2009(E) ISO 2009 All rights reserved iiiForeword 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 carried out through ISO te

17、chnical 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. ISO collaborates close

18、ly 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 International Standards. Draft

19、 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 elements of this docume

20、nt may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16931 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. This second edition cancels and replaces the first

21、edition (ISO 16931:2001), which has been technically revised. BS EN ISO 16931:2009BS EN ISO 16931:2009INTERNATIONAL STANDARD ISO 16931:2009(E) ISO 2009 All rights reserved 1Animal and vegetable fats and oils Determination of polymerized triacylglycerols by high-performance size-exclusion chromatogra

22、phy (HPSEC) 1 Scope This International Standard specifies a method using high-performance size-exclusion chromatography (HPSEC) to determine the contents, as mass fractions, of polymerized triacylglycerols (PTAGs) in oils and fats which contain at least 3 % (from peak areas) of these polymers. PTAGs

23、 (strictly speaking dimeric and oligomeric triacylglycerols) are formed during the heating of fats and oils, and thus, the method serves to assess the thermal deterioration of frying fats after use. This method is applicable to frying fats and fats and oils that have been thermally treated, provided

24、 that the content of PTAGs is at least 3 %. It can also be applied to the determination of polymers in fats for animal feedstuffs, although in this case, the extraction method used can have an influence on the result. NOTE For further details, see ISO 64924. 2 Normative references The following refe

25、renced 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 document (including any amendments) applies. ISO 661, Animal and vegetable fats and oils Preparation of test sam

26、ple 3 Terms and definitions For the purposes of this International Standard, the following terms and definitions apply. 3.1 polymerized triacylglycerols PTAGs constituents of heated fats and oils that are determined by HPSEC under the conditions specified in this International Standard NOTE The cont

27、ent of PTAGs is expressed as a percentage mass fraction, in grams per 100 g, of all peaks from eluted polymerized and mono-, di-, and triacylglycerols (PTAGs, MAGs, DAGs, and TAGs). 4 Principle The sample is homogeneously mixed with tetrahydrofuran (THF) and PTAGs separated by gel permeation chromat

28、ography according to molecular size. The compounds are detected by means of a refractive index detector. NOTE For better resolution, two columns in series (2 300 mm) are used. BS EN ISO 16931:2009ISO 16931:2009(E) 2 ISO 2009 All rights reserved5 Reagents Use only reagents of recognized analytical gr

29、ade. WARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed. 5.1 Tetrahydrofuran (THF), for HPLC, possibly stabilized with butylhydroxytoluene (BHT), degassed, free of water. Ensure th

30、at the THF used as a diluent for the sample has the same water content as the eluent; otherwise a negative peak can be observed. 5.2 Toluene, for HPLC. 5.3 Extra virgin olive oil, as standard reference: add 100 mg to 300 mg of oil to 10 ml THF and homogenize the mixture. NOTE Extra virgin olive oil

31、does not contain PTAGs and can be used to determine the retention time of the monomeric TAGs. 6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 Solvent reservoir, capacity 500 ml to 1 000 ml, with a polytetrafluoroethylene mobile-phase line filter. 6.2 HPLC pump, pulseles

32、s, with a volume flow rate of 0,5 ml/min to 1,5 ml/min. 6.3 Injection valve, with a 20 l loop and a suitable syringe with a volume of 50 l to 100 l, or a suitable autosampler. 6.4 Stainless-steel columns, length 2 300 mm, internal diameter 7,5 mm to 7,8 mm, packed with spheres, diameter 5 m, of high

33、-performance styrene-divinylbenzene co-polymer gel; of pore size 10 nm or 50 nm1). NOTE The chromatograms in Annex A show the required resolution with two columns. The use of a guard column is recommended. The efficiency of the column, determined as the number, n, of theoretical plates for monomeric

34、 TAGs, should be at least 6 000. Maintain the temperature of the column between 30 C and 35 C by means of a control device. If necessary, the column can be stored in toluene (5.2), although experience has shown that it is better to keep the system running with THF. 6.5 Detector, temperature-controll

