《ISO-10727-2002.pdf》由会员分享，可在线阅读，更多相关《ISO-10727-2002.pdf（14页珍藏版）》请在三一文库上搜索。
1、INTERNATIONAL STANDARD ISO 10727 Second edition 2002-07-15 Reference number ISO 10727:2002(E) ISO 2002 Tea and instant tea in solid form Determination of caffeine content Method using high-performance liquid chromatography Th et th soluble sous forme solide Dtermination de la teneur en cafine Mthode
2、 par chromatographie liquide haute performance ISO 10727:2002(E) ii ISO 2002 All rights reserved PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded
3、 are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobes licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Det
4、ails of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem
5、relating to it is found, please inform the Central Secretariat at the address given below. ISO 2002 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm,
6、without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.ch Web www.iso.ch Printed in Switzerland -,-,- ISO 10727:200
7、2(E) ISO 2002 All rights reserved iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body
8、 interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotec
9、hnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publ
10、ication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or al
11、l such patent rights. International Standard ISO 10727 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Tea. This second edition cancels and replaces the first edition (ISO 10727:1995), which has been technically revised. Annex A of this International Standard is for
12、information only. -,-,- -,-,- INTERNATIONAL STANDARDISO 10727:2002(E) ISO 2002 All rights reserved 1 Tea and instant tea in solid form Determination of caffeine content Method using high-performance liquid chromatography 1Scope This International Standard specifies a method for the determination by
13、high-performance liquid chromatography (HPLC) of the caffeine content of teas and instant teas. It is applicable to green tea, black tea and decaffeinated tea products. 2Normative references The following normative documents contain provisions which, through reference in this text, constitute provis
14、ions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the
15、normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 1572:1980, Tea Preparation of ground sample of known dry matter content ISO 3696:19
16、87, Water for analytical laboratory use Specification and test methods ISO 7513:1990, Instant tea in solid form Determination of moisture content (loss in mass at 103 C) 3Principle The caffeine from a test portion is extracted by reflux with water in the presence of magnesium oxide. After filtration
17、, the caffeine content is quantified using high-performance liquid chromatography with ultraviolet detection. 4Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 4.1Water, in accordance with grade 1 of ISO 3696. 4.2Methanol, HPLC grade. 4.3Mobile phase, methanol/w
18、ater mixture. Add of the methanol (4.2) to a 2 litre one-mark volumetric flask. Dilute to the mark with water and mix. Filter the mixture through a filter of pore size (5.3). By adjusting the methanol concentration, the retention time of the caffeine can be modified to optimize the HPLC separation.
19、This can also be done by increasing the column temperature, but a temperature of should not be exceeded. 4.4Ethanol/water mixture, (volume fraction). 600 ml 0,45m 60 C 1/4 -,-,- ISO 10727:2002(E) 2 ISO 2002 All rights reserved 4.5Magnesium oxide (so-called “dense magnesium oxide”). NOTELight magnesi
20、um oxide may result in inaccurate caffeine content values. 4.6Caffeine stock standard solution, corresponding to . Weigh, to the nearest , of caffeine into a one-mark volumetric flask. Add sufficient ethanol/water mixture (4.4) to half fill the flask. Swirl to dissolve the caffeine and then dilute t
21、o the mark with the ethanol/water mixture. This solution will remain stable for up to 1 month if stored in a refrigerator. 4.7Caffeine standard solutions. Standard solutions A, B, C and D shall be prepared on the day of use. 4.7.1Caffeine standard solution A, corresponding to . Allow the caffeine st
22、ock standard solution (4.6) to warm up to room temperature. Using a pipette, transfer of this solution to a one-mark volumetric flask. Dilute to the mark with water and mix. 4.7.2Caffeine standard solution B, corresponding to . Follow the procedure given in 4.7.1 but take, using a pipette, of the st
23、ock standard solution (4.6). 4.7.3Caffeine standard solution C, corresponding to . Follow the procedure given in 4.7.1 but take, using a pipette, of the stock standard solution (4.6). 4.7.4Caffeine standard solution D, corresponding to . Using a pipette, transfer of the standard solution B (4.7.2) t
24、o a one-mark volumetric flask. Dilute to the mark with water and mix. 5Apparatus Usual laboratory apparatus, and, in particular, the following. 5.1High-performance liquid chromatograph, equipped with an ultraviolet detector, allowing measurements to be made at a wavelength between and , and a data c
25、ollection/integration system or chart recorder. NOTEA wavelength close to is preferred since the maximum UV absorption of caffeine is at . Where diurnal variations in temperature are wide, a means of ensuring a constant column temperature should be provided, for example a column oven or water jacket
26、. 5.2Chromatographic column for HPLC, a reversed-phase C18 type, preferably with spherical particles and having an efficiency of at least theoretical plates1). The theoretical plate number of a column can be calculated as follows, from the shape of the peak obtained by injection of one of the caffei
27、ne standard solutions (4.7): 1)Spherisorb 5 ODS, Spherisorb 10 ODS, Nucleosil 5 C18, Nucleosil 7 C18, Nucleosil 10 C18, Zorbax BP C18, Hypersil ODS, CP-Sper C18, Bondapak C18, Supelcosil L C18 and Partisphere C18 are examples of suitable products available commercially. This information is given for
