BRITISH STANDARD Petroleum products and fat and oil derivates — Fatty acid methyl esters (FAME) for diesel engines — Determination of polyunsaturated (≥ double bonds) fatty acid methyl esters (PUFA) by gas chromatography ICS 75.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN 15779:2009 +A1:2013 BS 2000-578:2009 BS EN 15779:2009+A1:2013 National foreword This British Standard is the UK implementation of EN 15779:2009 +A1:2013 It supersedes BS EN 15779:2009, which is withdrawn The start and finish of text introduced or altered by amendment is indicated in the text by tags Tags indicating changes to CEN text carry the number of the CEN amendment For example, text altered by CEN amendment A1 is indicated by !" The UK participation in its preparation was entrusted to Technical Committee PTI/13, Petroleum Testing and Terminology A list of organizations represented on this committee can be obtained on request to its secretary Energy Institute, under the brand of IP, publishes and sells all Parts of BS 2000, and all BS EN petroleum test methods that would be Part of BS 2000, both in its annual publication “Standard methods for analysis and testing of petroleum and related products and British Standard 2000 Parts” and individually Further information is available from: Energy Institute, 61 New Cavendish Street, London W1G 7AR Tel: 020 7467 7100 Fax: 020 7255 1472 This publication does not purport to include all the necessary provisions of a contract Users are responsible for its correct application Compliance with a British Standard cannot confer immunity from legal obligations This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 November 2009 © The British Standards Institution 2014 Published by BSI Standards Limited 2014 ISBN 978 580 81876 Amendments/corrigenda issued since publication Date Comments 31 January 2014 Implementation of CEN amendment A1:2013 to BS EN 15779:2009, still dual numbered as BS 2000-578:2009 EUROPEAN STANDARD EN 15779:2009+A1 NORME EUROPÉENNE EUROPÄISCHE NORM September 2013 ICS 75.160.20 English Version Petroleum products and fat and oil derivates - Fatty acid methyl esters (FAME) for diesel engines - Determination of polyunsaturated (≥4 double bonds) fatty acid methyl esters (PUFA) by gas chromatography Produits pétroliers et produits dérivés des corps gras Esters méthyliques d'acides gras (EMAG) pour moteurs diesel (gazole) - Détermination de la teneur en esters méthyliques d'acides gras polyinsaturés (≥ doubles liaisons) (PUFA) par chromatographie en phase gazeuse Mineralölerzeugnisse und Erzeugnisse aus pflanzlichen und tierischen Fetten und Ölen - Fettsäure-Methylester (FAME) für Dieselmotoren - Bestimmung von mehrfach ungesättigten (≥ Doppelbindungen) Fettsäuremethylestern (PUFA) mittels Gaschromatographie This European Standard was approved by CEN on 22 September 2009 and includes Amendment approved by CEN on August 2013 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 obtained on application to the CEN-CENELEC 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-CENELEC Management Centre has the same status as the official versions CEN members are the national 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, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels © 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Ref No EN 15779:2009+A1:2013: E BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Foreword This document (EN 15779:2009+A1:2013) has been prepared by Technical Committee CEN/TC 19 “Gaseous and liquid fuels, lubricants and related products of petroleum, synthetic and biological origin”, the secretariat of which is held by NEN 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 March 2014, and conflicting national standards shall be withdrawn at the latest by March 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 identifying any or all such patent rights This document includes Amendment approved by CEN on August 2013 This document supersedes EN 15779:2009 The start and finish of text introduced or altered by amendment is indicated in the text by tags !" According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: 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, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Contents Page Introduction Scope Normative references Principle Apparatus .5 Reagents and materials 6 Sampling Procedure .7 Calculation Expression of results 10 10.1 10.2 10.3 Precision General Repeatability, r .8 Reproducibility, R 11 Test report Bibliography 10 BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Introduction Polyunsaturated fatty acid methyl esters are considered as a critical component in FAME since they show a low stability against oxidation and polymerization reactions The specification on polyunsaturated ester content is needed in FAME and biodiesel products