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!$op“1957677www.din.deDDIN EN 16466-3Vinegar Isotopic analysis of acetic acid and water Part 3: 18O-IRMS analysis of water in wine vinegar;English version EN 16466-3:2013,English translation of DIN EN 16466-3:2013-03Essig Isotopenanalyse von Essigsure und Wasser Teil 3: 18O-IRMS-Analyse von
3、Wasser in Weinessig;Englische Fassung EN 16466-3:2013,Englische bersetzung von DIN EN 16466-3:2013-03Vinaigre Analyse isotopique de lacide actique et de leau Partie 3: Analyse SMRI- O de leau dans le vinaigre de vin;18Version anglaise EN 16466-3:2013,Traduction anglaise de DIN EN 16466-3:2013-03www.
4、beuth.deDocument comprises 12 pagesIn case of doubt, the German-language original shall be considered authoritative.02.13 DIN EN 16466-3:2013-03 2 A comma is used as the decimal marker. National foreword This document (EN 16466-3:2013) has been prepared by Technical Committee CEN/TC C01 “Food produc
5、ts” (Secretariat: CCMC). This document 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 re
6、sponsible German body involved in its 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 docu
7、ment is as follows: ISO 5725-4 DIN 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 EUROPEN
8、NE EUROPISCHE NORM EN 16466-3 January 2013 ICS 67.220.10 English Version Vinegar - Isotopic analysis of acetic acid and water - Part 3: 18O-IRMS analysis of water in wine vinegar Vinaigre - Analyse isotopique de lacide actique et de leau - Partie 3: Analyse SMRI-18O de leau dans le vinaigre de vin E
9、ssig - Isotopenanalyse von Essig und Wasser - Teil 3: 18O-IRMS-Analyse von Wasser in Weinessig 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
10、 status of a national standard without any alteration. Up-to-date 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, Frenc
11、h, German). A version in any other language made by translation 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,
12、Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former 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 a
13、nd United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT 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
14、-3:2013: E EN 16466-3:2013 (E) 2 Contents Page Foreword . 3 Introduction 4 1 Scope 5 2 Normative references . 5 3 Principle 5 4 Reagents . 5 4.1 Carbon dioxide . 5 4.2 Carbon dioxide used for equilibration 5 5 Apparatus . 5 6 Procedure . 6 6.1 General . 6 6.2 Manual method . 6 6.3 Use of an automati
15、c exchange apparatus . 6 6.4 Manual preparation manual and automatic equilibration and analysis with a dual inlet IRMS 7 6.5 Use of an automatic equilibration apparatus coupled to a continuous flow system 7 6.6 Calculation and expression of the results 7 7 Precision . 8 7.1 Repeatability . 8 7.2 Rep
16、roducibility . 8 8 Test report 8 Annex A (informative) Results of the collaborative study (2009) 9 Bibliography 10 DIN EN 16466-3:2013-03 EN 16466-3:2013 (E) 3 Foreword This document (EN 16466-3:2013) has been based on an international collaborative study of the method published in Analytica Chimica
17、 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 publication of an identical text or by endorsement, at the latest by July 2013, and conflict
18、ing 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 shall not be held responsible for identifying any or all such patent rights. The European sta
19、ndard, 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. According to the CEN/CENELEC Internal Regulations, the national standards organisations of
20、 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, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Neth
21、erlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16466-3:2013-03 EN 16466-3:2013 (E) 4 Introduction Wine vinegar is defined by the European Regulations 479/2008 and 491/2009 as the product obtained exclusively from the
22、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 accordance with this, it is clear that the production of wine vinegar by fermenting dried grapes and rehydrating wi
23、th tap water is not allowed by European Regulations. The isotopic analysis of water from vinegar by 18O-IRMS enables the distinction of wine vinegar and vinegars from fermented dried grapes which have been rehydrated with water 1, and more generally helps to check the authenticity of wine vinegar. D
24、IN EN 16466-3:2013-03 EN 16466-3:2013 (E) 5 1 Scope This European Standard specifies an isotopic method to control the authenticity of wine vinegar. This method is applicable on wine vinegar in order to characterise the 18O/16O ratio of water, and allows differentiating wine vinegar from vinegars ma
25、de from raisins or alcohol vinegar. NOTE The Oxygen 18 isotopic analysis of water from vinegar 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 f
26、or its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. Not applicable. 3 Principle The 18O/16O ratio of water from vinegar is determined on CO2gas after equilibration of refer
27、ence CO2gas with vinegar water according to the following isotopic exchange reaction: OHOOCOHOC1621816182216+(1)After equilibration the carbon dioxide in the gaseous phase is used for analysis by means of Isotopic Ratio Mass Spectrometry (IRMS) where the 18O/16O isotopic ratio is determined on the C
28、O2resulting from the equilibration. 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 Carbon dioxide For analysis,
29、 used as secondary reference gas for the determination of 13C/12C ratio. Purity 5.2 minimum. 4.2 Carbon dioxide used for equilibration Depending on the instrument, this gas could be the same as 4.1 or in the case of continuous flow systems cylinders containing gas mixture helium-carbon dioxide can a
