1、DRAFT FOR DEVELOPMENT DD ENV 12140:1997 Method for Determination of stable carbon isotope ratio( 13 C/ 12 C) of sugars from fruit juices, using isotope ratio mass spectrometry ICS67.160.20DD ENV 12140:1997 This Draft for Development, having been prepared under the direction of the Consumer Products
2、and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 February1997 BSI 12-1998 The following BSI reference relates to the work on this Draft for Development: Committee reference AW/21 ISBN0580270181 Committees responsible for this Draftfor De
3、velopment The preparation of this Draft for Development was entrusted to Technical Committee AW/21, Fruit and vegetable juices, upon which the following bodies were represented: British Fruit Juice Importers Association British Retail Consortium British Soft Drinks Association Limited Campden and Ch
4、orleywood Food Research Association Department of Trade and Industry (Laboratory of the Government Chemist) Leatherhead Food Research Association Ministry of Agriculture, Fisheries and Food Royal Society of Chemistry Amendments issued since publication Amd. No. Date CommentsDD ENV 12140:1997 BSI 12-
5、1998 i Contents Page Committees responsible Inside front cover National foreword ii Foreword 2 Text of ENV 12140 3 List of references Inside back coverDD ENV 12140:1997 ii BSI 12-1998 National foreword This Draft for Development has been prepared by Technical Committee AW/21, and is the English lang
6、uage version of ENV12140:1996 Fruit and vegetable juices Determination of the stable carbon isotope ratio ( 13 C/ 12 C) of sugars from fruit juices Method using isotope ratio mass spectrometry, published by the European Committee for Standardization (CEN). ENV12140:1996 was produced as a result of i
7、nternational discussions in which the United Kingdom took an active part. This publication is not to be regarded as a British Standard It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis so that informa
8、tion and experience of its practical application may be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the European organization responsible for its conversion to a European Standard. A review of this publication will be i
9、nitiated2 years after its publication by the European organization so that a decision can be taken on its status at the end of its3-year life. The commencement of the review period will be notified by an announcement in BSI Update. According to the replies received by the end of the review period, t
10、he responsible BSI Committee will decide whether to support the conversion into a European Standard, to extend the life of the prestandard or to withdraw it. Comments should be sent in writing to the Secretary of BSI Technical Committee AW/21 at BSI,389 Chiswick High Road, London W44AL, giving the d
11、ocument reference and clause number and proposing, where possible, an appropriate revision of the text. ISO5725:1986, to which informative reference is made in the text, has been superseded by ISO5725-1:1994, ISO5725-2:1994, ISO5725-3:1994, ISO5725-4:1994 and ISO5725-6:1994, which are identical with
12、 the following Parts of BS ISO5725 Accuracy (trueness and precision) of measurement methods and results: BS ISO5725-1:1994, General principles and definitions. BS ISO5725-2:1994, Basic method for the determination of repeatability and reproducibility of a standard measurement method. BS ISO5725-3:19
13、94, Intermediate measures of the precision of a standard measurement method. BS ISO5725-4:1994, Basic method for the determination of the trueness of a standard measurement method. BS ISO5725-6:1994, Use in practice of accuracy values. Cross-references Publication referred to Corresponding British S
14、tandard ISO3696:1987 BS EN ISO3696:1995 Specification for water for laboratory use Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theENV title page, pages2 to8, an inside back cover and a back cover. This standard has been updated (see copyright date)
15、and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. EUROPEAN PRESTANDARD PRNORME EUROPENNE EUROPISCHE VORNORM ENV12140 October1996 ICS67.160.20 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determi
16、nation, ratios, isotopes, carbon mass spectrometry English version Fruit and vegetable juices Determination of the stable carbon isotope ratio ( 13 C/ 12 C) of sugars from fruits juices Method using isotope ratio mass spectrometry Jus de fruits et de lgumes Dtermination du rapport des isotopes stabl
17、es du carbone ( 13 C/ 12 C) des sucres contenus dans les jus de fruits Mthode utilisant la spectromtrie de masse des rapports isotopiques Frucht- und Gemesfte Bestimmung des Verhltnisses der stabilen Kohlenstoff-Isotope ( 13 C/ 12 C) im Zuckeranteil von Fruchtsften Verfahren unter Verwendung der Iso
