1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 14105:2011Fat and oil derivatives Fatty Acid Methyl Esters (FAME) Determination of free and total glycerol and mono-, di-, triglyceride contentsBS EN 14105:2011 BRITISH STA
2、NDARDNational forewordThis British Standard is the UK implementation of EN 14105:2011. It supersedes BS EN 14105:2003, which is withdrawn.The UK participation in its preparation was entrusted to T e c h n i c a l C o m m i t t e e A W / 3 0 7 , O i l s e e d s , a n i m a l a n d v e g e t a b l e f
3、ats and oils and their by products.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 provisions of a contract. Users are responsible for its correct application. BSI 2011 ISBN 978 0 580 685
4、66 8 ICS 67.200.10 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 31 May 2011.Amendments issued since publicationDate T e x t a f f e c t e dBS EN 14105:2011EUR
5、OPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14105 April 2011 ICS 67.200.10 Supersedes EN 14105:2003English Version Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents Produits drivs des corps gras - Esters m
6、thyliques dacides gras (EMAG) - Dtermination de la teneur en glycrols libre et total et en mono-, di- et triglycrides Erzeugnisse aus pflanzlichen und tierischen Fetten und len - Fettsure-Methylester (FAME) - Bestimmung des Gehaltes an freiem und Gesamtglycerin und Mono-, Di- und Triglyceriden This
7、European Standard was approved by CEN on 10 March 2011. 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
8、 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 memb
9、er 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, France, Germany, Greece, Hungary, Iceland,
10、 Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, 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 Mar
11、nix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14105:2011: EBS EN 14105:2011EN 14105:2011 (E) 2 Contents Page Foreword 31 Scope 42 Principle 43 Reagents .44 Apparatus .55 Preparation of solutions .65.1
12、 1,2,4-Butanetriol stock solution, 1 mg/ml .65.2 Glycerol stock solution, 0,5 mg/ml 65.3 Standard glycerides stock solution, 2,5 mg/ml 65.4 Commercial mixture of monoglycerides .65.5 Calibration solutions .66 Sampling .77 Procedure .77.1 Operating conditions .77.2 Analysis of the calibration solutio
13、ns .77.3 Analysis of the commercial mixture of monoglycerides .77.4 Preparation and analysis of the samples 87.5 Identification .87.6 Calibration 87.7 Column performance control .88 Determination of results .98.1 Integration of the peaks 98.2 Glycerol calibration function 98.3 Free glycerol .98.4 Gl
14、ycerides 108.5 Total glycerol . 109 Expression of results . 1010 Precision 1010.1 Interlaboratory test . 1010.2 Repeatability 1110.3 Reproducibility 1111 Test report . 11Annex A (informative) Sample chromatogram 12Annex B (normative) Calibration function calculation . 16Annex C (informative) Worked
15、example. 18Annex D (informative) Results of the interlaboratory trial . 19Bibliography . 21BS EN 14105:2011EN 14105:2011 (E) 3 Foreword This document (EN 14105:2011) has been prepared by Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods of s
16、ampling 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 endorsement, at the latest by October 2011, and conflicting national standards shall be withdrawn at the latest
17、by October 2011. 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 supersedes EN 14105:2003. The main modifications of the
18、 standard are: - the utilization of representative internal standards for monoglycrides, diglycerides and triglycerides in order to avoid using calibration solutions for these families of compounds; - the introduction of a performance criteria for the gas chromatography column calculated with the re
19、sponse factors for the diglyceride and triglyceride internal standards. The method has been updated to obtain better precision in general, needed for the limits required by European FAME specifications for automotive use 1. This has been done by introducing separate internal standards for mono- (C19
20、), di- (C38) and triglycerides (C57). Next an improvement of the integration has been incorporated and some evaluation of interference with minor components (i.e. dimers) has been done. Via a new Round Robin study, improvement of the precision of free glycerol and diglyceride measurement has been pr
