DIN EN 16466-1-2013 Vinegar - Isotopic analysis of acetic acid and water - Part 1 H-NMR analysis of acetic acid German version EN 16466-1 2013《醋 水和醋酸的同位素分析 第1部分 醋酸的H-NMR分析》.pdf

1、March 2013 Translation by DIN-Sprachendienst.English price group 9No 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!$ol“1957673www.din.deDDIN EN 16466-1Vinegar Isotopic analysis of acetic acid and water Part 1: H-NMR analysis of acetic acid;English version EN 16466-1:2013,English translation of DIN EN 16466-1:2013-03Essig Isotopenanalyse von Essigsure und Wasser Teil 1: 2H-NMR-Analyse von Essigsure;Engli

3、sche Fassung EN 16466-1:2013,Englische bersetzung von DIN EN 16466-1:2013-03Vinaigre Analyse isotopique de lacide actique et d leau Partie 1: Analyse RMN-2H de lacide actique;Version anglaise EN 16466-1:2013,Traduction anglaise de DIN EN 16466-1:2013-03www.beuth.deIn case of doubt, the German-langua

4、ge original shall be considered authoritative.Document comprises 13 pages 02.132eDIN EN 16466-1:2013-03 2 A comma is used as the decimal marker. National foreword This document (EN 16466-1:2013) has been prepared by Technical Committee CEN/TC C01 “Food products” (Secretariat: CCMC). This document ha

5、s 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 its pre

6、paration 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 ISO 57

7、25-4 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 16466-1 January 201

8、3 ICS 67.220.10 English Version Vinegar - Isotopic analysis of acetic acid and water - Part 1: 2H-NMR analysis of acetic acid Vinaigre - Analyse isotopique de lacide actique et de leau - Partie 1: Analyse RMN-2H de lacide actique Essig - Isotopenanalyse von Essigsure und Wasser - Teil 1: 2H-NMR-Anal

9、yse 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-date lists and b

10、ibliographical 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 under the res

11、ponsibility 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, Former Yugoslav R

12、epublic 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 EUROPEN DE NO

13、RMALISATION 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 16466-1:2013: E EN 16466-1:2013 (E) 2 Contents Page Foreword . 3 Introduction 4 1

14、Scope 5 2 Normative references . 5 3 Principle 5 4 Reagents . 5 5 Apparatus . 6 6 Procedure . 6 7 Precision . 8 8 Test report 9 Annex A (informative) Results of the collaborative study (2009) 10 Bibliography 11 DIN EN 16466-1:2013-03 EN 16466-1:2013 (E) 3 Foreword This document (EN 16466-1:2013) has

15、 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 standard, eithe

16、r 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 and/or CENE

17、LEC 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 analysis of

18、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 Macedonia, Fran

19、ce, 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 16466-1:2013-03 EN 16466-1:2013 (E) 4 Introduction Vinegar is defined in

20、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 by the Euro

21、pean 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, both the

22、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 2H-SNIF-NM

23、R and 13C-IRMS enables the distinction of grape origin from other sources, such as beet, cane, malt, apple and synthesis 1. DIN EN 16466-1:2013-03 EN 16466-1:2013 (E) 5 1 Scope This European Standard specifies an isotopic method to control the authenticity of vinegar. This method is applicable on ac

24、etic acid of vinegar (from wine, cider, agricultural alcohol, 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 a non-allowed origin (together with the method described in EN 16466-2). The isotopi

25、c analysis of the extracted acetic acid by 2H-NMR is based on a similar method already normalised for wine analysis 2. This European Standard is not applicable to complex matrices made with vinegar as an ingredient, such as balsamic vinegar. 2 Normative references The following documents, in whole o

26、r in part, are normatively referenced in this document and are indispensable for 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 acetic acid

27、 from vinegar is first extracted with diethyl ether (or alternatively another solvent with similar properties such as tert-butyl methyl ether), using a liquid-liquid extractor, during at least 5 h. The solvent is then eliminated by distillation. The water content of the residue can be determined by

28、the Karl Fischer method, or alternatively the acetic acid content may be determined by 1H-NMR 3, 4. The presence of organic impurities in the residue shall be checked e.g. on the basis of 1H-NMR analysis or by GC analysis. The isotopic ratio of hydrogen atoms at the methyl site of acetic acid, (D/H)

29、CH3, is then determined by Nuclear Magnetic Resonance analysis of the Deuterium in the acetic acid extracted from the vinegar. In case a correction is applied to the (D/H)CH3result to correct for organic impurities, this should be stated in the analytical report. 4 Reagents All reagents and consumab

30、les 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 Standard N,N-tetramethylurea (TMU) Standard TMU with a calibrated isot

31、ope ratio D/H. 4.3 Hexafluorobenzene (C6F6) Used as field-frequency stabilisation substance (lock). DIN EN 16466-1:2013-03 EN 16466-1:2013 (E) 6 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

