1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12630:1999 The Euro
2、pean Standard EN 12630:1999 has the status of a British Standard ICS 67.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Fruit and vegetable juices Determination of glucose, fructose, sorbitol and sucrose contents Method using high-performance liquid chromatographyThis B
3、ritish Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 August 1999 BSI 08-1999 ISBN 0 580 32213 0 BS EN 12630:1999 Amendments issued since publication A
4、md. No. Date Comments National foreword This British Standard is the English language version of EN 12630:1999. The UK participation in its preparation was entrusted to Technical Committee AW/21, Fruit and vegetables juices, which has the responsibility to: aid enquirers to understand the text; pres
5、ent to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on
6、request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility
7、of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations
8、. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover. The BSI copyright notice displayed throughout this document indicates when the document was last issued.CEN European Committee for Standardization
9、Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1999 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12630:1999 E EUROPEAN STANDARD EN 12630 NORME EUROPE
10、 ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, glucose, fructose, sorbitol, chromatographic analysis, high-performance liquid chromatography English version Fruit and vegetable juices Determination of glucose,
11、 fructose, sorbitol and sucrose contents Method using high-performance liquid chromatography Jus de fruits et de le gumes Dosage du glucose, du fructose, du sorbitol et du saccharose Me thode par chromatographie liquide haute performance Frucht- und Gemu sesa fte Bestimmung des Glucose-, Fructose-,
12、Sorbit- und Saccharosegehaltes Hochleistungs-flu ssigchromatographisches Verfahren This European Standard was approved by CEN on 8 January 1999. 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
13、national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version i
14、n any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, G
15、ermany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12630:1999 BSI 08-1999 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174, Fruit and vegetable juices Methods of analysis, the
16、Secretariat of which is held by AFNOR. 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 August 1999, and conflicting national standards shall be withdrawn at the latest by August 1999. According to
17、 the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain
18、, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Symbols and abbreviations 3 4 Principle 3 5 Reagents 3 6 Apparatus 3 7 Procedure 3 8 Calculation 4 9 Precision 4 10 Test report 5 Annex A (informative) Bibliography 6 Annex B (informative) Sta
19、tistical results of the interlaboratory test 6 Annex C (informative) Example chromatogram for the determination of sugars and sorbitol by HPLC 8Page 3 EN 12630:1999 BSI 08-1999 1 Scope This European Standard specifies a high-performance liquid chromatographic method for the determination of the gluc
20、ose, fructose, sorbitol and sucrose contents in fruit and vegetable juices and related products. This method does not allow the determination of sucrose in the presence of maltose, due to overlapping of the peaks. 2 Normative references This European Standard incorporates by dated or undated referen
21、ce, provisions from other publications. These normative 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 Standard only when incorporate
22、d in it by amendment or revision. For undated references, the latest edition of the publication referred to applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987). 3 Symbols and abbreviations 3.1 Symbols For the purposes of this standard, the fo
23、llowing symbols apply: c substance concentration; r mass concentration; g acceleration due to gravity at the surface of the earth (9,81 m/s 2 ). 3.2 Abbreviations For the purposes of this standard, the following abbreviations apply: HPLC High-performance liquid chromatography; EDTA Ethylenediaminete
24、tra-acetic acid. 4 Principle The sugars and sorbitol are separated on a cation-exchange resin (sulfonated polystyrene- divinylbenzene copolymer in the Ca 2+ form) by isocratic elution, using an aqueous solution of calcium disodium-EDTA as the mobile phase. The sugars and sorbitol are detected using
25、a differential refractive index detector and quantified using the external standard method. 5 Reagents 5.1 General. Use only reagents of recognized analytical grade and only water in accordance with at least grade 3 of EN ISO 3696:1995, unless otherwise stated. 5.2 Calcium disodium-EDTA solution, c(
26、C 10 H 12 N 2 O 8 CaNa 2 .xH 2 O) = 0,1 mmol/l, in 1 l of HPLC-grade water (5.1). 5.3 Standard solution. Prepare a standard solution of glucose, fructose, sorbitol and sucrose, at a concentration of 10 g/l in water (5.1). 6 Apparatus Usual laboratory apparatus, and, in particular, the following. 6.1
27、 HPLC equipment, consisting of HPLC pump, HPLC column (6.2) and differential refractive index detector, and column heater. 6.2 HPLC column. Polystyrene-divinylbenzene sulfonated cation-exchange column in the calcium form, particle size 10mm, length 30 cm, internal diameter 6,5 mm. A column with othe
28、r dimensions can be used if it gives similar results. 6.3 Syringe filter. Hydrophilic syringe filter, non-sterile, with 0,45mm pore size. 6.4 Centrifuge, capable of producing a centrifugal acceleration of 1 400g. NOTE The rotational frequency required to give correct centrifugal acceleration can be
29、calculated from the following equation: (1) a = 11,183 r3 2 n 1 000 where a is the centrifugal acceleration; r is the radius of the centrifuge in centimetres, measured from the midpoint (the centrifuge axis) to the bottom of the centrifuge tube when swung outwards; n is the rotational frequency per