18、topenverhltnis-Massenspektrometrie This European Prestandard (ENV) was approved by CEN on1996-02-29 as a prospective standard for provisional application. The period of validity of this ENV is limited initially to three years. After two years the members of CEN will be requested to submit their comm
19、ents, particularly on the question whether the ENV can be converted into an European Standard (EN). CEN members are required to announce the existance of this ENV in the same way as for an EN and to make the ENV available promptly at national level in an appropriate form. It is permissible to keep c
20、onflicting national standards in force (in parallel to the ENV) until the final decision about the possible conversion of the ENV into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg,
21、Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart,36 B-1050 Brussels 1996 Copyright reserved to CEN members Ref. No. ENV12140:1996
22、 EENV 12140:1996 BSI 12-1998 2 Foreword This European Prestandard has been prepared by the Technical Committee CEN/TC174 “Fruit and vegetable juices Methods of analysis” of which the secretariat is held by AFNOR. According to the CEN/CENELEC Internal Regulations, the national standards organizations
23、 of the following countries are required to announce the existence of this European Prestandard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom. Contents Page Foreword 2
24、 1 Scope 3 2 Normative references 3 3 Symbols 3 4 Principle 3 5 Reagents 3 6 Apparatus 3 7 Procedure 4 8 Calculation 4 9 Precision 5 10 Test report 5 Annex A (informative) Bibliography 7 Annex B (informative) Statistical results of the interlaboratory test 7 Table B.1 7ENV 12140:1996 BSI 12-1998 3 1
25、 Scope This European Prestandard specifies a method for the determination of the stable carbon isotope ratio( 13 C/ 12 C) of sugars from fruit juices by isotope ratio mass spectrometry (IRMS). 2 Normative references This European Prestandard incorporates by dated or undated reference, provisions fro
26、m other publications. These normatives references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Prestandard only when incorporated in it by ame
27、ndment or revision. For undated references the latest edition of the publication referred to applies. EN ISO3696:1995, Water for analytical laboratory use Specification and test methods. ISO5725:1986, Precision of the test methods Determination of repeatability and reproducibility for a standard tes
28、t method by inter-laboratory tests. 3 Symbols For the purposes of this standard the following symbols apply: 4 Principle 13 C/ 12 C isotope ratio in the carbon dioxide obtained from total and careful combustion of the sugars is determined by an isotope ratio mass spectrometer. 5 Reagents Use only re
29、agents of recognized analytical grade and only water in accordance with at least grade3 of ENISO3696:1995. 5.1 Calcium hydroxide 5.2 Sulfuric acid, 95% to97% (m/m) 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Isotope ratio mass spectrometer, with the ability to deter
30、mine the 13 C content of CO 2gas at natural abundance with an internal precision of0,05 or better (expressed in relative d value (see8). The internal precision is here defined as the difference between two measurements of the sameCO 2sample. The mass spectrometer will generally be fitted with a trip
31、le collector to simultaneously register at mass numbers44,45 and46. The mass spectrometer should either be fitted with a dual inlet system, for alternatively measuring the unknown sample and a standard, or use an on-line system which combusts the sample in an elemental analyser(6.2) followed by GC s
32、eparation of the combustion products prior to isotopic mass spectrometric determination. The former method offers the highest accuracy for the determination of variations in the isotope contents in the range of the natural abundance. However, correct results can also be obtained using the on-line me
33、thod provided a secondary standard is used. 6.2 Combustion apparatus (elemental analyser), which can quantitatively convert all carbon of the sample into carbon dioxide (CO 2 ), and which is able to remove all other combustion products mainly water from the CO 2 . ( 13 C/ 12 C) Isotope ratio of carb
34、on13 to carbon12 for a considered sample; d 13 C Carbon13( 13 C) content expressed in parts per thousand (); g Acceleration due to gravity at the surface of the earth (9,81m/s 2 );ENV 12140:1996 4 BSI 12-1998 6.3 Centrifuge, capable of producing a centrifugal acceleration of1400g at the base of the
