1、BS EN 15608:2008ICS 71.100.40NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDSurface active agents Quantitativedetermination offree fatty acid inalkylamidopropylbetaines Gas-chromatographicmethodThis British Standardwas published under theauthority of the Standa
2、rdsPolicy and StrategyCommittee on 31 January2009 BSI 2009ISBN 978 0 580 58044 4Amendments/corrigenda issued since publicationDate CommentsBS EN 15608:2008National forewordThis British Standard is the UK implementation of EN 15608:2008.The UK participation in its preparation was entrusted to Technic
3、alCommittee CII/34, Methods of test for surface active agents.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Com
4、pliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15608:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15608May 2008ICS 71.100.40English VersionSurface active agents - Quantitative determination of free fattyacid in alkylamidopropylbetaines - Gas-chromatograph
5、ic methodAgents de surface - Dosage quantitatif des acides graslibres dans les alkylamidopropylbtanes - Mthode parchromatographie en phase gazeuseGrenzflchenaktive Stoffe - Quantitative Bestimmung freierFettsure in Alkylamidopropylbetainen -Gaschromatographisches VerfahrenThis European Standard was
6、approved by CEN on 11 April 2008.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 nationa
7、lstandards may be obtained 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 translationunder the responsibility of a CEN member into its own language and notifi
8、ed to the CEN Management Centre has the same status as theofficial 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,
9、Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitatio
10、n in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15608:2008: EBS EN 15608:2008EN 15608:2008 (E) 2 Contents Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Reagents.4 5 Apparatus .5 6 Sampling and preparation of the test sample.5 7 Procedure .5 8 Calcu
11、lation and expression of results.6 9 Precision.6 10 Test report 7 Annex A (informative) Reference GLC Chromatogram .8 A.1 GC Conditions8 Annex B (informative) Method validation9 B.1 Calibration 9 B.2 Statistical and other data derived from the results of interlaboratory tests .9 B.3 Remarks10 Biblio
12、graphy 11 BS EN 15608:2008EN 15608:2008 (E) 3 Foreword This document (EN 15608:2008) has been prepared by Technical Committee CEN/TC 276 “Surface active agents”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication
13、of an identical text or by endorsement, at the latest by November 2008, and conflicting national standards shall be withdrawn at the latest by November 2008. 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
14、 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 following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Eston
15、ia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15608:2008EN 15608:2008 (E) 4 1 Scope This European Standard spec
16、ifies a procedure for the determination of the content of free fatty acid, FFA, in alkylamidopropylbetaines, which is defined as being the amount of fatty acid expressed in grams per 100 g of product. This method has been validated for the determination of fatty acids from C6to C20in a total concent
17、ration range from 0,02 g to more than 3,0 g fatty acid per 100 g of product. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the refer
18、enced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) ISO 607, Surface active agents and detergents Methods of sample division 3 Principle The free fatty acids are extracted with petroleum ether at acidic pH
19、. Then the extracted fatty acids are derivatised and subsequently analysed by GLC-FID. The chromatogram resolves the different acids according to their alkyl chain length. For quantification the sum of the peak areas of all fatty acid homologues is related to the peak area of the internal standard t
20、ridecanoic acid. 4 Reagents 4.1 General WARNING Your attention is drawn to the regulations covering the handling of hazardous substances. Technical, organisational and personal protection measures should be observed. During the analysis, unless otherwise specified, use only reagents of recognized an
