1、BS EN ISO11701:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDVegetable fats andoils Determinationof phospholipidscontent in lecithins byHPLC using a light-scattering detector(ISO 11701:2009)Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS
2、, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIThis British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 January2010 BSI 2010ISBN 978 0 580 65445 9Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 11701:2009National forewordThis Britis
3、h Standard is the UK implementation of EN ISO 11701:2009.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 secre
4、tary.This publication does not 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.Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Unco
5、ntrolled Copy, (c) BSIEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11701 December 2009 ICS 67.200.10 English Version Vegetable fats and oils - Determination of phospholipids content in lecithins by HPLC using a light-scattering detector (ISO 11701:2009) Corps gras dorigine vgtale - Dterm
6、ination de la teneur en phospholipides dans les lcithines par CLHP avec dtecteur diffusion de la lumire (ISO 11701:2009) Pfanzliche Fette und le - Bestimmung des Gehaltes an Phospholipiden in Lecithinen durch HPLC mittels eines Lichtstreudetektors (ISO 11701:2009) This European Standard was approved
7、 by CEN on 18 November 2009. 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 bibliographical references concerning such national s
8、tandards may be obtained on application 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 notifi
9、ed to the CEN Management Centre has the 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, Malt
10、a, Netherlands, Norway, Poland, Portugal, 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 explo
11、itation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11701:2009: ELicensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009EN ISO 11701:2009 (E) 3 Foreword This document (EN ISO 11701:2009) has
12、 been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration 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 European Standard shall be give
13、n the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 2010, and conflicting national standards shall be withdrawn at the latest by June 2010. Attention is drawn to the possibility that some of the elements of this document may be the
14、 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 organizations of the following countries are bound to implement this European Standard: Austria, Belgiu
15、m, 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 and the United Kingdom. Endorsement notice T
16、he text of ISO 11701:2009 has been approved by CEN as a EN ISO 11701:2009 without any modification. Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009ISO 11701:2009(E) ISO 2009 All rights reserved iiiContents Page Foreword iv 1 Sco
17、pe1 2 Normative references1 3 Terms and definitions .1 4 Principle .1 5 Reagents 2 6 Apparatus.2 7 Sampling 3 8 Preparation of test sample .3 9 Procedure.3 10 Calculation and expression of results 5 11 Precision of the method5 12 Test report6 Annex A (informative) HPLC chromatogram7 Annex B (informa
18、tive) Results of an interlaboratory test .8 Bibliography11 Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009ISO 11701:2009(E) iv ISO 2009 All rights reservedForeword ISO (the International Organization for Standardization) is a wo
19、rldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented o
20、n that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted
21、 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 International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standa
22、rd 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 document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11701 was prepared by
23、Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009INTERNATIONAL STANDARD ISO 11701:2009(E) ISO 2009 All rights reserved 1Vegetabl
24、e fats and oils Determination of phospholipids content in lecithins by HPLC using a light-scattering detector 1 Scope This International Standard specifies a method for the quantitative determination of phospholipids content by high performance liquid chromatography (HPLC) using a diol column and a
25、light-scattering detector. The method is applicable to crude, oil-containing lecithins, and to oil-free lecithins and lecithin fractions from vegetable fats and oils. The method is not applicable to animal and ruminant lecithins and enzymatically hydrolysed lecithins as the peak separation of lysoph
26、osphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidic acid (LPA) is insufficient. 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
27、, 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 content of an individual phospholipid m
28、ass fraction of N-acyl-phosphatidylethanolamine (N-acyl-PE) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or phosphatidylinositol (PI) or phosphatidic acid (PA) or lysophosphatidylcholine (LPC), determined in accordance with the method specified in this International Standard NOTE The
29、 content is expressed in grams per 100 g, numerically equal to a percentage mass fraction. 4 Principle The individual phospholipids are separated by HPLC using a diol column and a light-scattering detector. For the purpose of quantification, a certified reference mixture is used. Licensed Copy: Wang