35、centrifuge tube(6.4). NOTEThe rotational frequency required to give correct centrifugal acceleration can be calculated from the following equation: where: 6.4 Centrifuge tubes, of50ml capacity 7 Procedure 7.1 Preparation of the test sample Remove the solid constituent of a sample of approximately50m
36、l of natural or reconstituted fruit juice by centrifugation(6.3), at1400g for10min. 7.2 Purification and separation of sugars Purify the soluble substances remaining in the supernatant liquid after centrifugation by the addition of2g of powdered calcium hydroxide(5.1) to the solution whilst stirring
37、 it well (using, for example a magnetic stirrer) and heating in a water bath at90C for3min. During this stage of the procedure, organic acids, amino acids and other compounds are precipitated. Separate the precipitate by centrifugation(6.3) of the hot solution (for3min at1400g). Decant the clear sup
38、ernatant liquid and acidify it with0,1mol/l sulfuric acid(5.2) in order to obtain a pH of approximately5 when the colour of the solution changes. This solution contains mainly sugars, calcium sulfate and some colorants as minor ingredients. Partially remove residual calcium sulfate by storing the so
39、lution in a refrigerator at approximately4C overnight (approximately15h) followed by decantation. Freeze-dry the supernatant liquid and homogenize the lyophilisate to a fine powder before storing it in a glass vial with an air-tight plastics cap. 7.3 Combustion of sugars Combust the sample obtained
40、using the procedure given in7.2 in a circulating oxygen gas stream or in an elemental analyser(6.2). It is essential to effect complete conversion of organic carbon into carbon dioxide by a method that avoids any isotopic fractionation and allows the collection of the gas as a whole. A liquid nitrog
41、en trap is usually employed to collect the carbon dioxide before analysis by isotope ratio mass spectrometry. NOTESuitable microcombustion systems are commercially available. 7.4 Determination The 13 C/ 12 C isotope ratio in the carbon dioxide obtained from combustion of the sugars, as givenin7.3, i
42、s determined with the aid of an isotope ratio mass spectrometer (6.1). Determine the ratio for the isotopic species 13 CO 2 / 12 CO 2from the corresponding intensities. 8 Calculation In addition to the commonly used mass isotopic abundance (in of atoms), the so-called delta value(d) is also used as
43、an alternative system of units for indicating isotope content. Delta values are used exclusively for indicating variations (of the third decimal place) in the natural isotopic abundance. a =11,18 r (n/1000) 2 (1) a is the centrifugal acceleration; r is the radius of the centrifuge in centimetres, me
44、asured from the mid point (the centrifuge axis) to the bottom of the centrifuge tube when swung out; n is the rotational frequency per minute.ENV 12140:1996 BSI 12-1998 5 Express the d13 C values as the relative difference per thousand between the 13 C and 12 C ratios of a sample in relation to a st
45、andard, Pee Dee Belemnite from South Carolina in USA (the PDB standard). This is a fossil calcium carbonate with an isotope ratio ( 13 C/ 12 C) PDB =0,0112372 for the emitted CO 2 . This value is the reference point of the common international PDB scale for d 13 C values expressed in parts per thous
46、and () which are calculated using the following equation: A suitable secondary standard for routine use in this method is NBS22 (obtained from International Atomic Energy Agency (IAEA) 1) ), which has a value of 29,80 relative to PDB. 9 Precision Details of the interlaboratory test on precision of t
47、he method are summarized in annex B. The values derived from the interlaboratory test may not be applicable to analyte concentration ranges and matrices other than given in annex B. 9.1 Repeatability The absolute difference between two single results found on identical test material by one operator
48、using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than5% of the cases. The values are: 9.2 Reproducibility The absolute differences between two single test results on identical test material reported by two laboratories will exceed
49、the reproducibility limit R in not more than5% of the cases. The values are: 10 Test report The test report shall contain the following data: all information necessary for the identification of the sample (kind of sample, origin of sample, designation); a reference to this European Standard; the date and type of sampling procedure (ifknown); the date of receipt; (2) 1) International Atomic Energy Agency P.O. Box 100 A 1400 Wien AUSTRIA Orange juice r =0,26 ; Pineapple juice r =0,42 ; Beet sugar r =0,17 ; Cane suga