30、minute. 6.5 Centrifuge tubes. 7 Procedure 7.1 General Analysis by this method shall be on a volumetric basis, the results being expressed per litre of sample. The analysis of concentrated products may also be carried out on a volumetric basis, after dilution to a known relative density. In this case
31、, the relative density shall be indicated. Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be expressed per kilogram of product. In products with a high viscosity and/or a very high content of cells (for example pulp), determination on the bas
32、is of a weighed test sample is the usual procedure.Page 4 EN 12630:1999 BSI 08-1999 7.2 Preparation of test sample Mix cloudy samples well before dilution. The single-strength juice shall be diluted one volume part plus four volume parts of water and centrifuged at 1 400g for 15 min. The sample is t
33、hen filtered through the syringe filter (6.3) and is ready for HPLC analysis. 7.3 HPLC conditions Solvent: calcium disodium-EDTA solution (5.2). Flow rate: 0,5 ml/min. Column temperature: 908C (or as defined in the column manufacturers instructions). Injection volume: 10ml (typically). NOTE The diff
34、erential refractive index detector should be held at a constant temperature around ambient (258Ct o3 58 C). 7.4 HPLC analysis After equilibration of the HPLC system and the column, determine the retention times of the sugars and sorbitol by injection of solutions of the single compounds. This stage
35、shall also be used to confirm that no inversion of sucrose occurs on the column under the conditions chosen. Subsequent analysis can be carried out using a mixed standard. The standard solutions are injected at regular intervals between test samples (for example, typically every fifth or seventh inj
36、ection). A typical chromatogram for a mixed standard of the sugars and sorbitol is given in annex C. 8 Calculation The sugars and sorbitol concentrations are determined using the external standard method, by peak areas or peak heights. During the calculation, allow for any dilution factor and the re
37、lationship of the value to mass or volume. If a concentrated product has been diluted to single strength, report the relative density of the single-strength sample. Calculate the concentrations of the sugars and sorbitol, r, using the following equation: (2) r = 3 F P RF where P is the peak area or
38、peak height of the sugar or sorbitol under investigation, depending on which method is chosen; F is the dilution factor (5 for juices; for diluted concentrates the factor shall be calculated); RF is the appropriate response factor for the particular sugar or sorbitol, and is calculated using the fol
39、lowing equation: (3) RF = P s r s where P s is the peak area or peak height of the particular sugar or sorbitol in the standard chromatogram; r s is the mass concentration of the particular sugar or sorbitol in the standard solution. Report the concentrations of glucose, fructose, sorbitol and sucro
40、se in grams per litre to one decimal place. NOTE 1 Separate response factors should be determined for glucose, fructose, sorbitol and sucrose, using the equation above and the appropriate data. NOTE 2 When this method was assessed in a collaborative study, the concentrations of the sugars were also
41、assessed using enzymatic procedures. It was found that the enzymatic methods gave a better reproducibility for glucose and fructose, whereas the HPLC method gave a better reproducibility for sucrose. 9 Precision Details of the interlaboratory test on precision of the method are summarized in annex B
42、. The values derived from the interlaboratory test may not be applicable to analyse concentration ranges and matrices other than those given in annex B. 9.1 Repeatability The absolute difference between two single results found on identical test material by one operator using the same apparatus with
43、in the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of the cases. The values are given in Table 1. Table 1 Sample Glucose g/l Fructose g/l Sucrose g/l Sorbitol g/l Apple juice 1,3 1,6 0,9 0,3 Orange juice 1,1 1,1 1,4 nm 1) Cherry nectar 1,4 1,2 2,4 0,5 G
44、rape juice 2,0 1,7 nm 1) nm 1) 1) nm = not measured, as not present in this juice. 9.2 Reproducibility The absolute differences between two single test results on identical test material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. The value
45、s are given in Table 2. Table 2 Sample Glucose g/l Fructose g/l Sucrose g/l Sorbitol g/l Apple juice 2,9 6,1 3,0 0,9 Orange juice 3,1 3,1 2,5 nm 1) Cherry nectar 6,2 6,5 8,3 1,7 Grape juice 5,1 9,0 nm 1) nm 1) 1) nm = not measured, as not present in this juice.Page 5 EN 12630:1999 BSI 08-1999 10 Tes
46、t 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 (if known); the date of receipt; the date of test;
47、 test results and the units in which they have been expressed; whether the repeatability of the method has been verified; any particular points observed in the course of the test; any operations not specified in the method, or regarded as optional, which might have affected the results.Page 6 EN 126
48、30:1999 BSI 08-1999 Annex A (informative) Bibliography ISO 5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests. Determination of glucose, fructose, sucrose and sorbitol by HPLC: No. 67, 1995. In: Analyses Collec
49、tion/International Federation of Fruit Juice Producers. Loose-leaf edition, as of 1995. Zug: Swiss Fruit Union. Annex B (informative) Statistical results of the interlaboratory test In accordance with ISO 5725:1986, the following parameters have been defined in an interlaboratory test. (For literature on the method, see annex A). The test was conducted by the Max von Pettenkofer Institute of the Federal Health Office, Food Chemistry Department, Berlin, BRD: year of interlaboratory test: 1993; number of
