1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16466-3:2013Vinegar Isotopic analysis of acetic acid and waterPart 3: O-IRMS analysis of water in wine vinegar8BS EN 16466-3:2013 BRITISH STANDARDNational forewordThis Brit
2、ish Standard is the UK implementation of EN 16466-3:2013. 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
3、purport to 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 73668 1 ICS 67.220.10 Compliance with a British Standard cannot confer immunity from legal o
4、bligations.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 16466-3:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16466-3 January 2013 ICS 67.220.1
5、0 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 Essig - Isotopenanalyse von Essig und Wasser - Teil 3: 1
6、8O-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 status of a national standard without any alteration.
7、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, French, German). A version in any other language made by tra
8、nslation 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, Finl
9、and, 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 and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATI
10、ON 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 16466-3:2013: E BS EN 16466-3:2013EN 16466-3:2013 (E) 2 Cont
11、ents 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 automatic exchange apparatus . 6 6.4 Manual p
12、reparation 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 Reproducibility . 8 8 Test report 8 Anne
13、x A (informative) Results of the collaborative study (2009) 9 Bibliography 10 BS EN 16466-3:2013EN 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 Acta 649 (2009) 98-105, and organised und
14、er 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 conflicting national standards shall be withdrawn
15、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 standard, Vinegar Isotopic analysis of acetic
16、 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 the following countries are bound to impl
17、ement 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, Netherlands, Norway, Poland, Portugal, Romania
18、, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16466-3:2013EN 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 acetous fermentation of wine, which is in turn
19、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 with tap water is not allowed by European Regulat
20、ions. 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. BS EN 16466-3:2013EN 16466-3:2013 (E) 5 1 Scope
21、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 made from raisins or alcohol vinegar. NOTE The Oxygen
22、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 for its application. For dated references, only the e
23、dition 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 reference CO2gas with vinegar water according to the foll
24、owing 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 CO2resulting from the equilibration. 4 Reagents All r
25、eagents 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, used as secondary reference gas for the determinati
26、on 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 also be used 5 Apparatus All equipments and materials
27、 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. BS EN 16466-3:2013EN 16466-3:2013 (E) 6 5.2 Volumetric
28、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). 5.6 Syringes for sampling (if needed for the used system).
29、 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 generally used for the determination of the 18O/16O isotopic r
30、atios 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 equilibration devices are available, implying various co
31、nditions 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 conditions of operation is not possible. All values give
32、n 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 sample/standard is transferred into a flask using a pipette.
33、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 evacuated. After reaching a stable vacuum the gaseous CO2wor
34、king 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 water-bath is kept constant and homogeneous. After the equi
35、libration 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 the standard side (CO2reference standard gas) of the dua
36、l 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 attached to the equilibration system and cooled down to below
37、 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 the vials. Equilibrium is reached at a temperature of typic
38、ally (22 1) C after a minimum period of 5 h and with moderate agitation (if BS EN 16466-3:2013EN 16466-3:2013 (E) 7 available). Since the equilibration duration depends on various parameters (e.g. the vial geometry, temperature, applied agitation .), the minimum equilibrium time should be determined
39、 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 CO2contained in the sample side and the standard side (CO2refe
40、rence 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 sample / standard (e.g. 200 l) is introduced into a vial using a
41、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 changer. The equilibration is reached after at least 8 h at 40
42、 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 CO2contained in the sample side and the standard side (CO2reference s
43、tandard 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 is transferred into a vial using a pipette. The sample vials
44、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 1) C after a minimum period of 18 h. After the equilibration p
45、rocess 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
46、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 vinegar. The 18O/16O isotope ratio can be expressed by its deviation from a working reference. The isotopic deviation of oxygen 18 (18O) is then calculat
47、ed on a delta scale per thousand () by comparing the results obtained for the sample to be measured with those for a working reference previously calibrated on the basis of the primary international reference (V.SMOW2). The 18O values (in ) are expressed in relation to the working reference as follo
48、ws: ( ) ( )( )18 16 18 16sample standard (V.SMOW2)1818 16(V.SMOW2)standardO/ O O/ OOOO/RR R= (2) where Rsampleand Rstandardare respectively the 18O/16O isotope ratios of the sample and of the standard. Between two measurements of the standard working sample, the variation, and therefore the correcti
49、on to be applied to the results obtained from the samples, may be assumed to be linear. The standard working sample 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. BS EN 16466-3:2013EN 16466-3:2013 (E) 8 7 Precision 7.1 Repeatability In the collaborative study organised in 2009 (see Annex A: Results of the collaborative study (2009), the average repeatability limit (r = 2,8 x sr) of the 18
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