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1、INTERNATIONAL STANDARD ISO 15517 First edition 2003-04-15 Reference number ISO 15517:2003(E) ISO 2003 Tobacco Determination of nitrate content Continuous-flow analysis method Tabac Dtermination de la teneur en nitrates Mthode par analyse en flux continu Copyright International Organization for Stand
2、ardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) ii ISO 2003 All rights reserved PDF disclaimer This PDF file may contain embedded typefaces.
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6、of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, 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
7、CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo repr
8、oduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) ISO 2003 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 Standar
9、ds is normally carried out through ISO technical committees. 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
10、 part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is
11、to prepare International Standards. 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 member bodies casting a vote. Attention is drawn to the possibility
12、 that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 15517 was prepared by Technical Committee ISO/TC 126, Tobacco and tobacco products, Subcommittee SC 2, Leaf tobacco. Copyright Interna
13、tional Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- . iv Copyright International Organization for Standardization Provided by IHS und
14、er license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 15517:2003(E) ISO 2003 All rights reserved 1 Tobacco Determination of nitrate content Continuous-flow analysis met
15、hod 1Scope This International Standard specifies a method for the determination of the nitrate content in tobacco by continuous-flow analysis. This method is applicable to manufactured and unmanufactured tobacco. 2Principle An aqueous extract (see below) of the tobacco is prepared and the nitrate co
16、ntent of the extract is determined by reduction of the nitrate ions to nitrite ions with hydrazinium sulfate in the presence of a copper catalyst, followed by reaction with sulfanilamide to form the diazo compound. This is coupled with N-1-naphthyl- ethylenediamine dihydrochloride to form a coloured
17、 complex, which is measured at . If there is any nitrite content, it will be detected and included in the nitrate content result. Collaborative studies have shown that this method gives equivalent results for water and acetic acid extracts. It is recommended that acetic acid extracts should be used
18、if nitrate and reducing substances (see ISO 15153) or reducing carbohydrate analyses (see ISO 15154) are to be carried out simultaneously. 3Reagents All reagents shall be used according to good laboratory practice and existing national regulations. Use distilled water or water of at least equivalent
19、 purity. 3.1Polyoxyethylene lauryl ether (Brij 35 solution) Add 1 litre of water to of Brij 35. Warm and stir until dissolved. 3.2Sodium hydroxide solution (NaOH), p.a. Dissolve of sodium hydroxide in distilled water. Add of Brij 35 solution (3.1) and dilute to 1 litre. 3.3Copper sulfate stock solut
20、ion (CuSO4), p.a. Dissolve of copper(II) sulfate pentahydrate in distilled water and dilute to . 3.4Hydrazinium sulfate/copper sulfate reagent Dissolve the optimum amount of hydrazinium sulfate (N2H6SO4, p.a.) in water. Add of copper sulfate stock solution (3.3) and dilute to 1 litre with water. Sto
21、re in an amber glass bottle. Prepare a fresh solution every month. 3.5Sulfanilamide reagent (NH2C6H4SO2NH2) Add of concentrated orthophosphoric acid (H3PO4, volume fraction , of grade low in nitrate) to approximately of water. Dissolve of sulfanilamide in the solution followed by of N-1-naphthylethy
22、lenediamine dihydrochloride (C10H7NHCH2CH2NH22HCI). 520 nm 5 % 5 % 250 g 8,0 g1 ml 1,20 g100 ml 1,5 ml 25 ml85 % 175 ml2,5 g0,125 g Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo
23、reproduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) 2 ISO 2003 All rights reserved Dilute to with water and filter through a Whatman No. 401) (or equivalent) filter paper. Store in an amber glass bottle. Prepare a fresh solution every 2 days. 3.6Potassium nitrate (K
24、NO3), p.a. 3.7Standard nitrate solutions 3.7.1Stock solution Weigh, to the nearest , approximately of potassium nitrate (3.6). Dissolve in water and dilute to 1 litre in a volumetric flask. This solution contains approximately of nitrate. Store in a refrigerator. Prepare a fresh solution every month
25、. 3.7.2Working standards From the stock solution (3.7.1), produce a series of at least five calibration solutions whose concentrations cover the range expected to be found in the samples (e.g. to of nitrate). Calculate the exact concentration for each standard. Store in a refrigerator. Prepare fresh
26、 solutions every 2 weeks. If acetic acid (five times) is to be used to extract the nicotine from the test sample in 5.2, the working standards (3.7.2) shall be made from the stock solution every 2 weeks using acetic acid instead of water. 4Apparatus Usual laboratory apparatus and, in particular, the
27、 following items. 4.1Continuous flow analyser consisting of sampler, dialyser, heating bath, delay coils, colorimeter (or equivalent) with filter(s), and recorder. See Annex B for an example of a suitable layout. 5Procedure 5.1Preparation of samples for analysis Prepare the tobacco samples for analy
28、sis by grinding (the sample should totally pass through a sieve) and determine the moisture content. If the tobacco is too wet for grinding, it may be dried at a temperature not exceeding . Any contamination from nitrate and nitrite ions shall be minimized. 5.2Test portion Weigh, to the nearest , ap
