1、Automotive fuels and fat and oil derivates - Determination of steryl glycosides in fatty acid methyl esters (FAME) - Method by GC-MS with prior purification by SPEBS EN 16934:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPI
2、SCHE NORM EN 16934 August 2017 ICS 75.160.40 English Version Automotive fuels and fat and oil derivates - Determination of steryl glycosides in fatty acid methyl esters (FAME) - Method by GC-MS with prior purification by SPE Carburants pour automobiles et produits drivs des corps gras - Dtermination
3、 des strols glucosides dans les esters mthyliques dacides gras (EMAG) - Mthode par GC-MS avec purification pralable par SPE Brennstoffe und Erzeugnisse aus Fetten und len - Bestimmung des Gehaltes an Sterylglycosiden in Fettsure-Methylester (FAME) - Verfahren mittels GC-MS und vorausgehender Reinigu
4、ng mit SPE This European Standard was approved by CEN on 12 June 2017. 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 bibliograph
5、ical 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 responsibilit
6、y 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
7、 Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORM
8、ALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16934:2017 ENational forewordThis British Standard is the UK implementat
9、ion of EN 16934:2017.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.This publication does not purport to include all the necessary
10、 provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 91751 6ICS 75.160.20Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was pu
11、blished under the authority of the Standards Policy and Strategy Committee on 30 September 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16934:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16934 August 2017 ICS 75.160.40 English Version Automot
12、ive fuels and fat and oil derivates - Determination of steryl glycosides in fatty acid methyl esters (FAME) - Method by GC-MS with prior purification by SPE Carburants pour automobiles et produits drivs des corps gras - Dtermination des strols glucosides dans les esters mthyliques dacides gras (EMAG
13、) - Mthode par GC-MS avec purification pralable par SPE Brennstoffe und Erzeugnisse aus Fetten und len - Bestimmung des Gehaltes an Sterylglycosiden in Fettsure-Methylester (FAME) - Verfahren mittels GC-MS und vorausgehender Reinigung mit SPE This European Standard was approved by CEN on 12 June 201
14、7. 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 o
15、n 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-CENE
16、LEC 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, Ital
17、y, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Cent
18、re: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16934:2017 EBS EN 16934:2017EN 16934:2017 (E) 2 Contents PageEuropean foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reag
19、ents and materials . 4 5 Apparatus . 5 6 Sampling . 6 7 Procedure. 6 7.1 Sample purification 6 7.2 Sample preparation and analysis 7 7.3 Identification and integration peaks . 7 8 Calculation of results . 7 9 Expression of results 8 10 Precision . 8 10.1 General 8 10.2 Repeatability, r . 8 10.3 Repr
20、oducibility, R . 8 11 Test report . 8 Annex A (informative) Example chromatograms . 9 Bibliography . 12 BS EN 16934:2017EN 16934:2017 (E) 3 European foreword This document (EN 16934:2017) has been prepared by Technical Committee CEN/TC 19 “Gaseous and liquid fuels, lubricants and related products of
21、 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 February 2018, and conflicting national standards shall be wi
22、thdrawn at the latest by February 2018. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the nation
23、al 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, Lithu
24、ania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16934:2017EN 16934:2017 (E) 4 1 Scope This European Standard describes a procedure for the determination of steryl glycosides (SG) con
25、tent in fatty acid methyl esters (FAME) in a range between 20 mg/kg and 38 mg/kg. NOTE Steryl glycosides (SG) are mainly present in vegetable oils. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all of the safety
26、problems associated with its use. It is the responsibility of users of this standard to take appropriate measures to ensure the safety and health of personnel prior to application of the standard, and fulfil statutory and regulatory requirements for this purpose. 2 Normative references The following
27、 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, Petrole
28、um liquids - Manual sampling (ISO 3170) EN ISO 3171, Petroleum liquids - Automatic pipeline sampling (ISO 3171) 3 Principle The steryl glycosides (SG) content of a FAME sample is determined by gas chromatography with mass spectrometry detector (GC-MS) with cold on column (COC) or programmed temperat
29、ure vaporization (PTV) injection and measuring via selected ion monitoring (SIM). Before injection into the GC instrument the sample is purified via solid phase extraction (SPE) and derivatized with BSA+TMCS (4.2). The SG content is quantified by using cholesteryl-D-glucopyranoside (4.3) as internal
30、 standard (IS). 4 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise specified. 4.1 Pyridine, max. 0,1 % water. 4.2 N,O-bis(trimethylsilyl)acetamide + 5 % trimethylchlorosilane (BSA+TMCS) silylation reagent. 4.3 Cholesteryl-D-glucopyranoside (CAS number: 7073-6
31、1-2)1), which purity has been verified. 4.4 Methyl tert butyl ether (MTBE). 4.5 n-Heptane. 1)Cholesteryl-D-glucopyranoside (10 mg, PR463-XX-S10) provided by ASG Analytik-Service Gesellschaft mbH,Cholesterol -D-glucoside (28609-10MG) provided by Sigma-Aldrich, mixed SG standard, 98 % (soybean), provi