35、ed refractive index detector. The ideal temperature for the detector is just above that of the column (30 C to 35 C). 6.6 Chromatography data system (CDS), to allow display and accurate quantification of the peak areas. 6.7 Single-use syringes, capacity 1 ml. 1) 1 nm = 10 BS EN ISO 16931:2009ISO 169

36、31:2009(E) ISO 2009 All rights reserved 36.8 Nylon filters, pore size 0,45 m2). 6.9 HPLC syringe, capacity 50 l to 100 l. 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transportation or storage. Sampling is no

37、t part of the method specified in this International Standard. A recommended sampling method is given in ISO 55551. 8 Preparation of test sample The test sample shall be prepared in accordance with ISO 661. 9 Procedure 9.1 Starting up the HPLC equipment It is advisable to follow carefully the manufa

38、cturers recommendations. Switch on the system and pump THF at a volume flow rate of between 0,5 ml/min and 1 ml/min to purge the whole system up to the injection valve. Connect the column to the injection valve and wash it with about 30 ml of THF. Connect the column to the sample cell of the detecto

39、r. Fill the reference cell with THF. Adjust the mobile-phase flow to between 0,5 ml/min and 1,0 ml/min. Wait until a convenient stabilization of the system (no appreciable deviation of the baseline) is obtained. If the column recommended in this subclause is used, an acceptable stabilization of the

40、system should be obtained in about 15 min. With other column packings, the stabilization of the system may be more difficult. For example, changing the mobile phase should be done stepwise from toluene to THF, with different mixtures, each containing 25 % more THF. Acceptable stabilization is normal

41、ly obtained in about 12 h. The following conditions have been found to be suitable: Column: PLgel3)10 nm, 2 300 mm 7,6 mm, 5 m; Eluent: THF; Flow: 0,8 ml/min; Column oven: 35 C; Detector: RI set at 35 C; Injection volume: 20 l. 2) Nalgene 4 mm Syringe Filter is an example of a suitable product avail

42、able commercially. 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. 3) PLgel is an example of a suitable product avail

43、able commercially. 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. BS EN ISO 16931:2009ISO 16931:2009(E) 4 ISO 2009 A

44、ll rights reserved9.2 Preparation of test portion and analysis Add about 100 mg to 300 mg of the test sample (Clause 8) to 10 ml of THF and homogenize. If necessary, filter through a nylon filter (6.8). Inject, with the HPLC syringe (6.9), 20 l to 40 l of that solution. 10 Expression of results 10.1

45、 Qualitative analysis The chromatographic pattern of the determination shows a main peak representative of monomeric TAGs (relative molecular mass about 900) and one or several smaller peaks with shorter retention times representative of PTAGs (dimers and upper oligomers). Under suitable conditions,

46、 TAGs and PTAGs can be separated with good resolution (Figures A.1 and A.2) even at low levels of PTAGs. However, in some cases (which seem to be connected to complex degradation phenomena), the peak pattern preceding the triacylglycerol peaks can be less clear with consequent difficulties for the c

47、alculations (Figure A.2). 10.2 Quantitative analysis Calculation is carried out by the internal normalization method, assuming that all components of the sample which are eluted have the same response coefficient. It is important to have a straight baseline. The mass fraction, in grams per 100 g of

48、the PTAGs, wPTAG, is calculated using the following equation: PTAGPTAGtot100AwA=where APTAGis the sum of the peak areas of all PTAGs; Atotis the sum of the peak areas of all acylglycerols (PTAGs, TAGs, DAGs, and MAGs). Express the results to one decimal place. 11 Precision 11.1 Interlaboratory tests

49、 Details of interlaboratory tests on the precision of the method are summarized in Annex B. The values derived from these interlaboratory tests may not be applicable to concentration ranges and matrices other than those given. 11.2 Repeatability When the values of two independent test results, obtained 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, lie within the range of the mean values given in Table B.1,

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