30、 Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009ISO 11701:2009(E) 2 ISO 2009 All rights reserved5 Reagents WARNING Comply with any local regulations which specify the handling of hazardous substances. Technical, organizational and personal safety m
31、easures shall be followed. During the analysis, unless otherwise stated, use only reagents of recognized analytical grade. 5.1 Water, HPLC grade. 5.2 n-Hexane, HPLC grade. 5.3 2-Propanol, HPLC grade. 5.4 Acetic acid, wmin, 99,8 % mass fraction. 5.5 Triethylamine. 5.6 Solvent mixture: A mixture of 80
32、 ml n-hexane (5.2) and 20 ml 2-propanol (5.3) (volume fraction = 80 ml/100 ml for n-hexane and = 20 ml/100 ml for 2-propanol) is used to dissolve the standards and sample. 5.7 Reference substance (external standard) ILPS-LE011), mixed soy phospholipid reference standard, is a certified reference mix
33、ture with defined contents of N-acyl-PE, PA, PE, PC, PI, and LPC. 5.8 Mobile phase for the HPLC. 5.8.1 Eluent A. Mix 814,2 ml of n-hexane (5.2), 170,0 ml of 2-propanol (5.3), 15 ml of acetic acid (5.4), and 0,8 ml of triethylamine (5.5) (volume fraction = 81,42 ml/100 ml for n-hexane, = 17,00 ml/100
34、 ml for 2-propanol, = 1,50 ml/100 ml for acetic acid, and = 0,08 ml/100 ml for triethylamine). In order to obtain a reproducible eluent composition, it is recommended that the solvents be weighed out taking into account their densities. For a batch size of 2,5 l: 1 341,4 g of n-hexane, 331,5 g of 2-
35、propanol, 39,4 g of acetic acid, and 1,45 g (2,0 ml) of triethylamine. 5.8.2 Eluent B. Mix 844,2 ml of 2-propanol (5.3), 140 ml of water (5.1), 15,0 ml of acetic acid (5.4) and 0,8 ml of triethylamine (5.5) (volume fraction = 84,42 ml/100ml for 2-propanol, = 14,00 ml/100 ml for water, = 1,50 ml/100
36、ml for acetic acid and = 0,08 ml/100 ml for triethylamine). In order to obtain a reproducible eluent composition, it is recommended that the solvents be weighed out taking into account their densities. For a batch size of 2,5 l: 1 646,2 g of 2-propanol, 350,0 g of water, 39,4 g of acetic acid and 1,
37、45 g (2,0 ml) of triethylamine. 6 Apparatus 6.1 Analytical balance, capable of being read to the nearest 0,000 1 g. 6.2 HPLC basic equipment with gradient system and light-scattering detector. 6.3 HPLC column oven, adjustable to 55 C. 6.4 Degasser or similar equipment for degassing the eluent. 1) IL
38、PS-LE01 is the trade name of a product supplied by the International Lecithin and Phospholipid Society. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they ca
39、n be shown to lead to comparable results. Licensed Copy: Wang Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009ISO 11701:2009(E) ISO 2009 All rights reserved 36.5 HPLC column (250 mm 4,0 mm) with pre-column (20 mm 4,0 mm) packed with spherical microp
40、articles (5 m) of diol-bounded silica, e.g. LiChrospher 100 diol (5 m)2). The age and history of the column, the packaging of the column filling material and the temperature may influence the separation. 6.6 One-mark volumetric flasks, of capacities 50 ml, 100 ml and 2 500 ml, ISO 10421class A. 6.7
41、Microlitre syringe, of capacity 25 l, graduated in microlitres. 6.8 Filter for filtering the external standard and test sample solutions, e.g. Millex HV3). 6.9 Integration system. 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed d
42、uring transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 55552. 8 Preparation of test sample See ISO 661. The sample is heated to a maximum of 60 C to soften it (overheating shall be avoided) and then homog
43、enized by vigorous stirring. 9 Procedure 9.1 Preparation of standard reference solutions and test portions 9.1.1 Standard reference solutions R1, R2, and R3Prepare three different standard reference solutions. For this purpose, accurately weigh out in three different 100 ml one-mark volumetric flask
44、s approximately 550 mg, 850 mg and 1 150 mg of the certified reference mixture (5.7), dissolve in the solvent mixture (5.6) and make up to the mark with the same solvent. Filter (6.8) the standard reference solutions before injection into the HPLC. 9.1.2 Test sample solutions and test portions Weigh
45、, to the nearest 0,001 g, 425 mg of the sample in the case of crude lecithin or 255 mg of the sample in the case of deoiled or fractionated lecithin into separate 50 ml one-mark volumetric flasks, dissolve in solvent mixture (5.6) and make up to the mark with the same solvent. Filter (6.8) the test
46、sample solutions before drawing test portions S1aand S1bfrom them. 2) LiChrospher 100 diol is the trade name of a product supplied by Merck. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the product named. Equiv
47、alent products may be used if they can be shown to lead to the same results. 3) Example 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. Licensed Copy: Wa
48、ng Bin, ISO/EXCHANGE CHINA STANDARDS, 01/04/2010 02:08, Uncontrolled Copy, (c) BSIBS EN ISO 11701:2009ISO 11701:2009(E) 4 ISO 2009 All rights reserved9.2 HPLC analysis Adjust the working conditions in the equipment using the test samples and reference samples in order to get a separation according t
49、o the chromatogram in Figure A.1. Optimize the separation profile, depending on the type of column and gradient. The following conditions are recommended (see Table 1): Temperature of the oven: 55 C Sensitivity of the detector: 5 to 6 Temperature of the detector: 50 C Pressure of the detector: 0,20 MPa (2,0 bar) Flow: 1,0 ml/min Flow for column rinsing: 2,0 ml/min Table 1 Gradient programme for HPLC Time min Eluent A % Eluent B % Flow ml/min 0,0 95 5 1,0 5,0 80 20 1,0 8,5 60 40 1,0 15,0 0 100 1
