SIST EN 14105:2003

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.HWRGDErzeugnisse aus pflanzlichen und tierischen Fetten und Ölen - Fettsäure-Methylester (FAME) - Bestimmung des Gehaltes an freiem und Gesamtglycerin und Mono-, Di- und Triglyceriden (Referenzmethode)Produits dérivés des corps gras - Esters méthyliques d'acides gras (EMAG) - Détermination de la teneur en glycérols libre et total et en mono-, di- et triglycérides - Méthode de référenceFat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents (Reference method)67.200.10Animal and vegetable fats and oilsICS:Ta slovenski standard je istoveten z:EN 14105:2003SIST EN 14105:2003en01-november-2003SIST EN 14105:2003SLOVENSKI
STANDARD



SIST EN 14105:2003



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14105April 2003ICS 67.200.10English versionFat and oil derivatives - Fatty Acid Methyl Esters (FAME) -Determination of free and total glycerol and mono-, di-,triglyceride contents (Reference method)Produits dérivés des corps gras - Esters méthyliquesd'acides gras (EMAG) - Détermination de la teneur englycérols libre et total et en mono-, di- et triglycérides -Méthode de référenceErzeugnisse aus pflanzlichen und tierischen Fetten undÖlen - Fettsäure-Methylester (FAME) - Bestimmung desGehaltes an freiem und Gesamtglycerin und Mono-, Di- undTriglyceriden (Referenzmethode)This European Standard was approved by CEN on 2 January 2003.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the 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 translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14105:2003 ESIST EN 14105:2003



EN 14105:2003 (E)2ForewordThis document (EN 14105:2003) has been prepared by Technical Committee CEN /TC 307 "Oilseeds, vegetableand animal fats and oils and their by-products - Methods of sampling and analysis", the secretariat of which is heldby AFNOR.This European Standard
shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by October 2003, and conflicting national standards shall be withdrawn at the latestby October 2003.This document has been prepared under Mandate M/245 on Fatty Acid Methylester (FAME) given to CEN by theEuropean Commission and the European Free Trade Association.Annexes A to D are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,Slovakia, Spain, Sweden, Switzerland and the United Kingdom.SIST EN 14105:2003



EN 14105:2003 (E)31 ScopeThis European Standard specifies a method to determine the free glycerol and residual mono-, di- and triglyceridecontents in fatty acid methyl esters (FAME) intended for addition to mineral oils. The total glycerol content is thencalculated from the results obtained.This method is suitable for FAME from rapeseed, sunflower, soybean oils but is not suitable for FAME producedfrom or containing coconut and palm kernel oils because of overlapping of peaks.WARNING — The use of this method may involve hazardous equipment, materials and operations. Thismethod does not purport to address to all of the safety problems associated with its use, but it is theresponsibility of the user to search and establish appropriate safety and health practices and determinethe applicability of regulatory limitations prior to use.2 PrincipleTransformation of the glycerol and of the mono- and diglycerides into more volatile silylated derivatives in presenceof pyridine and of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA).Analysis of the silylated derivatives by gas chromatography on a short capillary column with thin film thickness, withan on-column injector or equivalent device, and flame ionization detection.After a calibration procedure, the quantification is carried out in the presence of two internal standards:¾ 1,2,4-butanetriol intended for the determination of the free glycerol;¾ 1,2,3-tricaproylglycerol (tricaprin) intended for the determination of the glycerides (mono-, di- and tri-).3 ReagentsUse only reagents of recognized analytical grade, unless otherwise specified.3.1N-methyl-N-trimethysilyltrifluoroacetamide (MSTFA).3.2Pyridine, stored on molecular sieve.3.3n-Heptane.3.41,2,4-Butanetriol, (internal standard No.1).3.51,2,3-Tricaproylglycerol (tricaprin), (internal standard No.2).3.6Reference substances : glycerol, 1-monooleoylglycerol (monoolein), 1,3-dioleoylglycerol (diolein),1,2,3-trioleoylglycerol (triolein), pure - GLC standard grade.3.7Internal standard No. 1 stock solution, 1 mg/ml.Accurately weigh approximately 50 mg (to the nearest 0,1 mg) of 1,2,4-butanetriol (3.4) in a 50 ml volumetric flask(4.4) and make up to the mark with pyridine (3.2).3.8Internal standard No. 2 stock solution, 8 mg/ml.Accurately weigh approximatively 80 mg (to the nearest 0,1 mg) of 1,2,3-tricaproylglycerol (3.5) in a 10 mlvolumetric flask (4.5) and make up to the mark with pyridine (3.2).SIST EN 14105:2003



