1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12742:1999 The Euro
2、pean Standard EN 12742:1999 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 free amino acids content Liquid chromatographic methodThis British Standard, having been prepared under th
3、e direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 June 1999 BSI 06-1999 ISBN 0 580 32215 7 BS EN 12742:1999 Amendments issued since publication Amd. No. Date Comments National foreword This Bri
4、tish Standard is the English language version of EN 12742:1999. The UK participation in its preparation was entrusted to Technical Committee AW/21, Fruit and vegetable juices, which has the responsibility to: aid enquirers to understand the text; present to the responsible European committee any enq
5、uiries 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 request to its secretary. Cross-references The Br
6、itish 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 of the BSI Standards Electronic Catalogue. A Brit
7、ish 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. Summary of pages This document comprises a fron
8、t cover, an inside front cover, the EN title page, pages 2 to 10, an inside back cover and a back cover.CEN European Committee for Standardization 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 expl
9、oitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12742:1999 E EUROPEAN STANDARD EN 12742 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, amino acids, ch
10、romatographic analysis English version Fruit and vegetable juices Determination of the free amino acids content Liquid chromatographic method Jus de fruits et de le gumes Dosage des teneurs en acides amine s libres Me thode par chromatographie en phase liquide Frucht- und Gemu sesa fte Bestimmung de
11、r Gehalte an freien Aminosa uren Flu ssigchromatographisches Verfahren This European Standard was approved by CEN on 17 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 st
12、andard 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 in any other
13、 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, Germany, Gre
14、ece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12742:1999 BSI 06-1999 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174, Fruit and vegetable juices Methods of analysis, the Secretariat
15、 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 the CEN/CE
16、NELEC 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, Sweden, S
17、witzerland and the United Kingdom. Contents Page Foreword 2 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 6 Annex B (informative) Statistical results of the inter
18、-laboratory test 6 Annex C (informative) Details of typical conditions and reagents used in the analysis of free amino acids in fruit and vegetable juices 9Page 3 EN 12742:1999 BSI 06-1999 1) These materials can be supplied by the Institute for reference materials and measurement, C.C.R Retieseweg,
19、2400 Geel, Belgium. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. 1 Scope This European Standard specifies a chromatographic
20、 procedure for the determination of the free amino acid content in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate pl
21、aces 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 incorporated in it by amendment or revision. For undated references the latest edition of the publication ref
22、erred to applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods. (ISO 3696:1987) 3 Symbols For the purpose of this European Standard, the following symbols apply: c substance concentration; r mass concentration; g acceleration due to gravity at the surface of t
23、he earth (9,81 m/s 2 ). 4 Principle Amino acids are separated on a cation exchange chromatography column by stepwise elution with a range of lithium citrate buffers of different molarities and pH values. After a colour reaction, with ninhydrin, the amino acids are quantified photometrically at a wav
24、elength of 570 nm (or 440 nm for proline). 5 Reagents 5.1 General Use only reagents of recognized analytical grade and only water in accordance with at least grade 1 of EN ISO 3696:1995. 5.2 Buffer solutions for amino acid elution Prepare the buffer solutions in accordance with the manufacturers ins
25、tructions. Ready-prepared lithium citrate buffer of different pH values and lithium ion molarities can also be used. The molarities and pH values of buffer solutions can differ in accordance with the equipment manufacturers instructions (e.g. the length and diameter of the analytical column and type
26、 of resin). Therefore it is impossible to give exact details for the preparation of the various buffer solutions. The description of the buffer solutions detailed in annex C are therefore only for use as a guide for the user of this standard. 5.3 Lithium hydroxide solution c(LiOH) = 0,2 mol/l to 0,4
27、 mol/l An example for the preparation of this solution for column regeneration is given in annex C. 5.4 Colour reagent Ninhydrin in a solution of 2-methoxyethanol and sodium acetate buffer. The stability of this mixture is very variable and ranges from a few days to a few weeks. An example for the p
28、reparation of the solution used in the colour development stage of this process is given in annex C. 5.5 Lithium citrate buffer (pH = 2,00 to 2,20) for sample dilution An example for the preparation of this dilution buffer is given in annex C. 5.6 Standard amino acids Weigh out individual portions o