21、oven. The precision statement of the former standard could be confirmed for triglyceride determination, but no improvement was made. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austr
22、ia, Belgium, Bulgaria, Croatia, 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 United Kingdom. BS EN
23、 14105:2011EN 14105:2011 (E) 4 1 Scope The purpose of this European Standard is to determine the free glycerol and residual mono-, di- and triglyceride contents in fatty acid methyl esters (FAME) intended for addition to mineral oils. The total glycerol content is then calculated from the obtained r
24、esults. Under the conditions described, the quantification limits are 0,001 % (m/m) for free glycerol, 0,10 % (m/m) for all glycerides (mono-, di- and tri-). This method is suitable for FAME prepared from rapeseed, sunflower, soybean, palm, animal oils and fats and mixture of them. It is not suitabl
25、e for FAME produced from or containing coconut and palm kernel oils derivatives because of overlapping of different glyceride peaks. NOTE For the purposes of this European Standard, the term “% (m/m)” is used to represent respectively the mass fraction. WARNING The use of this method may involve haz
26、ardous equipment, materials and operations. This method does not purport to address to all of the safety problems associated with its use, but it is the responsibility of the user to search and establish appropriate safety and health practices and determine the applicability of regulatory limitation
27、s prior to use. 2 Principle Transformation of the glycerol and of the mono- and diglycerides into more volatile and stable silyl derivatives in presence of pyridine and of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). Analysis of the sample after silylation, by gas chromatography on a short c
28、apillary column with thin film thickness, with an on-column injector or equivalent device, and flame ionization detection. After a calibration procedure, the quantification of glycerol is carried out in presence of the internal standard 1,2,4-butanetriol. Mono-, di- and triglycerides are directly ev
29、aluated in presence of an internal standard for each glyceride category: glyceryl monononadecanoate (Mono C19) for monoglycerides; glyceryl dinonadecanoate (Di C38) for diglycerides; glyceryl trinonadecanoate (Tri C57) for triglycerides. 3 Reagents Use only reagents of recognized analytical grade, u
30、nless otherwise specified. 3.1 N-methyl-N-trimethysilyltrifluoroacetamide (MSTFA) 3.2 Pyridine, max. 0,1 % water, stored on molecular sieve NOTE Pyridine silyl grade (3.10) can also be used. 3.3 Tetrahydrofurane (THF) 3.4 n-Heptane 3.5 Glycerol BS EN 14105:2011EN 14105:2011 (E) 5 3.6 1,2,4-Butanetri
31、ol 3.7 1 - Glyceryl monononadecanoate (Mono C19)1)3.8 1-3 Glyceryl dinonadecanoate (Di C38)2)3.9 Glyceryl trinonadecanoate (Tri C57)3)3.10 Pyridine, silyl grade 4 Apparatus Usual laboratory apparatus and, in particular, the following. 4.1 Gas chromatograph, equipped with an on-column injector or equ
32、ivalent device, a temperature-programmable oven and a flame ionization detector. 4.2 Capillary column, capable of being programmed up to 400 C (“high temperature“ type) for which the following characteristics are advised: 100 % dimethylpolysiloxane or 95 % dimethyl-5 % diphenylpolysiloxane stationar
33、y phase; length 15 m; internal diameter 0,32 mm; film thickness 0,1 m. 4.3 Volumetric flask, 50 ml capacity 4.4 Volumetric flasks, 20 ml capacity 4.5 Volumetric flasks, 10 ml capacity 4.6 Screw-cap vials with PTFE-faced septa, 10 ml capacity 4.7 Precision pipette, 1 ml capacity 4.8 Microsyringe, 100
34、 l capacity 4.9 Microsyringe, 500 l capacity 4.10 Microsyringe, 5 l or 10 l capacity specially designed for on-column operation 4.11 Graduated cylinder, 10 ml capacity 4.12 Analytical balance, with an accuracy of 0,1 mg 1)Monononadecaoin available from Larodan, ref. 31-1900-11 (www.larodan.se) 2)1,3
35、-dinonadecaoin available from Larodan, ref. 32-1903-8 (www.larodan.se) 3)Trinonadecaoin available from Larodan, ref. 33-1900-13 (www.larodan.se), or from Sigma , ref. T4632-1G () BS EN 14105:2011EN 14105:2011 (E) 6 4.13 Carrier gas, hydrogen or helium 4.14 Auxiliary gases, such as air, hydrogen and