29、proximately of the tobacco in a dry conical flask. Add of distilled water from a dispenser. Stopper the flask and shake for . 1)Whatman No. 40 is an example of a suitable product available commercially. This information is given for the convenience of the users of this International Standard and doe
30、s not constitute an endorsement by ISO of this product. 250 ml 0,000 1 g3,3 g 2 mg/ml 10g/ml200g/ml 5 % 5 % 520 nm 1 mm 40 C 0,1 mg250 mg50 ml25 ml 30 min Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/
31、23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) ISO 2003 All rights reserved 3 5.3Preparation of test extract Filter the extract through a Whatman No. 40 (or equivalent) filter paper. Reject the first few millilitres of the filtrate, then
32、collect the filtrate in an analyser cup. Run the samples and standards through the system in the normal manner (e.g. priming with six tobacco extracts, calibration standards and samples with one intermediate calibration solution after every six samples). If sample concentrations lie outside the rang
33、e of the standards, the samples shall be diluted and run again. 6Calculation 6.1Plot a graph of peak height against equivalent nitrate concentrations for all the standard solutions. 6.2Calculate the percentage of nitrate, , on a dry weight basis, in the tobacco using the formula: where is the nitrat
34、e concentration, expressed in micrograms per millilitre, obtained from the calibration curve (see 6.1); is the volume, in millilitres, of extract prepared (see 5.2) (normally ); is the mass, in milligrams, of the sample (see 5.2); is the moisture content, expressed as percentage by mass, of the toba
35、cco (see 5.1). The test result shall be expressed to two decimal places. When using acetic acid extracts, the standard nitrate solutions (3.7) shall be made up with acetic acid and the wash cycle shall be with acetic acid. 7Repeatability and reproducibility An international collaborative study invol
36、ving 12 laboratories and 3 samples conducted in 1993 showed that when single grades of tobacco were analysed by this method, the following values for repeatability limit ( ) and reproducibility limit () were obtained. The difference between two single results, found on different extractions by one o
37、perator using the same apparatus within a short time interval (the time it takes to analyse 40 sample cups) and without recalibration of the equipment during the time of analysis, will exceed the repeatability limit ( ) on average not more than once in 20 cases in the normal and correct operation of
38、 the method. Single results reported by two laboratories will differ by more than the reproducibility limit () on average not more than once in 20 cases in the normal and correct operation of the method. Data analysis gave the estimates as summarized in Tables 1 and 2. For the purposes of calculatin
39、g and , one test result was defined as the yield obtained from analysing a single extract once. w w = C V 100 m 1000 100 100 M C V25 ml m M 5 %5 % 5 % r R r R rR Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resa
40、le, 04/23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) 4 ISO 2003 All rights reserved Table 1 Extraction with water Tobacco typeMean content of nitrateRepeatability limitReproducibility limit (dry weight) Flue cured 0,110,030,12 Oriental0,
41、160,040,11 Burley2,430,120,41 Table 2 Extraction with acetic acid Tobacco typeMean content of nitrateRepeatability limitReproducibility limit (dry weight) Flue cured 0,110,030,20 Oriental0,160,040,21 Burley2,430,050,39 %rR 5 % %rR Copyright International Organization for Standardization Provided by
42、IHS under license with ISO Licensee=Aramco HQ/9980755100 Not for Resale, 04/23/2007 03:35:51 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15517:2003(E) ISO 2003 All rights reserved 5 Annex A (normative) Optimization of hydrazinium sulfate/copper sulfate A.1General Th
43、e optimization of the hydrazinium sulfate-copper sulfate reagent (see 3.4) shall be carried out when initially setting up the instrument. It should also be carried out when fresh batches of hydrazinium sulfate are purchased. A.2Reagents A.2.1Standard nitrite stock solution Dissolve of sodium nitrite
44、 (NaNO2, p.a.) in distilled water and dilute to volume in a 1 litre volumetric flask. This solution contains of nitrite. A.2.2Standard nitrite working solution Pipette a aliquot of the stock solution (A.2.1) into a volumetric flask and dilute to volume with distilled water. This solution contains of
45、 nitrite. A.3Optimization of the hydrazinium sulfate reagent A.3.1Dilute of the copper sulfate stock solution (3.3) to 1 litre with distilled water. A.3.2Dissolve of hydrazinium sulfate in distilled water and dilute to in a volumetric flask. A.3.3From a burette, dispense , , , ., aliquots of the hyd
46、razinium sulfate solution (refer to A.3.2) into volumetric flasks and dilute to volume with distilled water. These solutions contain , , , ., hydrazinium sulfate per litre. A.3.4Connect the hydrazinium/copper reagent line to the analyser sampler. Connect the water line to the dilute copper sulfate s
47、olution reservoir. Connect the sample line to the standard nitrite working solution reservoir (refer to A.2.2). A.3.5Start the analyser pump, pumping all other reagents as normal. A.3.6Place sample cups containing the hydrazinium solutions (A.3.3) in the sampler in ascending order of concentration.
48、A.3.7When the reaction colour reaches the flow cell, adjust the recorder response to full-scale deflection and start the sampler. A.3.8When all the hydrazinium solutions have been run, note the concentration which produces a loss in colour due to the reduction of the nitrite to nitrogen. A.3.9Replace the standard nitrite working solution (A.2.2) with the highest nitrate standard (see 3.7.2). Wait for the reagent base line to be re-established, then re-run the hydrazin