32、ded by Larodan (5 mg, ref. no. 60-1020-4) or Matreya (25 mg, ref. no. 1117) have been found suitable for use. Equivalent products may be used if they can be shown to lead to the same results. This information is given for the convenience of users of this European Standard and does not constitute an
33、endorsement by CEN of this product. BS EN 16934:2017EN 16934:2017 (E) 5 4.6 Acetone. 4.7 Methanol. 4.8 n-Heptane/MTBE 2:1 (V/V) mixture. 4.9 Toluene. 4.10 Internal standard solution of 100 mg/l Cholesteryl-D-glucopyranoside. Dissolve approximately 10 mg of cholesteryl-D-glucopyranoside (4.3) (accura
34、cy 0,1 mg) in a 100 ml calibrated flask with 10 ml of pyridine (4.1). Fill up to the mark using toluene (4.9) as solvent. Calculate the exact concentration in mg/ml (approximately 0,1 mg/ml). Alternatively, a commercially available IS solution may be used. 4.11 Carrier gas, Helium. 5 Apparatus Usual
35、 laboratory equipment and, in particular, the following: 5.1 Gas chromatograph, equipped with a cold on-column (COC) or programmed temperature vaporization (PTV) injector, a temperature programmable oven and a mass spectrometry detector. 5.2 Capillary column, high temperature (HT) capable for which
36、the following characteristics are suggested: 95 % dimethyl-5 % diphenyl polysiloxane stationary phase, length 15 m, internal diameter 250 m, film thickness 0,1 m. 5.3 Operating conditions: Injection Volume: 1 l, column temperature program: 100 C hold for 2 min, programmed at 10 C/min up to 370 C, fi
37、nal temperature hold for 6 min, carrier gas (Helium) constant flow: 1,2 ml/min, temperature MS Source: minimum 300 C, temperature MS Quad: minimum 180 C, solvent delay: 20 min, under above mentioned conditions, SIM Mode: The analysis is run in SIM mode, the following ions are used as sum for both an
38、alytes and IS: 147, 204, 217 m/z, recommended temperature of MS transfer line: minimum 370 C. 5.4 Analytical balance, accuracy 0,1 mg. BS EN 16934:2017EN 16934:2017 (E) 6 5.5 Precision pipette, 200 l capacity. 5.6 Precision pipette, 1 ml capacity. 5.7 SPE cartridge2), Silica gel, with no bonded phas
39、e, 1 g. 5.8 Volumetric flask, 100 ml capacity. 5.9 Graduated cylinder, 10 ml capacity. 5.10 Heating block, capable of keeping and maintaining the temperature of 80 C with an accuracy of 2 C. 5.11 Test tubes, 10 ml capacity, with plastic stoppers or screw caps. 6 Sampling Unless otherwise specified,
40、obtain samples in accordance with the procedures given in EN ISO 3170 or EN ISO 3171. 7 Procedure 7.1 Sample purification 7.1.1 Weigh 5,0 0,1 g of sample into a suitable bottle with accuracy of 0,01 g. 7.1.2 Add 500 l of pyridine (4.1) using a precision pipette (5.6). 7.1.3 Add 1 ml of the internal
41、standard solution (4.10) using a precision pipette (5.6). 7.1.4 Add 5 ml of n-heptane/MTBE (2:1 V/V) mixture (4.8) using a graduated cylinder (5.9). 7.1.5 Close the bottle and thoroughly shake the mixture. All elutions on the SPE cartridge shall be done using gravity elution only, do not apply vacuu
42、m. 7.1.6 Condition the SPE cartridge (5.7) by rinsing with 4 ml of n-heptane (4.5). 7.1.7 Gradually transfer all the sample mixture (7.1.5) onto the SPE cartridge. 7.1.8 When the entire sample has been transferred onto the cartridge rinse it with 5 ml of n-heptane/MTBE (2:1 V/V) mixture. 7.1.9 Disca
43、rd all collected eluent. 7.1.10 Elute the steryl glycosides by rinsing the SPE cartridge with 4 ml of acetone (4.6) followed by 4 ml of methanol (4.7) and collect the eluent in a 10 ml test tube (5.11). 2) SPE-Cartridges brand “Varian HF Mega BE-SI 1GM 6 ml (Bond Elute) order number 14256008 or Phen
44、omenex STRATA-SI (70 A) order number 8B-S012-JCH“ have been found suitable for use. Equivalent products may be used if they can be shown to lead to the same results. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this
45、 product.BS EN 16934:2017EN 16934:2017 (E) 7 7.1.11 Evaporate the eluent by heating to 60 C using the heating block (5.10) and applying a gentle flow of dry nitrogen until completely dry. 7.2 Sample preparation and analysis 7.2.1 Take the purified sample and add approximately 200 l of pyridine (4.1)
46、 and approximately 200 l of silylating reagent mixture (4.2). 7.2.2 Close the test tube, shake thoroughly and let the sample react in the heating block at 80 C, whilst shaking every 15 min. 7.2.3 Take the sample from the heating block and allow the sample to cool to room temperature. 7.2.4 Open the
47、test tube, add 1 ml of heptane, carefully close again and mix thoroughly. 7.2.5 Inject 1 l of this solution into the GC and start the GC and data collection system. 7.3 Identification and integration peaks The identification of IS as well as SG peaks is carried out by comparing the retention times o
48、f known compounds. It is therefore advised, in order to identify the IS and the SG peaks, to inject the pure compound and an isolated sample of SG from commercial FAME. Samples are subject to derivatization in any case. Typical chromatograms with internal standard and steryl glycoside peaks are give
49、n in Annex A. Record the area of the IS peak and the area of the SG mixture peak. 8 Calculation of results The SG content is calculated using a relative response factor of 1,00. Calculate the concentration of steryl glycosides in the sample using the formula: = SG IS ISIS S1 000 A CVSGAm(1) where SGis the concentration of steryl glycosides (mg/kg); ISAis the peak area of internal standard cholesteryl-D-glucopyranoside; SGAis the sum of the peak areas of the individu
