1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16466-2:2013Vinegar Isotopic analysis of acetic acid and waterPart 2: C-IRMS analysis of acetic acidBS EN 16466-2:2013 BRITISH STANDARDNational forewordThis British Standar
2、d is the UK implementation of EN 16466-2:2013. The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/2, Food Technical Committee Chairmen.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to
3、include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2013. Published by BSI Standards Limited 2013ISBN 978 0 580 73667 4 ICS 67.220.10 Compliance with a British Standard cannot confer immunity from legal obligations.
4、This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2013.Amendments issued since publicationDate T e x t a f f e c t e dBS EN 16466-2:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16466-2 January 2013 ICS 67.220.10 English V
5、ersion Vinegar - Isotopic analysis of acetic acid and water - Part 2: 13C-IRMS analysis of acetic acid Vinaigre - Analyse isotopique de lacide actique et de leau - Partie 2 : Analyse SMRI-13C de lacide actique Essig - Isotopenanalyse von Essigsure und Wasser - Teil 2: 13C-IRMS-Analyse von Essigsure
6、This European Standard was approved by CEN on 3 November 2012. 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 ref
7、erences 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 C
8、EN 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 Macedon
9、ia, 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 COMIT EUROPEN DE NORMALISATION EUROPI
10、SCHES KOMITEE FR NORMUNG 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 16466-2:2013: E BS EN 16466-2:2013EN 16466-2:2013 (E) 2 Contents Page Foreword . 3 Introduction 4 1
11、Scope 5 2 Normative references . 5 3 Principle 5 4 Reagents . 5 5 Apparatus . 6 6 Procedure . 6 7 Precision . 8 8 Test report 8 Annex A (informative) Results of the collaborative study (2009) 9 Bibliography 10 BS EN 16466-2:2013EN 16466-2:2013 (E) 3 Foreword This document (EN 16466-2:2013) has been
12、based on an international collaborative study of the methods published in Analytica Chimica Acta 649 (2009) 98-105, and organised under the auspices of the Permanent International Vinegar Committee (CPIV, Brussels). This European Standard shall be given the status of a national standard, either by p
13、ublication of an identical text or by endorsement, at the latest by July 2013, and conflicting national standards shall be withdrawn at the latest by July 2013. 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 sh
14、all not be held responsible for identifying any or all such patent rights. The European standard, Vinegar Isotopic analysis of acetic acid and water, consists of the following parts: Part 1: 2H-NMR analysis of acetic acid; Part 2: 13C-IRMS analysis of acetic acid; Part 3: 18O-IRMS analysis of water.
15、 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, Ge
16、rmany, 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 16466-2:2013EN 16466-2:2013 (E) 4 Introduction Vinegar is defined by EN 13188 as
17、 the acetic acid solution resulting from a double fermentation: a) transformation of sugars to ethanol and b) transformation of ethanol to acetic acid. Conversely EN 13189 defines acetic acid as “Product made from materials of non-agricultural origin“. Wine vinegar is defined by the European Regulat
18、ions 479/2008 and 491/2009 as the product obtained exclusively from the acetous fermentation of wine, which is in turn defined as the product exclusively obtained from the alcoholic fermentation of fresh grapes, whether crushed or not, or of grape must. In all types of vinegar, both the ethanol and
19、the acetic acid should be obtained by a biotechnological process, and the use of acetic acids obtained from either petroleum derivatives or the pyrolysis of wood is not permitted according to the above definitions. The isotopic analysis of acetic acid extracted from vinegar by 2H-SNIF-NMR and 13C-IR
20、MS enables the distinction of grape origin from other sources, such as beet, cane, malt, apple and synthesis 1. BS EN 16466-2:2013EN 16466-2:2013 (E) 5 1 Scope This European Standard specifies an isotopic method to control the authenticity of vinegar. This method is applicable on acetic acid of vine
