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1、A Reference number ISO 10519:1997(E) INTERNATIONAL STANDARD ISO 10519 Second edition 1997-11-01 Rapeseed Determination of chlorophyll content Spectrometric method Graines de colza Dtermination de la teneur en chlorophylle Mthode spectromtrique Copyright International Organization for Standardization
2、 Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10519:1997(E) ISO 1997 All rights reserved. Unless otherwise specified, no part of this publication
3、 may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from the publisher. International Organization for Standardization Case Postale 56 CH-1211 Genve 20 Switzerland Internetcentraliso.ch X.400c=ch; a
4、=400net; p=iso; o=isocs; s=central Printed in Switzerland ii 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 commi
5、ttees. Each member body 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 I
6、nternational Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the memb
7、er bodies casting a vote. International Standard ISO 10519 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 2, Oleaginous seeds and fruits. This second edition cancels and replaces the first edition (ISO 10519:1992), which has been technically revised. Annex
8、es A and B of this International Standard are for information only. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without li
9、cense from IHS -,-,- INTERNATIONAL STANDARD ISOISO 10519:1997(E) 1 Rapeseed Determination of chlorophyll content Spectrometric method 1Scope This International Standard specifies a spectrometric method for the determination of the chlorophyll content of rapeseed. It is not applicable to the determin
10、ation of chlorophyll in oils. 2Normative references The following standards contain provisions which, through reference in this test, constitute provisions of this International Standard. At the time of the publication, the editions indicated were valid. All standards are subject to revision, and th
11、e parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 648:1977, Laboratory glassware -
12、 One-mark pipettes. ISO 664:1990, Oilseeds - Reduction of laboratory sample to test sample. ISO 665:1977, Oilseeds - Determination of moisture and volatile matter content. 3Definition For the purposes of this International Standard, the following definition applies. 3.1 chlorophyll content mass frac
13、tion of substances in the sample contributing to the absorption band at a wavelength near 665 nm, as determined under the operating conditions specified in this International Standard and measured as chlorophyll A NOTE The chlorophyll content is expressed in milligrams per kilogram. 4Principle Extra
14、ction of a test portion in a suitable apparatus with a specified extraction solvent. Spectrometric determination of the chlorophyll content of the extracted solution 5Reagent Use only reagents of recognized analytical grade unless otherwise stated. Copyright International Organization for Standardiz
15、ation Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10519:1997(E) ISO 2 5.1Extraction solvent Transfer to a 500 ml beaker 100 ml of anhydrous etha
16、nol. Add to the contents of the beaker 300 ml of anhydrous iso-octane (2,2,5-trimethylpentane) or anhydrous technical n-hep- tane or anhydrous petroleum ether (essentially composed of C7 hydrocarbons, with a boiling range between 90 C and 100 C). 6Apparatus Usual laboratory apparatus and, in particu
17、lar, the following. 6.1Analytical balance, capable of weighing to the nearest 0,001 g. 6.2Mechanical grinder, blade type, or coffee mill or equivalent. 6.3Mechanical microgrinder (see figure 1), comprising stainless-steel tubes of approx- imately 50 ml volume which can be securely stoppered, stainle
18、ss-steel ball-bearings ( 16 mm), and an apparatus to shake the securely stoppered tubes horizontally at a frequency of 240 min1, with a horizontal displacement of 3,5 cm, or a Dangoumau ball mill1). Dimensions in millimetres Figure 1 Mechanical microgrinder 6.4Filter paper, medium speed, V-folded. 6
19、.5Spectrometer (preferably with wavelength scanning), suitable for carrying out absorbance measurements at wavelengths between 600 nm and 700 nm, with a spectral bandwidth of 2 nm. 6.6Optical cells, having a path length of at least 1 cm. 6.7Pipettes, of 30 ml capacity, complying with the requirement
20、s of ISO 648, class A, or a repetitive dispenser capable of dispensing 30 ml with an error of less than 1 %. 6.8Culture tubes, of 20 ml capacity, provided with stoppers. 1) Dangoumau ball mill is an example of a suitable apparatus available commercially. This information is given for the convenience
21、 of users of this International Standard and does not constitute an endorsement by ISO of this apparatus. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduct
22、ion or networking permitted without license from IHS -,-,- ISOISO 10519:1997(E) 3 7Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 542. It is important that the laboratory receive a sample which is truly representati
23、ve and has not been damaged or changed during transport and storage. 8Preparation of test sample Prepare a test sample in accordance with ISO 664 from the laboratory sample as received, after separation of impurities. Dry seeds with a moisture content of greater than 10 % (m/m) for 12 h at 45 C to r
24、educe the moisture level to 10 % (m/m) or less in order to reduce the risk of destroying chlorophyll pigments. Transfer 50 g of the test sample to the mechanical grinder (6.2) and grind to produce a uniformly ground seed. If a small grinder such as a coffee mill is used, grind several portions of 10