28、 the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. 500g/ml 0,001 g0,125 g250 ml 15g/ml 3,0 ml 100 ml 10g/ml 2,0 ml 5g/ml 1,0 ml 2g/ml 20 ml100 ml 254 nm280 nm 280 nm272 nm 5 000N N =5,54 ? t W0,5 ?2 ISO 10727:2002(E) ISO 2002 All
29、 rights reserved 3 where is the retention time of the peak; is the peak width at half peak height. NOTEIn this International Standard, the chromatographic conditions and the composition of the mobile phase (4.3) specified are suitable for a Partisphere C18 cartridge column of dimensions , fitted in
30、a Whatman HPLC cartridge system. If other types of column are used, an alternative mobile phase and alternative chromatographic conditions may be necessary. 5.3Filter, membrane filter unit of pore size. 5.4Electric heating mantle, to accommodate a 1 litre round-bottom flask. 5.5Flask, round bottom,
31、of capacity 1 litre, with ground glass neck. 5.6Anti-bumping granules. 5.7Condenser, water-cooled with ground glass joint to fit a 1 litre round-bottom flask. 5.8Analytical balance, capable of weighing to an accuracy of . 6Sampling It is important that the laboratory receive a sample that is truly r
32、epresentative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. Recommended sampling methods are given in ISO 1839 for regular green tea and black tea; ISO 7516 for instant tea. 7Preparation of test sample Gr
33、ind the sample of green tea or black tea in accordance with ISO 1572. Grinding is not required for instant tea samples. 8Procedure 8.1General If it is required to check whether the repeatability requirement is met, carry out two single determinations in accordance with 8.2 to 8.6 under repeatability
34、 conditions. 8.2Determination of dry matter content Calculate the dry matter content from the moisture content (loss in mass at ) determined on a portion of the test sample in accordance with: ISO 1572 for green tea or black tea; ISO 7513 for instant tea. t W0,5 110 mm4,6 mm 0,45m 0,001 g 103 C -,-,
35、- ISO 10727:2002(E) 4 ISO 2002 All rights reserved 8.3Test portion 8.3.1Green tea and black tea Weigh, to the nearest , of the test sample (clause 7) into a round-bottom flask (5.5). 8.3.2Instant tea Weigh, to the nearest , of the test sample (clause 7) into a round-bottom flask (5.5). 8.4Extraction
36、 of caffeine 8.4.1Add, to the tea in the flask, of magnesium oxide (4.5), a few anti-bumping granules (5.6) and of water. Weigh, to the nearest , the flask and contents. 8.4.2Mix the contents. Attach the condenser (5.7) and place the flask in the electric heating mantle (5.4) and rapidly bring to bo
37、iling. Reduce the heat and gently simmer for with occasional swirling. 8.4.3Remove the flask and contents from the heating mantle and cool to room temperature. Dry the outside of the flask and weigh to the nearest . The mass of the cooled flask and contents shall be equal to the mass determined in 8
38、.4.1. If the masses differ, add sufficient water to increase the mass to that determined in 8.4.1 and then mix contents. 8.4.4Leave the flask to stand to allow the contents to settle, then remove approximately of the supernatant solution and pass it through the filter (5.3). 8.5Dilution (for regular
39、 green tea and black tea and for regular instant tea only) Using a pipette, transfer of the filtrate obtained in 8.4.4 to a one-mark volumetric flask. Dilute to the mark with water and mix. For decaffeinated products, no dilution is necessary. 8.6Determination 8.6.1Adjustment of the apparatus Set up
40、 the chromatograph (5.1) in accordance with the manufacturers instructions and adjust it as follows: flow rate of the mobile phase (4.3): to depending upon the column used (see 5.2); temperature of the column (5.2): optimally (but see 4.3). 8.6.2HPLC analysis Once the flow rate of the mobile phase (
41、4.3) and the temperature are stable, inject onto the column of each of the caffeine standard solutions A, B, C and D (4.7.1 to 4.7.4) followed by an equal volume of the test solution (obtained using the procedure in 8.4.4 for decaffeinated tea and the procedure in 8.5 for regular leaf tea and regula
42、r instant tea). Repeat injection of standard solutions at regular intervals (typically after six test solutions). Collect and record the data for the peaks of all standards and test samples. 0,001 g 1,0 g 0,001 g 0,5 g 4,5 g0,5 g 300 ml0,1 g 20 min 0,1 g 10 ml 1,0 ml10 ml 0,5 ml/min1,5 ml/min 40 C 2
43、0l -,-,- ISO 10727:2002(E) ISO 2002 All rights reserved 5 9Calculation Identify and measure the peak areas or heights (area is preferable) for all standards and samples. Construct a linear calibration graph of caffeine standard concentration (micrograms per millilitre) against peak areas or heights
44、and obtain the response factor (RF) automatically using a data collection/integration system, or manually from a selected point on the calibration graph: where RFis the response factor for caffeine; is the concentration of caffeine in the caffeine standard solution, in micrograms per millilitre; is
45、the peak area for the caffeine standard solution; is the peak height for the caffeine standard solution. The caffeine content, , expressed as mass fraction in percent of the sample on a dry basis, is given by the formula or where is the peak area for the test sample; is the peak height for the test
46、sample; is the response factor for caffeine; is the dilution factor (10 for regular leaf teas and instant teas); is the mass, in grams, of the test sample; is the dry matter content (mass fraction, in percent), determined in accordance with 8.2. 10Precision 10.1Interlaboratory test Details of the in
47、terlaboratory test to determine the precision of the method are summarized in annex A. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. RF= Cstd Astd orRF= Cstd hstd Cstd Astd hstd w w = AsampRF 300d m 100 wDM 100 1
48、000 000 w = hsampRF 300d m 100 wDM 100 1 000 000 Asamp hsamp RF d m wDM -,-,- ISO 10727:2002(E) 6 ISO 2002 All rights reserved 10.2Repeatability The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than of cases be greater than the repeatability limit ( ) values given in Table A.1. 10.3Reproduci