21、gar (from cider, alcohol, wine, etc.) in order to characterise the botanical origin of acetic acid and to detect adulterations of vinegar using synthetic acetic acid or acetic acid from not allowed origin (together with the method described in EN 16466-1). The isotopic analysis of the extracted acet
22、ic acid by 13C-IRMS is based on a similar method already normalised for wine analysis 2. 2 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. F
23、or undated references, the latest edition of the referenced document (including any amendments) applies. Not applicable. 3 Principle The acetic acid from vinegar is first extracted with diethyl ether (or alternatively another solvent with similar properties such as tert-butyl methyl ether), using a
24、liquid-liquid extractor, during at least 5 h. The solvent is then eliminated by distillation. The 13C/12C ratio of acetic acid from vinegar is then determined by Isotope Ratio Mass Spectrometry (IRMS) on the CO2gas resulting from a complete combustion at high temperature in an Elemental Analyser. 4
25、Reagents All reagents and consumables used shall meet stated requirements of the used method/apparatus (as specified by the manufacturer). However, all reagents and consumables can be replaced by items with similar performance. 4.1 Diethyl ether For analysis. 4.2 Carbon dioxide For analysis, used as
26、 secondary reference gas for the determination of 13C/12C ratio. Purity 5.2 minimum. 4.3 Helium For analysis. Purity 5.6 minimum. 4.4 Oxygen For analysis. Purity 5.0 minimum. BS EN 16466-2:2013EN 16466-2:2013 (E) 6 4.5 Oxidation reagent For the furnace of the combustion system, like copper oxide, co
27、balt oxide. 4.6 Desiccant To eliminate water produced in combustion if necessary, such as magnesium perchlorate 5 Apparatus All materials listed below are commercially available and used in food control laboratories. 5.1 For the extraction of acetic acid from vinegar 5.1.1 Liquid-liquid extractor of
28、 400 ml or 800 ml. 5.1.2 Spinning band or Vigreux column. 5.1.3 Round bottom flask of 500 ml. 5.1.4 Erlenmeyer of 250 ml. 5.1.5 Condenser. 5.1.5 Heater. 5.2 For the determination of the isotopic ratio 13C/12C of acetic acid from vinegar 5.2.1 Isotope Ratio Mass spectrometer with an internal repeatab
29、ility of 0,05. 5.2.2 Triple collector for simultaneous recording of ions m/z 44, 45 and 46. 5.2.3 Dual Inlet or Conflo to introduce alternatively reference CO2gas and CO2produced by sample combustion. 5.2.4 Elemental Analyser to carry out the complete combustion of organic products into CO2gas and e
30、quipped with a water trap. 5.2.5 Tin or silver capsules for liquid samples or liquid injectors systems. 5.2.6 Tweezers for encapsulation. 5.2.7 Eppendorf pipette with plastic disposable tip. 6 Procedure 6.1 Extraction of acetic acid from vinegar 6.1.1 Liquid-liquid extraction Put 125 ml of diethyl e
31、ther into a 250 ml round bottom flask. Use a 400 ml or a 800 ml liquid-liquid extractor, depending on the acetic acid content of the vinegar (at least 6 ml of pure acetic acid shall be recovered at the end of the extraction). BS EN 16466-2:2013EN 16466-2:2013 (E) 7 Pour the vinegar into the extracto
32、r and complete with diethyl ether. Adapt the round bottom flask, open the water for the condenser and switch the heater on. The extraction shall last at least 5 h. Then, after this time, separate the aqueous and the organic solution. Recover the organic solution from the extractor and add it to the
33、extract in the round bottom flask. 6.1.2 Purification of the extract The round bottom flask containing the acetic acid in solution in diethyl ether is distilled on spinning band or Vigreux column. An appropriate 250 ml Erlenmeyer is used to collect the distillate. Open the water for the condenser an
34、d switch the heater on. The heating shall be weak during the distillation of the solvent (boiling point of diethyl ether 34 C). When the main part of the solvent has been distilled (no more vapours at the head of the column), increase the heating. The distillation is completed when the temperature i
35、s stable at about 98 C (pure acetic acid distils at 116 C to 117 C). 6.2 Determination of the isotopic ratio 13C/12C of acetic acid from vinegar 6.2.1 Experimental determinations Place the samples in capsules (the appropriate quantity of acetic acid shall be calculated according to the quantity of c
