1、August 2009DEUTSCHE 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!$Y9“1542204ww
2、w.din.deDDIN EN ISO 16931Animal and vegetable fats and oils Determination of polymerized triacylglycerols by high-performancesize-exclusion chromatography (HPSEC) (ISO 16931:2009)English version of DIN EN ISO 16931:2009-08Tierische und pflanzliche Fette und le Bestimmung von polymerisierten Triacylg
3、lycerinen mitHochleistungs-Ausschlusschromatographie (HPSEC) (ISO 16931:2009)Englische Fassung DIN EN ISO 16931:2009-08SupersedesDIN EN ISO 16931:2002-04www.beuth.deDocument comprises 16 pagesDIN EN ISO 16931:2009-08 2 National foreword This standard has been prepared by Technical Committee ISO/TC 3
4、4 “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 and animal fats and oils and their by-products Methods of sampling and analysis” (Secre
5、tariat: 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 Deutsche Gesellschaft fr Fettwissenschaft e. V. (German Fat Research Society) in the J
6、oint Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen 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 572
7、5-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 16931:2002-04 as follows: a) The former two notes have been incorporated in the text of the Scope. b) A note has been added to clause 4 “Principle”. c) In subclause 5.3 extra virgin olive oil instead of sod
8、ium sulfate has been used as standard reference. d) In subclause 6.1 the capacity of the solvent reservoir has been extended (now 500 ml to 1 000 ml). e) A note has been added to subclause 6.4. f) Subclauses 6.7 and 6.9 have been included. g) HPLC conditions have been specified in subclause 9.1 “Sta
9、rting up the HPLC equipment”. h) The substance volumes used have been changed in subclause 9.2 “Preparation of test portion and analysis”. i) Subclause 10.2 “Quantitative analysis” has been revised. j) Different chromatograms have been included in Annex A. k) Results of interlaboratory tests carried
10、 out in 2007 have been included in Annex B. l) The bibliography has been updated. m) The standard has been editorially revised to reflect the current CEN rules for drafting publications. Previous editions DIN EN ISO 16931: 2002-04 DIN EN ISO 16931:2009-08 3 National Annex NA (informative) Bibliograp
11、hy DIN EN ISO 661, Animal and 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 (true
12、ness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN EN ISO 16931:2009-08 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16931 April 200
13、9 ICS 67.200.10 Supersedes EN ISO 16931:2001English Version Animal and vegetable fats and oils - Determination of polymerized triacylglycerols by high-performance size-exclusion chromatography (HPSEC) (ISO 16931:2009) Corps gras dorigines animale et vgtale - Dtermination de la teneur en triacylglycr
14、ols polymriss par chromatographie liquide dexclusion haute performance Tierische und pflanzliche Fette und le - Bestimmung von polymerisierten Triacylglycerinen mit Hochleistungs-Ausschlusschromatographie (HPSEC) (ISO 16931:2009) This European Standard was approved by CEN on 16 March 2009. CEN membe
15、rs 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 national standards may be obtained on applicatio
16、n to the CEN 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 language and notified to the CEN Management Centre has th
17、e same status as the official 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, Portuga
18、l, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means re
19、served worldwide for CEN national Members. Ref. No. EN ISO 16931:2009: E(CLHP dexclusion) (ISO 16931:2009) 2 Contents Page Foreword .3 1 Scope4 2 Normative references4 3 Terms and definitions .4 4 Principle .4 5 Reagents 5 6 Apparatus.5 7 Sampling 6 8 Preparation of test sample .6 9 Procedure.6 9.1
20、Starting up the HPLC equipment .6 9.2 Preparation of test portion and analysis.7 10 Expression of results7 10.1 Qualitative analysis.7 10.2 Quantitative analysis.7 11 Precision 7 11.1 Interlaboratory tests7 11.2 Repeatability 7 11.3 Reproducibility 8 12 Test report8 Annex A (informative) Chromatogra
21、ms 9 Annex B (informative) Results of interlaboratory tests 11 Bibliography12 EN ISO 16931:2009 (E) DIN EN ISO 16931:2009-08 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 3
22、07 “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 the latest by
23、 October 2009, and conflicting 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 p
24、atent rights. This document supersedes 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, F
25、rance, Germany, Greece, Hungary, 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 16
26、931:2009 without any modification. EN ISO 16931:2009 (E) DIN EN ISO 16931:2009-08 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
27、 which contain at least 3 % (from peak areas) of these polymers. PTAGs (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 fr
28、ying fats and fats and oils that have been thermally treated, provided 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 fur
29、ther details, see ISO 64924. 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 document (including any amendments) applie
30、s. 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 polymerized triacylglycerols PTAGs constituents of heated fats and oils that are determined by HPSEC under
31、 the conditions specified in this International Standard NOTE The content 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 wi
32、th tetrahydrofuran (THF) and PTAGs separated by gel permeation chromatography 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. 4 DIN EN ISO 16931:2009-08 EN ISO 16931:2009 (E) 5 Rea
33、gents Use only reagents of recognized analytical grade. 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 butylhydro
34、xytoluene (BHT), degassed, free of water. Ensure that 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 h
35、omogenize the mixture. NOTE Extra virgin olive oil 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 polytetrafluoroethylen
36、e mobile-phase line filter. 6.2 HPLC pump, pulseless, 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
37、 7,8 mm, packed with spheres, diameter 5 m, of high-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
38、 the number, n, of theoretical plates for monomeric 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 r
39、unning with THF. 6.5 Detector, temperature-controlled 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, capa
40、city 1 ml. 1) 1 nm = 10 5 EN ISO 16931:2009 (E) DIN EN ISO 16931:2009-08 6.8 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 transport
41、ation or storage. Sampling is not 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 advisabl
42、e to follow carefully the manufacturers 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 t
43、o the sample cell of the detector. 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
44、acceptable stabilization of the 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. Ac
45、ceptable stabilization is normally 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 exa
46、mple of a suitable product available 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 exa
47、mple of a suitable product available 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. 6 DIN EN ISO 16931
48、:2009-08 EN ISO 16931:2009 (E) 9.2 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
49、 results 10.1 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, 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 pat