30、lso be used 5 Apparatus All equipments and materials used shall meet stated requirements of the used method/apparatus (as specified by the manufacturer). However, all equipments and materials can be replaced by items with similar performance. 5.1 Vials with septa appropriate for the used system. DIN
31、 EN 16466-3:2013-03 EN 16466-3:2013 (E) 6 5.2 Volumetric pipettes with appropriate tips. 5.3 Temperature controlled system to carry out the equilibration at constant temperature, typically within 1 C. 5.4 Vacuum pump (if needed for the used system). 5.5 Autosampler (if needed for the used system). 5
32、.6 Syringes for sampling (if needed for the used system). 5.7 GC Column to separate CO2from other elementary gases (if needed for the used system). 5.8 Water removal device (e.g. cryo-trap, selective permeable membranes). 6 Procedure 6.1 General The descriptions that follow refer to procedures gener
33、ally used for the determination of the 18O/16O isotopic ratios by means of equilibration of water with a CO2working standard and the subsequent measurement by IRMS. These procedures can be altered according to changes of equipment and instrumentation provided by the manufacturers as various kinds of
34、 equilibration devices are available, implying various conditions of operation. Two main technical procedures can be used for introduction of CO2into the IRMS either through a dual inlet system or using a continuous flow system. The description of all these technical systems and of the corresponding
35、 conditions of operation is not possible. All values given for volumes, temperatures, pressures and time periods are only indicative. Appropriate values shall be obtained from specifications provided by the manufacturer and/or determined experimentally. 6.2 Manual method A defined volume of the samp
36、le/standard is transferred into a flask using a pipette. The flask is then attached tightly to the manifold. Each manifold is cooled down to below 80 C to deep-freeze the samples (manifold equipped with capillary opening tubes do not require this freezing step). Subsequently, the whole system is eva
37、cuated. After reaching a stable vacuum the gaseous CO2working standard is allowed to expand into the various flasks. For the equilibration process each manifold is placed in a temperature controlled waterbath typically at 25 C ( 1 C) for 12 h (overnight). It is crucial that the temperature of the wa
38、ter-bath is kept constant and homogeneous. After the equilibration process is completed, the resulting CO2is transferred from the flasks to the sample side bellow of the dual inlet system. The measurements are performed by comparing several times the ratios of the CO2contained in the sample side and
39、 the standard side (CO2reference standard gas) of the dual inlet. This approach is repeated till the last sample of the sequence has been measured. 6.3 Use of an automatic exchange apparatus A defined volume of the sample/standard is transferred into a vial using a pipette. The sample vials are atta
40、ched to the equilibration system and cooled down to below 80 C to deep-freeze the samples (systems equipped with capillary opening tubes do not require this freezing step). Subsequently, the whole system is evacuated. After reaching a stable vacuum the gaseous CO2working standard is expanded into th
41、e vials. Equilibrium is reached at a temperature of typically (22 1) C after a minimum period of 5 h and with moderate agitation (if DIN EN 16466-3:2013-03 EN 16466-3:2013 (E) 7 available). Since the equilibration duration depends on various parameters (e.g. the vial geometry, temperature, applied a
42、gitation .), the minimum equilibrium time should be determined experimentally. After the equilibration process is completed, the resulting CO2is transferred from the vials to the sample side bellow of the dual inlet system. The measurements are performed by comparing several times the ratios of the
43、CO2contained in the sample side and the standard side (CO2reference standard gas) of the dual inlet. This approach is repeated till the last sample of the sequence has been measured. 6.4 Manual preparation manual and automatic equilibration and analysis with a dual inlet IRMS A defined volume of sam
44、ple / standard (e.g. 200 l) is introduced into a vial using a pipette. The open vials are then placed in a closed chamber filled with the CO2used for equilibration (4.2). After several purges to eliminate any trace of air, the vials are closed and then placed on the thermostated plate of the sample
45、changer. The equilibration is reached after at least 8 h at 40 C. Once the process of equilibration completed, the CO2obtained is dried and then transferred into the sample side of the dual inlet introduction system. The measurements are performed by comparing several times the ratios of the CO2cont
46、ained in the sample side and the standard side (CO2reference standard gas) of the dual inlet. This approach is repeated until the last sample of the sequence has been measured. 6.5 Use of an automatic equilibration apparatus coupled to a continuous flow system A defined volume of the sample/standard
47、 is transferred into a vial using a pipette. The sample vials are placed into a temperature controlled tray. Using a gas syringe the vials are flushed with mixture of He and CO2. The CO2remains in the headspace of the vials for equilibration. Equilibrium is reached at a temperature typically of (30
48、1) C after a minimum period of 18 h. After the equilibration process is completed the resulting CO2is transferred by means of the continuous flow system into the ion source of the mass spectrometer. CO2reference gas is also introduced into the IRMS by means of the continuous flow system. The measurement is carried out according to a specific protocol for each kind of equipment. 6.6 Calculation and expression of the results The purpose of the method is to measure the 18O/16O ratio of water extracted from vine