EN 14105:2003 (E)43.9Glycerol stock solution, 0,5 mg/ml.Accurately weigh approximately 50 mg (to the nearest 0,1 mg) of glycerol (3.6) in a 10 ml volumetric flask (4.5) andmake up to the mark with pyridine (3.2). Using a pipette (4.7) transfer 1 ml of this solution into a 10 ml volumetricflask (4.5) and make up to the mark with pyridine (3.2).3.10Glyceride stock solution, 5 mg/ml.For each reference glyceride, mono-, di- and triolein (3.6), accurately weigh approximately 50 mg
(to the nearest0,1 mg) in a 10 ml volumetric flask (4.5) and make up to the mark with pyridine (3.2).3.11Monoglycerides1), commercial mixture.Made up of monopalmitoylglycerol (monopalmitin), monostearoylglycerol (monostearin) and of monooleoylglycerol(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 (4.5) andmake up to the mark with pyridine (3.2).3.12Calibration solutionsPrepare daily four calibration solutions by transferring into a series of vials (4.6) the volumes of stock solutions ofreference substances (3.9 and 3.10) and of internal standards (3.7 and 3.8) given in Table 1, using microsyringes(4.8 and 4.9). The choice of the appropriate syringe shall be done according to Table 1. Do not use syringe atmaximum capacity, but dispense the half volume twice (i. e.: in case of 100 ml dosing using a 100 ml syringe, load50 ml twice). Be sure that needle and body of syringe are free from air bubbles, and measure volumes only bydifference (i. e.: when dispensing 80 ml, fill syringe up to 100 ml and supply solution up to the 20 ml mark).NOTEThe silylated standard solutions are only stable one day.Table 1 — Preparation of calibration solutionsCalibration solution1234Syringe, mlml of glycerol solution104070100100ml of monoolein solution50120190250500ml of diolein solution104070100100ml of triolein solution10306080100ml of internal std sol. No. 180808080100ml of internal std sol. No. 21001001001005003.13Carrier gas, hydrogen or helium.3.14Auxiliary gases:¾ air;¾ hydrogen.
1)Products available commercially from SIGMA, reference 178-8 (for example). This information is given for the convenience of users of thisEuropean Standard and does not constitute an endorsement by CEN of these products.SIST EN 14105:2003



EN 14105:2003 (E)54 ApparatusUsual laboratory apparatus and, in particular, the following.4.1Gas chromatograph, equipped with an on-column injector or equivalent device, a temperature-programmable oven and a flame ionization detector.4.2Capillary column, capable of being programmed up to 400 °C ("high temperature" type) for which thefollowing characteristics are advised:¾ 100 % dimethylpolysiloxane or 95 % dimethyl-5 % diphenyl polysiloxane stationary phase;¾ length 10 m;¾ internal diameter 0,32 mm;¾ film thickness 0, 1 mm.4.3Operating conditionsThe chromatographic analysis conditions will be chosen taking into account the characteristics of the column beingused and the type of carrier gas (hydrogen or helium). It is however recommended to observe an analysis time ofabout 30 min to ensure triglycerides elution.By way of indication, an example of analysis conditions is described below:¾ column temperature:50 °C hold for 1 min, programmed at 15 °C/min up to 180 °C, programmedat 7 °C/min up to 230 °C, programmed at 10 °C/min up to 370 °C, finaltemperature hold for 5 min;¾ detector temperature:380 °C;¾ carrier gas pressure (hydrogen):80 kPa;¾ volume injected:1 ml.4.4Volumetric flask, 50 ml capacity.4.5Volumetric flasks, 10 ml capacity.4.6Screw-cap vials with PTFE-faced septa, 10 ml capacity.4.7Precision pipette, 1 ml capacity.4.8Microsyringe, 100 ml capacity.4.9Microsyringe, 500 ml capacity.4.10Microsyringe, 1 ml capacity specially designed for on-column operation.4.11Graduated cylinder, 10 ml capacity.4.12Analytical balance, with an accuracy of ± 0,1 mg.SIST EN 14105:2003



EN 14105:2003 (E)65 Procedure5.1 Preparation and analysis of the calibration solutionsUsing a microsyringe (4.8), add 100 ml of MSTFA (3.1) to each of the four calibration solutions (3.12), closehermetically the vials and shake vigorously and avoid contact with moisture. Store 15 min at room temperature,then add 8 ml of heptane (3.3) using a graduated cylinder (4.11).Analyse 1 ml of each reaction mixture by gas chromatography under the conditions defined above (4.3). Eachreaction mixture gives rise to two chromatographic analysis. Samples are stable for some hours after derivatisation.5.2 Preparation and analysis of the commercial mixture of monoglyceridesUsing microsyringes (4.8 and 4.9), transfer 200 ml of commercial mixture of monoglyceride dissolved in pyridine(3.11) and 100 ml of MSTFA (3.1) into a 10 ml vial (4.6). Avoid contact with moisture. Close hermetically the vialand shake vigorously. Store 15 min at room temperature, then add 8 ml of heptane (3.3). Analyse 1 ml of thereaction mixture by gas chromatography according to the conditions described above (4.3).5.3 SamplingSampling is not part of the method specified in this European Standard. A recommended sampling method is givenin EN ISO 5555 [1].5.4 Preparation and analysis of the samplesAccurately weigh approximately 100 mg of homogenized sample in a 10 ml vial (4.6). Using a syringe (4.8 and 4.9),add 80 ml of internal standard N° 1 stock solution (3.7), 100 ml of internal standard No. 2 stock solution (3.8) and100 ml of MSTFA (3.1). Avoid contact with moisture. Close hermetically the vial and shake vigorously. Store 15 minat room temperature, then add 8 ml of heptane (3.3). Analyse 1 µl of the reaction mixture by gas chromatographyaccording to the conditions described in (4.3).For each sample, two test portions are submitted to the derivatisation reaction and give rise, each one, to twochromatographic analyses. Samples are stable for some hours after derivatisation.5.5 IdentificationThe analysis of the calibration solutions under the same operating conditions as those used for the analysis of thesample allows the identification of the peaks by comparison of the retention times. Due to the overlapping of theelution zones of the methyl esters and of the monoglycerides, it is therefore advised, in order to identify themonoglyceride peaks, to inject the commercial mixture composed of monopalmitine, monostearin and monooleine(3.11), the latter having been previously submitted to the derivatisation reaction (5.2).A chromatogram of a rapeseed oil methyl ester sample, obtained under the operating conditions and preparationdescribed above (4.3 and 5.4) is presented in annex C (
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