36、nitrogen 5 Preparation of solutions 5.1 1,2,4-Butanetriol stock solution, 1 mg/ml Accurately weigh approximately 50 mg (accuracy 0,1 mg) of 1,2,4-butanetriol (3.6) in a 50 ml volumetric flask (4.3) and make up to the mark with pyridine (3.2). 5.2 Glycerol stock solution, 0,5 mg/ml Accurately weigh a
37、pproximately 50 mg (accuracy 0,1 mg) of glycerol (3.5) in a 10 ml volumetric flask (4.5) and make up to the mark with pyridine (3.2). Using a pipette (4.7), transfer 1 ml of this solution into a 10 ml volumetric flask (4.5) and make up to the mark with pyridine (3.2). 5.3 Standard glycerides stock s
38、olution, 2,5 mg/ml For each reference glyceride, monononadecanoate (3.7), dinonadecanoate (3.8) and trinonadecanoate (3.9), accurately weigh approximately 50 mg (accuracy 0,1 mg) in a unique 20 ml volumetric flask (4.4) and make up to the mark with tetrahydrofurane (3.3). The solution shall be perfe
39、ctly limpid at ambient temperature. After storage in refrigerator at 4 C the solution might show a precipitate that must re-dissolve spontaneously when restored at ambient temperature, without any external heating. NOTE If stored at 4 C the solution is stable for almost 3 months. 5.4 Commercial mixt
40、ure of monoglycerides Made up of mono-palmitoylglycerol (monopalmitin), mono-stearoylglycerol (monostearin) and of mono-oleoylglycerol (monoolein), present in quantities having an identical mass. Prepare a stock solution of this mixture by weighing approximately 100 mg in a 10 ml volumetric flask (4
41、.5) and make up to the mark with pyridine (3.2). This solution may be used to locate the relevant peaks in GC paths. 5.5 Calibration solutions Prepare four calibration solutions by transferring into a series of vials (4.6) the volumes of stock solutions of glycerol (5.2) and of 1,2,4-butanetriol (5.
42、1) given in Table 1, using the 100 l microsyringes (4.8). Do not use syringe at maximum capacity, but dispense the half volume twice (i.e.: in case of 100 I dosing using a 100 I syringe, load 50 I twice). Make sure that needle and body of the syringe are free from air bubbles, and measure volumes on
43、ly by difference (i.e.: when dispensing 80 I, fill syringe up to 100 I and supply solution up to the 20 I mark). BS EN 14105:2011EN 14105:2011 (E) 7 Table 1 Preparation of calibration solutions Stock solution 1 2 3 4 Syringe l l l l l glycerol solution (5.2) 10 40 70 100 100 internal butanetriol sol
44、. (5.1) 80 80 80 80 100 6 Sampling Samples shall be taken in accordance with the requirements of national standards or regulations for the sampling of the product under test. A recommended sampling method is given in EN ISO 5555 2 or EN ISO 3170 3. 7 Procedure 7.1 Operating conditions The chromatogr
45、aphic analysis conditions shall be chosen taking into account the characteristics of the column being used and the type of carrier gas (hydrogen or helium). It is however recommended to observe an analysis time of about 30 min to 35 min to ensure triglycerides elution. EXAMPLE By way of indication,
46、an example of analysis conditions is described below: column temperature: 50 C hold for 1 min, programmed at 15 C/min up to 180 C, programmed at 7 C/min up to 230 C, programmed at 10 C/min up to 370 C, final temperature hold for 15 min; detector temperature: 380 C; carrier gas pressure (hydrogen): 8
47、0 kPa; volume injected: 1 l. 7.2 Analysis of the calibration solutions Using a microsyringe (4.10), add 150 l of MSTFA (3.1) to each of the four calibration solutions (5.5), close hermetically the vials and shake vigorously. Store 15 min at room temperature, then add 8 ml of n-heptane (3.4) using a
48、graduated cylinder (4.11). Analyse 1 l of each reaction mixture by gas chromatography under the conditions defined under 7.1, using only the first part of temperature programme, stopping the analysis when the temperature of 230 C has been reached. Each reaction mixture gives rise to two chromatograp
49、hic analyses. Samples are stable for some hours after derivatisation. NOTE The silylated standard solutions are only stable one day. 7.3 Analysis of the commercial mixture of monoglycerides Using microsyringes (4.10), transfer 200 l of commercial mixture of monoglycerides dissolved in pyridine (3.10) and 150 l of MSTFA (3.1) into a 10 ml vial (4.6). Avoid contact with humidity. Hermetically close the vial and sh