29、f amino acids so that, after appropriate dilution, the absolute levels in the analyser fall within the linear range for each amino acid. It is useful to use a standard solution of similar concentration to that expected in the sample. The use of certified reference materials 1) is recommended for thi
30、s procedure. An example for the preparation of the amino acid test solution is given in annex C. 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Amino acid analyser, with one of the following column dimensions: 9m m3500 mm, 6 mm3 200 mm, 4,6 mm3 200 mm or 270 mm, 4 mm3
31、200 mm, 4 mm3 100 mm or 3,2 mm3 140 mm The columns can be made of glass or steel. The various columns, described above, give significant differences in peak resolution and analysis times.Page 4 EN 12742:1999 BSI 06-1999 2) Durrum, Biotronik, Beckman r is the radius of the centrifuge in centimetres,
32、measured from the mid point (the centrifuge axis) to the bottom of the centrifuge tube when swung out; n is the rotational frequency per minute. 6.4 Centrifuge tubes 6.5 Membrane filter, non sterile hydrophilic syringe filter(s) with a pore size of 0,45mm. 7 Procedure 7.1 Preparation of the test sam
33、ple Normally products shall not be pre-treated and their analysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The analysis of concentrated samples may also be carried out on a volumetric basis, after dilution to a known relative density. In this case,
34、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 basis
35、 of a weighed test sample is the usual procedure. Mix cloudy samples well before dilution. Mix the juice sample with the dilution buffer (5.5) and the internal standard solution (c(nor-leucine or nor-valine) = 1 mmol/l, as detailed in annex C). Centrifuge this solution at 4 000 g for 15 min. Finally
36、 clarify the sample by passing the solution through the syringe filter (6.5) and the sample is ready for analysis. NOTE The sample dilution required depends on the expected composition of the juice and loading mixture. However, this normally lies between 1:1 and 1:10. For orange juice a dilution of
37、1:10 is typically used. In special cases, e.g. cherry juice, it is recommended that two separate dilutions be used (for example 1:5 and 1:20). This allows for the wide variation in the concentrations of the various amino acids (for example in the case of cherry the very high level of asparagine). Th
38、e sample loading varies from one system to another and also depends on the sample dilution. However, this is generally between 20ml to 300ml. 7.2 Test procedure Carry out the amino acid separation according to the particular instrumental conditions. Elution with 4 to 6 different buffers (5.2) of dif
39、ferent pH values and ionic strengths can be used. For example a normal initial buffer would have a lithium ion concentration of c(LiOH) = 0,10 mol/l and a pH of 2,94 (buffer A in annex C). This buffer strength would typically increase to c(LiOH) = 1,40 mol/l and this buffer would have a pH of 3,65 (
40、buffer E in annex C). Maintain the column at a constant temperature between approximatively 308C and 808C dependent on the elution step carried out and on the actual system employed. Choose the pH, lithium molarity, the running time of the different buffer and the column temperature to give the opti
41、mum separation conditions, which may vary from one laboratory to another. Regenerate the column with a lithium hydroxide solution (c(LiOH) = 0,2 mol/l to 0,4 mol/l) between samples and prior to re-equilibration of the column in the initial buffer run. The temperature at which the colour reaction, wi
42、th ninhydrin, is carried out is usually between 1008C and 1308C. The reaction products are measured photometrically at a wavelength of 570 nm for all the amino acids except proline which is detected at 440 nm. 8 Calculation Carry out the calibration of the system using an amino acid standard solutio
43、n, for example, that detailed in annex C. The quantification of the individual amino acids is carried out using the internal standard procedure and peak areas. If a concentrated product has been diluted to a single strength, report the relative density of the single strength sample. The results are
44、given in mmol per litre to two decimal places.Page 5 EN 12742:1999 BSI 06-1999 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 analyte concentration ranges and matrices oth
45、er 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 value r in not more than 5 % of the cases. The value
46、s are: for amino acids present at low concentrations c 0,1 mmol/l: r = 2,8 s r ; where: s r = 0,0026 + 0,01323 c mmol/l. 9.2 Reproducibility The absolute difference between single test results on identical test material reported by two laboratories will exceed the reproducibility value R in not more
47、 than 5 % of the cases. The values are: for amino acids present at low concentrations c 0,1 mmol/l: R = 2,8 S R ; where: S R = 0,007 + 0,04733 c mmol/l. 10 Test Report The test report shall contain the following data: all information necessary for the identification of the sample (kind of sample, or
48、igin 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 units in which they have been expressed; whether the repeatability has been verified; any particular points observed in the
49、 course of the test; any operations not specified in the method or regarded as optional, which might have affected the results.Page 6 EN 12742:1999 BSI 06-1999 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. Moore, S. and Stein, W.H. Chromatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. (1951), 192, 663-681. Determination of free amino acids: No.