to limit the content of polyunsaturated FAME with more than three double bonds At the time of the first FAME fuel specifications no test method was available for such a complicated determination in terms of identification and quantification, so technical work has been done in a joint working group with CEN/TC 307 before any standardisation steps could be taken The method has been prepared by the partners of the project "BIOScopes" (Lot 1, Task a) funded by the European Commission, DG TREN, with the purpose to execute a Pan-European round robin test to determine the precision data and the usability of this new and other revised determination methods for FAME BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Scope This European Standard specifies a method for the determination of the polyunsaturated (≥ double bonds) fatty acid (PUFA) methyl esters content of fatty acid methyl ester (FAME) as a whole between 0,6 % (m/m) and 1,5 % (m/m) The method covers the predominant four polyunsaturated fatty acid methyl esters of eicosatetraenoic acid (C 20:4 (n-6)), eicosapentaenoic acid (C 20:5 (n-3)), docosapentaenoic acid (C 22:5 (n-3), and docosahexaenoic acid (C 22:6 (n-3)) Studies have indicated that based on the linearity of results from this European Standard, PUFA methyl esters can be determined in FAME in the range between 0,3 % (m/m) to 3,0 % (m/m) However, the precision was not established in that range, as no samples within the upper ranges where included in the final interlaboratory test (see 10.1) Although the method is applicable to all uses, it is predominantly for FAME for use in diesel engines NOTE For the purposes of this document, the term “% (m/m)” is used to represent the mass fraction of a material NOTE This European Standard is based on A.O.C.S Official Method Ce 1b-89 [1] Normative references !The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application For dated references, only the edition cited applies For undated references, the latest edition of the referenced document (including any amendments) applies." EN ISO 3170, Petroleum liquids — Manual sampling (ISO 3170:2004) EN ISO 3171, Petroleum liquids — Automatic pipeline sampling (ISO 3171:1988) Principle Determination of the percentage of polyunsaturated (≥ double bonds) fatty acid (PUFA) methyl ester present in FAME is done by gas chromatography/FID detection using internal calibration with C 23:0 methyl ester The theoretical detector correction factors relative to C 23:0 internal standard for different poly-unsaturated ester types are applied to the analytical data for optimum accuracy Apparatus 4.1 Capable gas chromatograph, consisting of a capillary injection system (preferable split mode at a split ratio of 1:50), a flame ionization FID detector and the following: 4.1.1 Injector, temperature 220 °C 4.1.2 Detector, temperature 275 °C 4.1.3 Oven temperature profile, initial temperature 150 °C, initial hold time min; program rate 15 °C/min up to 200 °C; °C/min up to 250 °C final temperature 4.1.4 Capillary column, fused silica; 30 m in length, 0,25 µm film thickness and 0,20 mm to 0,32 mm internal diameter The liquid phase shall be bonded Carbowax or an equivalent polyethylene glycol type BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) NOTE 4.1.5 HP–INNOWax, SUPELCOWAX 10, Carbowax, CP-WAX 52CB and DBwax have been found satisfactory Carrier gas, hydrogen or helium of 99,99 % purity or better 4.1.6 Additional gas, depending on instrumentation (compressed air, oxygen), but of high purity An oxygen scrubber is mandatory with Wax-type columns 4.1.7 Data analysis, according to the corresponding instrumentation software 4.2 Screw-cap vials, of 1,5 ml content, with leak-tight (Teflon) lined caps 4.3 Volumetric pipette or syringe, of ml content 4.4 Pasteur type pipettes 4.5 Volumetric flask, of 25 ml content 4.6 Analytical balance, capable to weigh at ± 0,1 mg Reagents and materials 5.1 Tricosanoic acid methyl ester (C 23:0), !minimum purity of 99,0 % (m/m)"; internal standard (IS) solution, to be stored at + °C and to be warmed up to room temperature before use !NOTE 1" If the storage conditions are maintained carefully, the solution has been found to be stable up to one month without any degradation losses !NOTE Standards are available from Nu Chek Prep® (www.nu-chekprep.com) 1)" 5.2 Heptane, analytical grade 5.3 Reference PUFA methyl esters, C 20:4 (n-6) (eicosatetraenoic acid), C 20:5 (n-3) (eicosapentaenoic acid), C 22:5 (n-3) (docosapentaenoic acid, and C 22:6 (n-3) (docosahexaenoic acid), !with a minimum purity of 99,0 % (m/m)" !NOTE Standards are available from Nu Chek Prep® (www.nu-chekprep.com).1)" Sampling Unless otherwise specified in the commodity specification, samples shall be taken as described in EN ISO 3170 or EN ISO 3171 and/or in accordance with the requirements of national