32、 5.1.1 Liquid-liquid extractor of 400 ml or 800 ml. 5.1.2 Spinning band or Vigreux distillation column. 5.1.3 Round bottom flask of 500 ml. 5.1.4 Erlenmeyer of 250 ml. 5.1.5 Condenser. 5.1.6 Heater. 5.2 For 2H-SNIF-NMR determination of acetic acid from vinegar 5.2.1 Analytical balance, precision 0,1

33、 mg. 5.2.2 Filter 0,45 m. 5.2.3 NMR spectrometer fitted with a specific “deuterium“ probe tuned to a frequency vo, characteristic of channel Bo (e.g. Bo = 7,05 T, vo = 46,05 MHz and for Bo = 9,4 T, vo = 61,4 MHz) having a decoupling channel (B2) and a field-frequency stabilisation channel (lock) at

34、the fluorine frequency. The resolution measured on the spectrum, transformed without exponential multiplication (i.e. LB = 0) and expressed by the width at the half-height of the methyl signals of acetic acid and the methyl signal of TMU shall be less than 0,5 Hz. The sensitivity (signal-to-noise ra

35、tio), measured with an exponential multiplying factor LB equal to 2 shall be greater than or equal to 150 for the methyl signal of acetic acid containing less than 25 % of water. For example, using a NMR spectrometer of field Bo = 7,05 T, 400 scans are necessary to reach this value. 5.2.4 Automatic

36、sample changer (optional). 5.2.5 Data-processing software enabling lorentzian integration. 5.2.6 10 mm sample tubes of sufficient quality for NMR spectrometer 400 MHz. 5.2.7 Fume hood. 6 Procedure 6.1 Extraction of acetic acid from vinegar 6.1.1 Liquid-liquid extraction Put 125 ml of diethyl ether i

37、nto 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). Pour the vinegar into the extractor and complete with diethyl ether. Adapt the r

38、ound 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 extract in the round bottom flask. DIN EN 1646

39、6-1:2013-03 EN 16466-1:2013 (E) 7 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 and

40、 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 a

41、t the top of the column is at least 90 (pure acetic acid distils at 116 C 117 C). 6.1.3 Determination of the residual water content First, the traces of solvent in the acetic acid are removed by blowing dry N2on the cold residue for 10 min. The water content is determined by the Karl Fischer method.

42、 In case more than 25 % (w/w) residual water is found, the extraction should be performed again. 6.2 2H-SNIF-NMR determination of acetic acid from vinegar 6.2.1 NMR preparation Weigh approximately 3,25 g of acetic acid (solution obtained from the extraction) to the nearest 0,1 mg into a previously w

43、eighed bottle. Add approximately 1,1 g of N,N-tetramethylurea (TMU) as internal standard to the nearest 0,1 mg. Add 150 l of hexafluorobenzene (C6F6) as lock substance. Homogenise by shaking. The samples should be filtered on 0,45 m syringe filters while transferring into 10 mm NMR tube. Cap on the

44、tube tightly to avoid evaporation during measurement. CAUTION It is strongly recommended to perform the NMR tube preparation under a fume hood, wearing safety glasses and gloves. 6.2.2 Acquisition of 2H-SNIF-NMR spectra Spectrometer shall be checked for sensitivity and resolution according to specif

45、ications given above (5.2). Place a sample of acetic acid prepared as in 6.1 in a 10 mm tube and introduce it into the probe. Typical conditions for obtaining 2H-SNIF-NMR spectra are as follows: a constant probe temperature (e.g. 303 K) acquisition time of at least 5,5 s for 1 200 Hz spectral width

46、(16 Kb memory) (i.e. about 20 x 10-6at 61,4 MHz or 27 x 10-6at 46,1 MHz) 90 pulse adjustment of acquisition time: its value shall be of the same order as the dwell time parabolic detection: fix the offset O1 between the OD and CH2D reference signals for acetic acid determine the value of the decoupl

47、ing offset O2 from the 1H-NMR spectrum measured by the decoupling coil on the same tube. Good decoupling is obtained when O2 is located in the middle of the frequency interval existing between the CH3 and TMU groups. Use the wide band-decoupling mode. DIN EN 16466-1:2013-03 EN 16466-1:2013 (E) 8 For

48、 each spectrum, carry out a number of accumulations NS sufficient to obtain the signal-to-noise ratio given in 5.2 and repeat this set of NS accumulations NE = 5 times. The values of NS depend on the types of spectrometer and probe used. 6.2.3 Calculations and expression of the result Appropriate so

49、ftware based on a complex least square curve fitting algorithm should be used to determine the signal area (phasing and baseline correction are sensitive parameters to be correctly adjusted). Calculate for each spectrum the D/HCH3(x 10-6) as follows: stHDxSstSaaxpuritymaamstxMstMaaxPaaPstCHHD )/(*3)/( = where aa is the acetic acid; st is the internal standard TMU; P is the number of equivalent deuterium positions for the considered molecular site; M is the molecular weight, in g.mol-1; m is the weighted mass in g, we