1、April 2010 Translation by DIN-Sprachendienst.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
2、67.200.10!$a=“1625826www.din.deDDIN EN ISO 11701Vegetable fats and oils Determination of phospholipids content in lecithins by HPLC using alight-scattering detector (ISO 11701:2009)English translation of DIN EN ISO 11701:2010-04Pflanzliche Fette und le Bestimmung des Gehaltes an Phospholipiden in Le
3、cithinen durch HPLC mittels einesLichtstreudetektors (ISO 11701:2009)Englische bersetzung von DIN EN ISO 11701:2010-04Corps gras dorigine vgtale Dtermination de la teneur en phospholipides dans les lcithines par CLHP avecdtecteur diffusion de la lumire (ISO 11701:2009)Traduction anglaise de DIN EN I
4、SO 11701:2010-04www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 16 pages04.10 DIN EN ISO 11701:2010-04 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food pro
5、ducts” (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” (Secretariat: AFN
6、OR, 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 (German Fat Research Society), Joint Committee Gemeinsc
7、haftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standards corresponding to the International Standards referred to in the EN are as follows: ISO 661 DIN EN ISO 661 ISO 1042 DIN EN ISO 1042 ISO 5555 DIN EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 IS
8、O 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy
9、(trueness 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 1042, Laboratory glassware One-mark volumetric flasks DIN EN ISO 5555, Animal and vegetable fats and oils Sampling E
10、UROPEAN 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 - Dtermination de la teneur en
11、 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 by CEN on 18 November
12、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 standards may be obtaine
13、d 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 notified to the CEN Managemen
14、t 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, Malta, Netherlands, Norway,
15、 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 exploitation in any form and
16、 by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11701:2009: EContents DIN EN ISO 11701:2010-04 EN ISO 11701:2009 (E) 2 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
17、sample .6 9 Procedure.6 10 Calculation and expression of results 8 11 Precision of the method8 12 Test report9 Annex A (informative) HPLC chromatogram10 Annex B (informative) Results of an interlaboratory test .11 Bibliography14 Foreword in collaboration with Technical Committee CEN/TC 307 “Oilseeds
18、, vegetable and animal fats and oils and their 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 June 2010, and conflicting national standards shall be withdrawn at the latest by June 2010. Attentio
19、n 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. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the
20、 following countries are bound to implement this European Standard: 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
21、, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 11701:2009 has been approved by CEN as a EN ISO 11701:2009 without any modification. DIN EN ISO 11701:2010-04 EN ISO 11701:2009 (E) 3 This document (EN ISO 11701:2009) has been prepared by Technical Com
22、mittee ISO/TC 34 “Food products” by-products - Methods of sampling and analysis” the secretariat of which is held by AFNOR. 1 Scope This International Standard specifies a method for the quantitative determination of phospholipids content by high performance liquid chromatography (HPLC) using a diol
23、 column and a 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 separa
24、tion of lysophosphatidylethanolamine (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 unda
25、ted references, 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
26、phospholipid mass 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 Sta
27、ndard NOTE The 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. DIN E
28、N ISO 11701:2010-04 EN ISO 11701:2009 (E) 4 5 Reagents WARNING Comply with any local regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed. During the analysis, unless otherwise stated, use only reagents of recognized
29、 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 ml n-hexane (5.2) and 20 ml 2-propanol (5.3) (volume fraction = 80 ml/100 ml for n-hexane and = 20 ml/1
30、00 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 mixture with defined contents of N-acyl-PE, PA, PE, PC, PI, and LPC. 5.8 Mobile phase for the HPLC. 5.8.1 E
31、luent 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 ml for 2-propanol, = 1,50 ml/100 ml for acetic acid, and = 0,08 ml/100 ml for triethylamine). In order
32、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-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
33、-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 ml for acetic acid and = 0,08 ml/100 ml for triethylamine). In order to obtain a reproducible eluent com
34、position, 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 1,45 g (2,0 ml) of triethylamine. 6 Apparatus 6.1 Analytical balance, capable of being read to the nearest
35、 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) ILPS-LE01 is the trade name of a product supplied by the International Lecithin and Phospholipid Society.
36、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 can be shown to lead to comparable results. DIN EN ISO 11701:2010-04 EN ISO 11701:2009 (E) 5 6.5 HPLC colu
37、mn (250 mm 4,0 mm) with pre-column (20 mm 4,0 mm) packed with spherical microparticles (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 volum
38、etric flasks, of capacities 50 ml, 100 ml and 2 500 ml, ISO 10421class A. 6.7 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
39、have been sent to the laboratory. It should not have been 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 55552. 8 Preparation of test sample See ISO 661. The sample is heated t
40、o a maximum of 60 C to soften it (overheating shall be avoided) and then homogenized 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 pur
41、pose, accurately weigh out in three different 100 ml one-mark volumetric flasks 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 befor
42、e injection into the HPLC. 9.1.2 Test sample solutions and test portions Weigh, 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 mixtu
43、re (5.6) and make up to the mark with the same solvent. Filter (6.8) the test 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 Stan
44、dard and does not constitute an endorsement by ISO of the product named. Equivalent 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 an
45、d does not constitute an endorsement by ISO of this product. DIN EN ISO 11701:2010-04 EN ISO 11701:2009 (E) 6 9.2 HPLC analysis Adjust the working conditions in the equipment using the test samples and reference samples in order to get a separation according to the chromatogram in Figure A.1. Optimi
46、ze 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
47、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 1,0 17,5 0 100 1,0 17,6 95 5 1,0 21,0 95 5 1,0 22,0 95 5 2,0 27,0 95 5 2,0 29,0 95 5 1,0 9.3 Calibration Use 20 l injection volumes for calcul
48、ation of the linear regression lines and for determination of the test sample. Plot peak area against concentration to obtain a calibration curve. NOTE Light-scattering detectors are not linear over the whole range (S-shaped calibration curves). The concentrations of the standard reference solutions
49、 have been chosen to be in the linear range. The following analysis sequence is recommended for the quantitative determination of phospholipids: R1, R2, R3(one injection each), S1a, S1b(test portions drawn from the test sample, two injections each), R1, R2, R3(one injection each). 9.4 Determination Inject 20 l of the test sample solution into the HPLC and register the peak areas. Identify the peaks by comparing the retention times of the substance in the chromatograms of the standar
