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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 chromatography Licen
3、sed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI This British 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 ef
4、fect on 15 August 1999 BSI 08-1999 ISBN 0 580 32213 0 BS EN 12630:1999 Amendments issued since publication Amd. No.DateComments 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
5、, Fruit and vegetables juices, which has the responsibility to: aid enquirers to understand the text; present 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 development
6、s and promulgate them in the UK. A list of organizations represented on this committee can be obtained on 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 u
7、nder the section entitled International Standards Correspondence Index, or by using the Find facility 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 app
8、lication. Compliance with a British Standard does not of itself confer immunity from legal obligations. 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
9、 this document indicates when the document was last issued. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat:
10、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 STANDARDEN 12630 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices
11、, chemical analysis, determination of content, glucose, fructose, sorbitol, chromatographic analysis, high-performance liquid chromatography English version Fruit and vegetable juices Determination of glucose, fructose, sorbitol and sucrose contents Method using high-performance liquid chromatograph
12、y 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-, Sorbit- und Saccharosegehaltes Hochleistungs-flu ssigchromatographisches Verfahren This Eur
13、opean 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 national standard without any alteration. Up-to-date lists and bibliographical references c
14、oncerning 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 in any other language made by translation under the responsibility of a CEN member into its
15、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, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain,
16、Sweden, Switzerland and United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 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
17、of analysis, the 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 1
18、999. According to 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
19、, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword2 1Scope3 2Normative references3 3Symbols and abbreviations3 4Principle3 5Reagents3 6Apparatus3 7Procedure3 8Calculation4 9Precision4 10Test report5 Annex A (informative) Bibliography6 Annex B (informative) Statisti
20、cal results of the interlaboratory test6 Annex C (informative) Example chromatogram for the determination of sugars and sorbitol by HPLC8 Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 3 EN 12630:1999 BSI 08-1999 1 Scope This European
21、Standard specifies a high-performance liquid chromatographic method for the determination of the glucose, 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
22、 of the peaks. 2 Normative references This European Standard incorporates by dated or undated reference, 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 amendmen
23、ts to or revisions of any of these publications apply to this European Standard only when incorporated 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 metho
24、ds (ISO 3696:1987). 3 Symbols and abbreviations 3.1 Symbols For the purposes of this standard, the following symbols apply: csubstance concentration; rmass concentration; gacceleration due to gravity at the surface of the earth (9,81 m/s2). 3.2 Abbreviations For the purposes of this standard, the fo
25、llowing abbreviations apply: HPLC High-performance liquid chromatography; EDTA Ethylenediaminetetra-acetic acid. 4 Principle The sugars and sorbitol are separated on a cation-exchange resin (sulfonated polystyrene- divinylbenzene copolymer in the Ca2+form) by isocratic elution, using an aqueous solu
26、tion of calcium disodium-EDTA as the mobile phase. The sugars and sorbitol are detected using 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 leas
27、t grade 3 of EN ISO 3696:1995, unless otherwise stated. 5.2 Calcium disodium-EDTA solution, c(C10H12N2O8CaNa2.xH2O) = 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.
28、1). 6 Apparatus Usual laboratory apparatus, and, in particular, the following. 6.1 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 fo
29、rm, particle size 10 mm, length 30 cm, internal diameter 6,5 mm. A column with other dimensions can be used if it gives similar results. 6.3 Syringe filter. Hydrophilic syringe filter, non-sterile, with 0,45 mm pore size. 6.4 Centrifuge, capable of producing a centrifugal acceleration of 1 400g. NOT
30、EThe rotational frequency required to give correct centrifugal acceleration can be calculated from the following equation: (1)a = 11,18 3 r 3 2 n 1 000 where ais the centrifugal acceleration; ris the radius of the centrifuge in centimetres, measured from the midpoint (the centrifuge axis) to the bot
31、tom of the centrifuge tube when swung outwards; nis the rotational frequency per 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 ou
32、t on a volumetric basis, after dilution to a known relative density. In this case, 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
33、 and/or a very high content of cells (for example pulp), determination on the basis of a weighed test sample is the usual procedure. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 4 EN 12630:1999 BSI 08-1999 7.2 Preparation of test sam
34、ple 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 then filtered through the syringe filter (6.3) and is ready for HPLC analysis. 7.3 HPLC conditions Solvent: calc
35、ium disodium-EDTA solution (5.2). Flow rate: 0,5 ml/min. Column temperature: 90 8C (or as defined in the column manufacturers instructions). Injection volume: 10 ml (typically). NOTEThe differential refractive index detector should be held at a constant temperature around ambient (25 8C to 35 8C). 7
36、.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 shall also be used to confirm that no inversion of sucrose occurs on the column under the conditions chosen.
37、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 injection). A typical chromatogram for a mixed standard of the sugars and sorbitol is given in annex C. 8 Calcul
38、ation 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 relationship of the value to mass or volume. If a concentrated product has been diluted to single strength, rep
39、ort 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 Pis the peak area or peak height of the sugar or sorbitol under investigation, depending on which method is chosen; Fis the dilution
40、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 following equation: (3)RF = Ps rs where Psis the peak area or peak height of the particular sugar or sorbitol in the
41、 standard chromatogram; rsis the mass concentration of the particular sugar or sorbitol in the standard solution. Report the concentrations of glucose, fructose, sorbitol and sucrose in grams per litre to one decimal place. NOTE 1Separate response factors should be determined for glucose, fructose,
42、sorbitol and sucrose, using the equation above and the appropriate data. NOTE 2When this method was assessed in a collaborative study, the concentrations of the sugars were also assessed using enzymatic procedures. It was found that the enzymatic methods gave a better reproducibility for glucose and
43、 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. The values derived from the interlaboratory test may not be applicable to analyse concentration ranges and matrices othe
44、r 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 within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of the cases. The values
45、 are given in Table 1. Table 1 SampleGlucose g/l Fructose g/l Sucrose g/l Sorbitol g/l Apple juice1,31,60,90,3 Orange juice1,11,11,4nm1) Cherry nectar 1,41,22,40,5 Grape juice2,01,7nm1)nm1) 1) nm = not measured, as not present in this juice. 9.2 Reproducibility The absolute differences between two s
46、ingle 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 values are given in Table 2. Table 2 SampleGlucose g/l Fructose g/l Sucrose g/l Sorbitol g/l Apple juice2,96,13,00,9 Orange juice3,13,12,5nm1) Che
47、rry nectar 6,26,58,31,7 Grape juice5,19,0nm1)nm1) 1) nm = not measured, as not present in this juice. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 5 EN 12630:1999 BSI 08-1999 10 Test report The test report shall contain the following
48、 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; test results and the units in which they have been
49、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. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 6 EN 12630:1999 BSI 08-1999 Annex A (informative) Bibliography ISO 5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard
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