standards or regulations for the sampling of the product under test If it is necessary to determine PUFA methyl esters directly out of marine oils, the sample preparation should be in accordance with EN ISO 5509 [2] ! ® 1) Nu Chek Prep (www.nu-chekprep.com) is an example of a suitable product available commercially This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products." BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Procedure 7.1 Weigh approx 25 mg to the nearest 0,1 mg of internal standard (5.1) into a 25 ml volumetric flask (4.5) and fill up to mark with heptane (5.2) 7.2 Weigh approx 100 mg to the nearest 0,1 mg of FAME into a 1,5 ml vial (4.2) 7.3 Pipette ml aliquot of the Internal Standard (5.1) into the vial, cap and mix thoroughly 7.4 Inject µl of solution under appropriate gas chromatographic conditions (see 4.1) Calculation The PUFA content, C, expressed in mass percentage, is calculated according: C × VIS (A ) × CF × 100 C = ∑ PUFAAIS PUFA × IS m (1) where: AIS is the peak area of the internal standard C 23:0; APUFA are the peak areas of the polyunsaturated fatty acid methyl esters (see Figure 1); CIS is the concentration in mg/ml of the internal standard; CFPUFA are the theoretical correction factors of the corresponding PUFA methyl esters (see below); VIS is the volume in ml of the internal standard added; m is the mass in mg of the sample (see 7.2) Identification of the polyunsaturated fatty acid methyl esters has to be done by using the reference PUFA methyl esters (5.3) The following theoretical detector correction factors (CFPUFA) relative to C 23:0 shall be applied to the calculation for optimum accuracy (see also Figure 1): a) 20:4 n-6: 0,99; b) 20:5 n-3: 0,98; c) 22:5 n-3: 0,98; and d) 22:6 n-3: 0,97 BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) ! " Key C 20:4 (n-6) methyl ester C 20:5 (n-3) methyl ester Internal standard C 23:0 methyl ester C 22:5 (n-3) methyl ester C 22:6 (n-3) methyl ester Figure — Reference chromatogram based on salmon oil methyl esters Expression of results Report the PUFA content as mass percentage, in % (m/m), rounded to the nearest 0,01 10 Precision 10.1 General The precision given was derived from statistical analysis by EN ISO 4259 [3] of the results of interlaboratory testing of a matrix of biodiesel fuels including types PUFA methyl esters from fish oil blended in FAME in the range of 0,1 % (m/m) to 1,5 % (m/m) NOTE The interlaboratory testing and the statistical evaluation are detailed in [4] 10.2 Repeatability, r The difference between two test results, obtained by the same operator with the same apparatus under constant operating conditions on identical test material would in the long run, in the normal and correct operation of the test method, exceed the following value in absolute value in only one case in twenty: r = 0,07 % (m/m) BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) 10.3 Reproducibility, R The difference between two single and independent test results, obtained by different operators working in different laboratories on identical test material, would in the long run, in the normal and correct operation of the test method, exceed the following value in only one case in twenty: R = 0,27 % (m/m) 11 Test report The test report shall contain at least the following information: a) reference to this European Standard, i.e EN 15779; b) type and complete identification of the product tested; c) used method of sampling (see Clause 6); d) result of the test (see Clause 9); e) any deviation, by agreement or otherwise, from the procedure specified in this European Standard, or regarded as optional; f) details of any incidents which may have influenced the test results; g) date of the test BS EN 15779:2009+A1:2013 EN 15779:2009+A1:2013 (E) Bibliography [1] American Oil Chemists´ Society Official Method, Fatty Acid Composition by GLC – Marine Oils, Official Methods and Recommended Practices of the American Oil Chemists´ Society, Champaign, 1989, Method Ce 1b-89 [2] EN ISO 5509, Animal and vegetable fats and oils — Preparation of methyl esters of fatty acids (ISO 5509:2000) [3] EN ISO 4259, Petroleum products — Determination and application of precision data in relation to methods of test (ISO 4259:2006) [4] BIOScopes Final Report, available from Ecofys bv Kanaalweg 16-G; P.O Box 8408, Utrecht, NL-3503 RK, The Netherlands, www.ecofys.nl 10 This page deliberately set blank NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW British Standards Institution (BSI) BSI is the national body responsible for preparing British Standards and other standards-related publications, information and services BSI is incorporated by Royal Charter British Standards and other standardization products are published by BSI Standards Limited About us 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