25、 g and then combine them and mix thoroughly the ground portions. 9Procedure 9.1Test portions Weigh, to the nearest 0,001 g, 2 g of the test sample (clause 8) into a stainless-steel tube or the extraction vessel of the Dangoumau ball mill (6.3). 9.2Extraction 9.2.1 Add, using a pipette (6.7), 30 ml o
26、f the extraction solvent (5.1) to the tube or vessel. If using a tube, add three stainless-steel balls to the tube and shake for 1 h. For Dangoumau ball mills, add at least four medium-sized steel balls to the vessel and extract for 20 min. 9.2.2 Allow the extract to settle for 10 min and then decan
27、t a sufficient volume of the extract through the filter paper (6.4) into a culture tube (6.8) to fill the optical cell (6.6). Stopper the tube as soon as possible to minimize evaporation. NOTE The presence of more than one phase in the extraction solvent indicates the presence of excessive moisture,
28、 either in the sample which should contain less than 10 % (m/m) moisture or in the solvents (which should be anhydrous). 9.3Determination Transfer the filtered extract to a cell (6.6) and determine by means of the spectrometer (6.5) the absorbance at wavelengths of 665 nm, 705 nm and 625 nm. (The re
29、adings at 705 nm and 625 nm are used to calculate a baseline correction.) Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted with
30、out license from IHS -,-,- ISO 10519:1997(E) ISO 4 10Expression of results The chlorophyll content, w, in milligrams per kilogram of the product as received, is given by the formula w kAV ml = corr where Acorr (the corrected absorbance) is equal to A665 - (A705 + A625)/2; A665is the absorbance at 66
31、5 nm; A705is the absorbance at 705 nm; A625is the absorbance at 625 nm; kis a constant which is equal to 13; lis the path length, in centimetres, of the optical cell; mis the mass, in grams, of the test portion; Vis the volume, in millilitres, of solvent added to the tube (9.2.1). If it is desired t
32、o express the chlorophyll content relative to the dry product, take into account in the calculation the moisture content of the sample, determined in accordance with ISO 665. 11Precision Details of an interlaboratory test on the precision of the method are summarized in annex A. The values derived f
33、rom this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. 11.1 Repeatability 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 operat
34、or using the same equipment within a short interval of time, should not be greater than 10 % of the arithmetic mean of the two results. 11.2 Reproducibility The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories wi
35、th different operators using different equipment should not be greater than 20 % of the arithmetic mean of the two results. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:
36、17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISOISO 10519:1997(E) 5 12Test report The test report shall specify the method in accordance with which sampling was carried out, if known, the method used, the test result(s) obtained, and if the repeatability has been chec
37、ked, the final quoted result obtained. It shall also mention all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the result(s). The test report shall include all information necessary for the co
38、mplete identification of the sample. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10519:
39、1997(E) ISO 6 Annex A (informative) Results of interlaboratory test An interlaboratory test was carried out by 16 laboratories in accordance with ISO 57252). The repeatability and reproducibility values shown in table A.1 were obtained. Table A.1 - Statistical results of interlaboratory test Sample
40、1) Parameter ABCDEFHIJMean Number of labs. participating 161616161616161616 Number of outliers 2) 111111114 Mean value (mg/kg) 25,831,040,449,875,782,910,520,431,2 Repeatability standard deviation sr, mg/kg 1,131,471,451,752,742,990,490,680,99 Reproducibility standard deviation sR, mg/kg 1,932,153,0
41、23,444,95,681,021,632,19 Repeatability coefficient of variation, % 4,394,763,593,523,623,614,663,353,163,85 Reproducibility coefficient of variation, % 7,476,947,496,916,476,859,798,007,047,44 Repeatability limit (r), mg/kg 3,174,134,064,907,688,381,381,932,79 Reproducibility limit (R), mg/kg 5,396,
42、038,479,6313,7115,902,894,616,21 Repeatability (r), % 12,313,310,19,910,110,113,19,58,910,81 Reproducibility (R),% 20,919,42119,318,119,227,522,519,920,88 1) Samples A through E were from the 1992 study; samples F through H were from the 1990 study. A: 25 mg/kg chlorophyll B: 30 mg/kg chlorophyll C:
43、 40 mg/kg chlorophyll D: 50 mg/kg chlorophyll E: 75 mg/kg chlorophyll F: 80 mg/kg chlorophyll H: 10 mg/kg chlorophyll I: 20 mg/kg chlorophyll J: 30 mg/kg chlorophyll 2) One laboratory in each of the studies was eliminated due to failure to carry out the method correctly. With sample J, three additio
44、nal laboratories were eliminated because of a difference greater than 3 mg/kg between two values. 2) ISO 5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests (now withdrawn), was used to evaluate the precision da
45、ta. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISOISO 10519:1997(E) 7 Annex B (informative
46、) Bibliography 1 ISO 542:1990, Oilseeds Sampling. 2 ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technica
47、l Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10519:1997(E) ISO ICS 67.200.20 Descriptors: agricultural products, plant products, oilseeds, rape, chemical analysis, determination of content, chlorophyll, spectrometric, method. Price based on 6 pages Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/24/2007 23:09:17 MDTNo reproduction or networking permitted without license from IHS -,-,-
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