1、October 2016 English price group 11No 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 67.200.10!%n=“2577526www.din.deD
2、IN EN ISO 29841Vegetable fats and oils Determination of the degradation products of chlorophylls a and a (pheophytins a, a and pyropheophytins) (ISO 29841:2009 + Amd 1:2016);English version EN ISO 29841:2014 + A1:2016,English translation of DIN EN ISO 29841:2016-10Pflanzliche Fette und le Bestimmung
3、 thermischer Abbauprodukte des Chlorophyll a und a (Pheophytin a, a und Pyropheophytin) (ISO 29841:2009 + Amd 1:2016);Englische Fassung EN ISO 29841:2014 + A1:2016,Englische bersetzung von DIN EN ISO 29841:2016-10Corps gras dorigine vgtale Dtermination des produits de dcomposition des chlorophylles
4、a et a (phophytines a, a et pyrophophytines) (ISO 29841:2009 + Amd 1:2016);Version anglaise EN ISO 29841:2014 + A1:2016,Traduction anglaise de DIN EN ISO 29841:2016-10SupersedesDIN EN ISO 29841:201407www.beuth.deDocument comprises 19 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the Germ
5、an-language original shall be considered authoritative.10.16 DIN EN ISO 29841:2016-10 2 A comma is used as the decimal marker. National foreword This document (EN ISO 29841:2014 + A1:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboratio
6、n with Technical Committee CEN/TC 307 “Oil seeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte
7、(DIN Standards Committee Food and Agricultural Products) and the Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Joint Working Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. This standard in
8、cludes EN ISO 29841:2014 and Amendment 1 approved by CEN on 15 January 2016. The start and finish of text introduced or altered by amendment is indicated in the text by tags !“. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 661 DIN EN
9、 ISO 661 ISO 5555 DIN EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 29841:2014-07 as follows: a) in Subclause 6.2, the capacity of the taper-shaped flask has been changed from “10 ml or 25 ml” to “25 ml or 50 ml” since a 10 ml flask
10、is not large enough as part of the sample could be lost when the flask is inserted into the rotary evaporator; b) Subclause 6.6 has been replaced since a more detailed explanation of the home-made silica mini-columns has been included in the NOTE to 6.6; c) the heading of Subclause 9.2 has been amen
11、ded to indicate that the relative content of the analytes is to be calculated. Previous editions DIN EN ISO 29841: 2014-07 DIN EN ISO 29841:2016-10 3 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN EN ISO 5555, Animal and
12、 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 measurement methods and results Part 2: Basic method for the determination of repeata
13、bility and reproducibility of a standard measurement method DIN EN ISO 29841:2016-10 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 29841 April 2014 + A1 February 2016 ICS 67.200.10 English Version Vegetable fats and oils Determination of the degradation
14、products of chlorophylls a and a (pheophytins a, a and pyropheophytins) (ISO 29841:2009 + Amd 1:2016) Corps gras dorigine vgtale Dtermination des produits de dcomposition des chlorophylles a et a (phophytines a, a et pyrophophytines) (ISO 29841:2009 + Amd 1:2016) Pflanzliche Fette und le Bestimmung
15、thermischer Abbauprodukte des Chlorophyll a und a (Pheophytin a, a und Pyropheophytin) (ISO 29841:2009 + Amd 1:2016) EN ISO 29841:2014 was approved by CEN on 2014-04-11 and Amendment A1:2016 on 2016-01-15. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the
16、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 application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard e
17、xists 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-CENELEC Management Centre has the same status as the official versions. CEN members are the national
18、standards bodies of 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, Romania, Slovakia,
19、 Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means rese
20、rved worldwide for CEN national Members. Ref. No. EN ISO 29841:2014 + A1:2016 E2 Contents Page Foreword to EN ISO 29841:2014 . 3 !European foreword to EN ISO 29841:2014/A1:2016“ 4 1 Scope 5 2 Normative references 5 3 Terms and definitions . 5 4 Principle . 5 5 Reagents . 5 6 Apparatus . 6 7 Sample .
21、 7 7.1 Sampling . 7 7.2 Preparation of the test sample . 7 8 Procedure. 7 9 Results of the determination 8 9.1 Peak identification 8 9.2 !Calculation of the relative pyropheophytin a content“ 8 10 Precision of the method 8 10.1 Interlaboratory test 8 10.2 Repeatability . 8 10.3 Reproducibility 8 11
22、Test report . 9 (informative) Example of a typical chromatogram 10 Annex A(informative) Results of an interlaboratory test . 12 Annex B(informative) Direct HPLC method with fluorescence detection . 13 Annex CC.1 Principle 13 C.2 Solvents for HPLC . 13 C.3 Apparatus 13 C.4 Procedure 13 Bibliography .