21、alytical grade and that have been checked in advance as to not interfere with the analytical results and water complying with grade 1 as defined in EN ISO 3696. 4.2 Tridecanoic acid, purity 99 % (m/m) (CAS number: 638-53-9). 4.3 Petroleum ether (40 C to 60 C) (CAS number: 101316-46-5). 4.4 Ethanol (
22、CAS number: 64-17-5). 4.5 HCl, c = 37% (m/m) (CAS number: 7647-01-0). 4.6 Internal Standard Solution Weigh to the nearest 0,1 mg, 0,3 g of pure tridecanoic acid (4.2) in a 25 ml volumetric flask and make up to the mark with petroleum ether. This is the Internal Standard Solution. 4.7 TMPAH, Trimethy
23、lphenylammonium hydroxide solution, c(TMPAH) about 0,5 M in methanol (CAS number: 1899-02-1). BS EN 15608:2008EN 15608:2008 (E) 5 4.8 Methanol water free (CAS number: 67-56-1). 4.9 Methyl tert-butyl ether water free (CAS number: 1634-04-4). 4.10 Phenolphthalein (CAS number: 77-09-8), 1 % in MeOH. 5
24、Apparatus Ordinary laboratory apparatus and the following. 5.1 15 ml glass vial with threaten cap. 5.2 Gas-chromatograph, equipped with capillary split injector and a flame ionisation detector. 5.3 Capillary fused silica column, capable of the separation characteristics shown in the chromatogram of
25、Annex A. 6 Sampling and preparation of the test sample The test sample shall be prepared and stored in accordance with ISO 607. 7 Procedure 7.1 Fatty acid extraction Weigh to the nearest 0,1 mg, 1,0 g of sample in a 15 ml glass vial with threaten cap. For samples having an expected content lower tha
26、n 1 g of free fatty acid per 100 g of sample, weigh about 2 g of sample. Add 5 ml of a water-ethanol 1:1 (v/v) mixture, and homogenize. Add with a precision pipette, 500 l aliquot of Internal Standard Solution (4.6). Add 0,5 ml of HCl (4.5) to acidify the media, and homogenize. The pH-value of the s
27、olution shall be below 2. Add 5 ml of Petroleum Ether. Shake the tube vigorously (about 1 min) and let stand until phase separation (2 min are usually enough). 7.2 Preparation of the Methyl Ester (Methylation by TMPAH) NOTE Prior to analysis, the fatty acids extracted from the sample are methylated
28、to ensure good GC peak allowing a good quantitative analysis. Methylation can easily be done by TMPAH with the reaction being performed in the GLC injector. However, as this procedure requires high temperatures the subsequent use of an apolar GLC column (100% dimethyl polysiloxane) is recommended. A
29、lternatively the methylation can be performed as liquid reaction with methanol with boron trifluoride as catalyst. Transfer 500 l of the extract (7.1) to an autosampler vial (2 ml). Add 500 l of a mixture of methanol/methyl tert-butyl ether (4.8/4.9) 1:1 and mix carefully. Subsequently add two drops
30、 of the phenolphthalein solution (4.10). BS EN 15608:2008EN 15608:2008 (E) 6 Finally TMPAH (4.7) is added drop wise until the solution is no longer discoloured and remains magenta. Analyse the solution directly by injecting 2 l into the GC at an injector temperature of at least 310 C. 7.3 Gas Chroma
31、tography GC The analysis of the derivatives is performed by capillary gas chromatography. There are various separation columns of different polarity capable to separate fatty acid methyl esters. However their application may be restricted by the methylation procedure (see 7.2 and Annex B.3). In eith
32、er case the retention times of the individual fatty acids (C6 to C20 including all of the relevant unsaturated species) have to be determined by a reference analysis of a standard FAME (fatty acid methyl esters) mixture. An appropriate GC analysis on a non polar separation column including all param
33、eters is shown in Annex A. 8 Calculation and expression of results The total free fatty acid, w, is obtained from the GLC chromatogram and calculated as follows in grams per 100 g sample: sistdistdistdiimRmAAw= (1) where Ai is the area of each one of the peaks corresponding to fatty acid homologues
34、except that of the internal standard; Aistdis the area of the internal standard peak; msis the sample weight (7.1), in grams; mistdis the weight of Internal standard in the aliquot (7.1), in grams; Ristdis the Internal standard purity in grams per 100 grams. The relative response factors of all the
35、fatty acid homologues respectively the tridecanoic acid internal standard are assumed to be equal for C6to C20fatty acids (see Annex B). 9 Precision 9.1 Repeatability limit The absolute difference between two independent single test results, obtained using the same method on identical test material
