1、March 2013 Translation by DIN-Sprachendienst.English price group 8No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 6
2、7.220.10!$om“1957674www.din.deDDIN EN 16466-2Vinegar Isotopic analysis of acetic acid and water Part 2: 13C-IRMS analysis of acetic acid;English version EN 16466-2:2013,English translation of DIN EN 16466-2:2013-03Essig Isotopenanalyse von Essigsure und Wasser Teil 2: 13C-IRMS-Analyse von Essigsure;
3、Englische Fassung EN 16466-2:2013,Englische bersetzung von DIN EN 16466-2:2013-03Vinaigre Analyse isotopique de lacide actique et de leau Partie 2: Analyse SMRI-13C de lacide actique;Version anglaise EN 16466-2:2013,Traduction anglaise de DIN EN 16466-2:2013-03www.beuth.deDocument comprises 12 pages
4、In case of doubt, the German-language original shall be considered authoritative.02.13 DIN EN 16466-2:2013-03 2 A comma is used as the decimal marker. National foreword This document (EN 16466-2:2012) has been prepared by Technical Committee CEN/TC C01 “Food products” (Secretariat: CCMC). This docum
5、ent has been based on an international collaborative study of the three methods (EN 16466 Parts 1 to 3) published in Analytica Chimica Acta 649 (2009) 98-105, and organised under the auspices of the Permanent International Vinegar Committee (CPIV, Brussels). The responsible German body involved in i
6、ts preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-05-03 AA Essig. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 5725-4 DIN
7、ISO 5725-4 National Annex NA (informative) Bibliography DIN ISO 5725-4, Accuracy (trueness and precision) of measurement methods and results Part 4: Basic methods for the determination of the trueness of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16466-2 Janua
8、ry 2013 ICS 67.220.10 English Version 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:
9、13C-IRMS-Analyse von Essigsure 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-dat
10、e lists and bibliographical 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
11、under the responsibility 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, Form
12、er Yugoslav Republic of Macedonia, 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
13、EUROPEN DE NORMALISATION EUROPISCHES 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 EN 16466-2:2013 (E) 2 Contents Page Foreword . 3 Intr
14、oduction 4 1 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 DIN EN 16466-2:2013-03 EN 16466-2:2013 (E) 3 Foreword This document (EN 16466
15、-2:2013) has been 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 sta
16、ndard, either by publication 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
17、 and/or CENELEC shall 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
18、analysis of water. 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 Mac
19、edonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16466-2:2013-03 EN 16466-2:2013 (E) 4 Introduction Vinegar is
20、 defined by EN 13188 as 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
21、by the European Regulations 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
22、, both the ethanol and 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
23、 2H-SNIF-NMR and 13C-IRMS enables the distinction of grape origin from other sources, such as beet, cane, malt, apple and synthesis 1. DIN EN 16466-2:2013-03 EN 16466-2:2013 (E) 5 1 Scope This European Standard specifies an isotopic method to control the authenticity of vinegar. This method is appli
24、cable on acetic acid of vinegar (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 an
25、alysis of the extracted acetic 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
26、the edition cited applies. For 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-b
27、utyl methyl ether), using a 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
28、in an Elemental Analyser. 4 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 d
29、ioxide For analysis, used as 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. DIN EN 16466-2:2013-03 EN 16466-2:2013 (E) 6 4.5 Oxidation reagent For the furnace of the combust
30、ion system, like copper oxide, cobalt 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
31、 5.1.1 Liquid-liquid extractor of 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 spect
32、rometer with an internal repeatability 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 o
33、rganic products into CO2gas and equipped 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 e
34、xtraction Put 125 ml of diethyl ether 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). DIN EN 16466-2:2013-03 EN 16466-2:2013 (E
35、) 7 Pour the vinegar into the extractor 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
36、 from the extractor and add it to the 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 distillat
37、e. Open the water for the condenser and 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 distillati
38、on is completed when the temperature is 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 cal
39、culated according to the quantity of carbon 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 auto
40、matic sampler of the elemental analyser. 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 flow
41、s. Check the system for leaks. Adjust 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
42、each sample combustion is eluted towards 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 r
43、atio of acetic acid extracted from vinegar. 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 f
44、or a working reference previously calibrated 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 Rstanda
45、rdare respectively the 13C/12C isotope 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 DIN E
46、N 16466-2:2013-03 EN 16466-2:2013 (E) 8 shall 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
47、 in 2009 (see Annex A: Results of the collaborative 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
48、limit (R = 2,8 x sR) of the 13C of acetic acid 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 type 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