23、 15 EN ISO 29841:2014 + A1:2016 (E) DIN EN ISO 29841:2016-103 Foreword to EN ISO 29841:2014 The text of ISO 29841:2009 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 29841:2014 by Techni
24、cal 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 by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsem
25、ent, 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 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 identify
26、ing any 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav
27、 Republic 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 the United Kingdom. Endorsement notice The text of ISO 29841:2009 has be
28、en approved by CEN as EN ISO 29841:2014 without any modification. EN ISO 29841:2014 + A1:2016 (E) DIN EN ISO 29841:2016-104 !European foreword to EN ISO 29841:2014/A1:2016“ !This document (EN ISO 29841:2014/A1:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration
29、with 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 by AFNOR. This Amendment to the European Standard EN ISO 29841:2014 shall be given the status of a national standard, either by p
30、ublication of an identical text or by endorsement, at the latest by August 2016, and conflicting national standards shall be withdrawn at the latest by August 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELE
31、C 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 to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic
32、, Denmark, Estonia, Finland, Former Yugoslav Republic 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 the United Kingdom. Endorsem
33、ent notice The text of ISO 29841:2009/Amd 1:2016 has been approved by CEN as EN ISO 29841:2014/A1:2016 without any modification.“ EN ISO 29841:2014 + A1:2016 (E) DIN EN ISO 29841:2016-105 1 Scope This International Standard specifies a procedure for the determination of the degradation products pheo
34、phytin a, a and pyropheophytin a of chlorophylls. The method is applicable to vegetable fats and oils only. 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,
35、the latest edition of the referenced document (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 document, the following terms and definitions apply. 3.1 pyropheophytin a (content in chlorophyll
36、degradation products) mass of pyropheophytin a divided by the sum of the masses of pheophytin a, a and pyropheophytin a 4 Principle Separation of the pigments (chlorophylls, pheophytins a and a, pyropheophytin a) using a miniaturized column chromatography on a silica gel column from the major part o
37、f the lipids (triglycerides). The eluate is analysed by RP18-HPLC, and the separated components are monitored at 410 nm using a photometric detector. The analysis of some seed oils (e.g. rapeseed oil) requires a higher sensitivity which can be achieved using fluorescence detection with an excitation
38、 wavelength of 430 nm and an emission wavelength of 670 nm. A direct HPLC determination with fluorescence detection is also possible and described in Annex C. 5 Reagents WARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and pe
39、rsonal safety measures shall be followed. During the analysis, unless otherwise stated, use only reagents of recognized analytical grade. 5.1 Chlorophyll a. NOTE Due to the lack of pheophytin and pyropheophytin reference materials, chlorophyll a is used to produce these degradation products. Pheophy
40、tins are obtained from a chlorophyll a solution by acidification; pyropheophytins by additional heat ( 100 C) (Reference 4). 5.2 Acetone. EN ISO 29841:2014 + A1:2016 (E) DIN EN ISO 29841:2016-106 5.3 Methanol. 5.4 Diethyl ether. 5.5 Petroleum ether, boiling point range 40 C to 60 C. 5.6 n-Heptane or
41、, if not available, n-hexane. CAUTION Owing to its toxicity, avoid using n-hexane if n-heptane is available. 5.7 Petroleum ether and diethyl ether solvent mixture, volume fractions: PetE= 90 ml/100 ml; Et2O= 10 ml/100 ml. 6 Apparatus Usual laboratory equipment, and in particular the following. 6.1 H
42、PLC setup. 6.1.1 HPLC system, consisting of a pump, a sample-injecting device (20 l loop) or auto sampler, either a photometric detector for measurements at 410 nm or a fluorescence detector (excitation wavelength, ex= 430 nm and emission wavelength, em= 670 nm), and an integration system. 6.1.2 HPL
43、C column, 250 mm length, 4,0 mm or 4,6 mm internal diameter, filled with reverse-phase type RP18 particles, size 5 m. The following conditions have been found to be suitable: Stationary phase: Partisil ODS 3, 5 m, 250 mm null 4,6 mm1); Mobile phase: Water, methanol, and acetone (6.1.3); Flow rate: 1
44、,0 ml/min. 6.1.3 Mobile phase, water, methanol, and acetone mixture, volume fractions: H2O= 4 ml/100 ml; MeOH= 36 ml/100 ml; Me2C=O= 60 ml/100 ml. All solvents shall be HPLC grade. 6.2 !deleted“ !Taper-shaped flask, of capacity 25 ml or 50 ml.“ 6.3 Beakers, of different capacities. 6.4 Rotary evapor
45、ator, with water bath. 6.5 Auto sampler vials, of suitable capacity. 6.6 !deleted“ !Silica cartridge, 500 mg/6 ml or 1 000 mg/6 ml, 55 m, 7 nm or diol cartridge, 3 ml. 1) Example, available commercially, of a C18 reverse phase with a 10,5 % mass fraction carbon load, end-capped for deactivation of s
46、ilanols to minimize the need for ion suppression or ion pairing agents. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. EN ISO 29841:2014 + A1:2016 (E) DIN EN ISO 29841:2016-107 NOTE Also, in-house s
47、ilica mini-columns can be used for the separation. For this, use Silica gel 60, for column chromatography (0,063 mm to 0,100 mm), adjusted to a moisture content of w = 5 % (mass fraction). Activate the silica gel by heating overnight at (160 null 5) C. After heating, place the silica gel in a desicc
48、ator for cooling and then transfer the silica gel to a stoppered flask. Add 5 % 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.2)“ 7 Sample 7.1 Sampling Sampling is not part of the
49、 method specified in this 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 the test sample Prepare the test sample in accordance with ISO 661. IMPORTANT Test samples shall be stored cold and protected from light in