36、arbon necessary given the sensitivity and the linearity of the mass spectrometry apparatus used). Each capsule shall be completely sealed. At least 2 capsules shall be prepared for every sample. Place the capsules in the appropriate place on the tray of the automatic sampler of the elemental analyse
37、r. Place systematically capsules containing working references at the beginning and at the end of the sample series, and insert regularly control samples. Check the IRMS instrument and adjust it for optimal combustion: furnace temperature, helium and oxygen flows. Check the system for leaks. Adjust
38、the IRMS to measure the ionic currents m/z = 44, 45 and 46. Check the accuracy of the system using known control samples before starting to measure the samples. The samples placed on the auto sampler of the elemental analyser are introduced in turn. The CO2from each sample combustion is eluted towar
39、ds the mass spectrometer which measures the ionic currents. The software records the 3 ionic currents and calculates the isotopic ratio 13C/12C for each sample. 6.2.2 Calculation and expression of the results The purpose of the method is to measure the 13C/12C ratio of acetic acid extracted from vin
40、egar. The 13C/12C isotope ratio can be expressed by its deviation from a working reference. The isotopic deviation of carbon 13 (13C) is then calculated on a delta scale per thousand () by comparing the results obtained for the sample to be measured with those for a working reference previously cali
41、brated on the basis of the primary international reference (V-PDB). The 13C values (in ) are expressed in relation to the working reference as follows: ( ) ( )( )13 12 13 12sample standard (V-PDB)1313 12(V-PDB)standardC/ C C/ CCC/ CRR R= where Rsampleand Rstandardare respectively the 13C/12C isotope
42、 ratios of the sample and of the standard. Between two measurements of the standard working sample, the variation, and therefore the correction to be applied to the results obtained from the samples, may be assumed to be linear. The standard working sample BS EN 16466-2:2013EN 16466-2:2013 (E) 8 sha
43、ll be measured at the beginning and at the end of all sample series. A correction can then be calculated for each sample according to its position in the sequence using linear interpolation. 7 Precision 7.1 Repeatability In the collaborative study organised in 2009 (see Annex A: Results of the colla
44、borative study (2009), the average repeatability limit (r = 2,8 x sr) of the 13C of acetic acid was 0,45 . 7.2 Reproducibility In the collaborative study organised in 2009 (see Annex A: Results of the collaborative study (2009), the average reproducibility limit (R = 2,8 x sR) of the 13C of acetic a
45、cid was 0,91 . 8 Test report The test report shall contain at least the following: a) all information necessary for the identification of the sample; b) a reference to this European Standard or to the method used; c) the results and the units in which the results have been expressed; d) date and typ
46、e of sampling procedure (if known); e) date of receipt; f) date of test; g) any particular points observed in the course of the test; h) any operations not specified in the method or regarded as optional which might have affected the results. BS EN 16466-2:2013EN 16466-2:2013 (E) 9 Annex A (informat
47、ive) Results of the collaborative study (2009) The samples have been prepared using the materials listed in Table A.1, plus a mixture of samples B and C. All samples have been analysed as blind duplicates, so in total twelve samples have been sent to each participating laboratories. Following the IU
48、PAC internal harmonised protocol for collaborative studies 3, this experimental design intends to have more than five “materials“, i.e. different matrix / sample pairs. Homogeneity tests have been performed by the coordinator before shipment of samples. The statistical tests performed according to 4
49、 confirmed a sufficient homogeneity for the six sample pairs. Statistical calculations have then been performed according to ISO 5725-4 5 and the IUPAC protocol 3. Outliers have been removed in the following way: a loop of Cochran tests for removal of laboratories with highest variance, single and pair value Grubbs tests for individual or paired individual outliers, then back to Cochran test, etc., keeping a proportion of outliers 2/9. Then the standard deviations of repeatability (sr) and of reproducibility (sR) for each material ha