36、in the same laboratory by the same operator using the same equipment within a short interval of time, will not exceed the repeatability limit, r, in more than 5 % of cases. BS EN 15608:2008EN 15608:2008 (E) 7 9.2 Reproducibility limit The absolute difference between two independent single test resul
37、ts, obtained using the same method on identical test material in different laboratories by different operators using different equipment, will not exceed the reproducibility limit, R, in more than 5 % of the cases. 10 Test report The test report shall include the following information: a) all inform
38、ation necessary for the identification of the sample tested; b) reference to this European Standard (EN 15608:2008); c) test results; d) details of any operation not specified in this European Standard or in the European Standards to which reference is made, and any operations regarded as optional,
39、as well as any incidents like to have affected the results. BS EN 15608:2008EN 15608:2008 (E) 8 Annex A (informative) Reference GLC Chromatogram Key 1 response 2 retention time Figure A.1 - Chromatogram of a betaine sample containing 2 % FFA A.1 GC Conditions Inject 2 l of sample extract into the ga
40、s chromatograph, the following conditions have been found suitable: Column HP-11)(30 m, 0,32 mm, 0,25 m)Head P 15 psi Tinlet/TFID310 C T1(t1) 80 C (2 min) Rate 8 C/min T2( t2) 300 C (20 min). Post run 305 C (10min) Split 20 ml/min Helium 1) HP1(100% dimethyl polysiloxane) is the trade name of produc
41、t supplied by Agilent Technologies This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. BS EN 15608:2008EN 15608:2008 (E) 9 Annex B (informative) Method validation B.1 Calibration The individual calibrati
42、on factors of C8- C20 acid versus C13 acid may differ from 0,85 l to 1,1 depending on the chain length and the cleanness of the GC equipment . For coconut fatty acids an overall average correction factor of 0,97 to 1,03 depending on the individual chain length distribution is achieved. For practical
43、 reasons a relative response factor of coconut fatty acids to tridecanoic acid is assumed as 1. This overcomes the necessity of calibration measurements for all individual fatty acids. This may be acceptable unless betaines with a fatty acid distribution differing from coconut fatty acid have to be
44、analysed. For a maximum precision or if the chain length distribution differs from coconut fatty acid, a suitable calibration is advised. B.2 Statistical and other data derived from the results of interlaboratory tests The interlaboratory test was carried out in 2006 by CESIO (Comit Europen des agen
45、ts de Surface et de leurs Intermdiaires Organiques)/AISE (International Association for Soaps, Detergents and Maintenance Products) WG “Surfactant Analysis“. The test samples were commercial alkylamidopropylbetaines. The results of interlaboratory test were evaluated in accordance with ISO 5725-2 (s
46、ee Table B.1). Table B.1 Results of interlaboratory test Tegobetaine F50 Tegobetain CKNumber of participating laboratories 5 5 Number of accepted test results 30 30 Mean value (% w/w) 2,02 0,64 Repeatability standard deviation (sr) 0,053 0,038 Repeatability coefficient of variation 2,6 5,9 Repeatabi
47、lity limit (r) (2,8 x sr) 0,149 0,105 Reproducibility standard deviation (sR) 0,111 0,072 Reproducibility coefficient of variation 5,5 11,3 Reproducibility limit (R) (2,8 x sR) 0,311 0,201 BS EN 15608:2008EN 15608:2008 (E) 10 B.3 Remarks The procedure described has been validated for its recovery. F
48、or one step extraction as is described in 7.1, the average recovery has been of 90 %. The extraction of free fatty acids in one step has been introduced for practical reasons although short chain fatty acids (C8-C12) are not fully extracted. A higher recovery ( 97 %) is achievable if the extraction
49、is performed in 3 steps, each with 2 ml of petroleum ether solvent. BS EN 15608:2008EN 15608:2008 (E) 11 Bibliography 1 ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results - Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method BS EN15608:2008BSI GroupHeadquarters 389Chiswick High Road,London, W4 4AL, UKTel +44 (0)20 8996 9001Fax +44 (0)20 8996 - British Standards InstitutionBSI is the independent national body responsible f
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