1、BS ISO20370:2009ICS 65.160NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMaterial used forproducing wrappingsfor cigarette filters,cigarettes and othertobacco products Determination ofacetate contentThis British Standardwas published under theauthority of the S
2、tandardsPolicy and StrategyCommittee on 31 October2009 BSI 2009ISBN 978 0 580 63202 0Amendments/corrigenda issued since publicationDate CommentsBS ISO 20370:2009National forewordThis British Standard is the UK implementation of ISO 20370:2009.The UK participation in its preparation was entrusted to
3、TechnicalCommittee AW/40, Tobacco and tobacco products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance
4、 with a British Standard cannot confer immunityfrom legal obligations.BS ISO 20370:2009Reference numberISO 20370:2009(E)ISO 2009INTERNATIONAL STANDARD ISO20370First edition2009-10-01Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of ac
5、etate content Matriaux utiliss pour la fabrication des enveloppes pour les filtres de cigarette, pour les cigarettes et pour les autres produits du tabac Dosage de lactate BS ISO 20370:2009ISO 20370:2009(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licens
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10、211 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 ii ISO 2009 All rights reservedBS ISO 20370:2009ISO 20370:2009(E) ISO 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwi
11、de 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 interested in a subject for which a technical committee has been established has the right to be represented on tha
12、t committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in a
13、ccordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is 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 re
14、quires 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 document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 20370 was prepared by Techn
15、ical Committee ISO/TC 126, Tobacco and tobacco products. BS ISO 20370:2009BS ISO 20370:2009INTERNATIONAL STANDARD ISO 20370:2009(E) ISO 2009 All rights reserved 1Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of acetate content WARNIN
16、G The use of this International Standard can involve hazardous materials, operations and equipment. This International Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this International Standard to establish appropriate sa
17、fety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard specifies a method for the determination of the acetate content of material used to produce wrappings for cigarette filters, cigarettes and other tobacco products. 2
18、Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 187, Paper, boards and p
19、ulps Standard atmosphere for conditioning and testing and procedure for monitoring the atmosphere and conditioning of samples ISO 287, Paper and board Determination of moisture content of a lot Oven-drying method ISO 3696, Water for analytical laboratory use Specification and test methods 3 Terms an
20、d definitions For the purposes of this document, the following terms and definitions apply. 3.1 acetate content materials for producing wrappings for cigarette filters, cigarettes and other tobacco products anhydrous acetic acid content determined by the enzymatic method NOTE Acetate is generally ad
21、ded to wrapping materials, in particular cigarette paper, as sodium acetate and potassium acetate to influence the burning rate of the cigarette and, consequently, the puff number. BS ISO 20370:2009ISO 20370:2009(E) 2 ISO 2009 All rights reserved4 Principle The acetate content is determined by an en
22、zymatic method in which acetate (acetic acid), catalyzed by the enzyme acetyl coenzyme A synthase (ACS) with adenosine 5-triphosphate (ATP) and coenzyme A (CoA), is first converted to acetyl coenzyme A, adenosine 5-monophosphate (AMP) and pyrophosphate in accordance with the following reaction: acet
23、ate + ATP + CoA ACSacetyl-CoA + AMP + pyrophosphate (1) Catalyzed by citrate synthase (CS), acetyl coenzyme A reacts with oxaloacetate to form citrate in accordance with the following reaction: acetyl-CoA + oxaloacetate + H2O CScitrate + CoA (2) The oxaloacetate required for the reaction (2) is form
24、ed from malate and nicotinamide-adenine dinucleotide (NAD+) by catalysis using L-malate dehydrogenase (L-MDH) in accordance with the following reaction: malate + NAD+L-MDH oxaloacetate + H+ NADH (3) The determination is based on the formation of NADH, which is measured by the increase in absorbance
25、at 340 nm. Since the determination reaction is preceded by an indicator reaction, the amount of NADH formed is not linearly proportional to the acetate content. 5 Reagents 5.1 General All reagents used shall be of recognized analytical grade. Water used shall be in accordance with at least grade 3 o
26、f ISO 3696. 5.2 Test kit for enzymatic acetate determination 5.2.1 General Commercially available test kits shall be used that generally contain two reagent mixtures Roche-Biopharm 10.148.261.035, or equivalent1). Optionally, the determination may be performed using individual reagents. In that case
27、, the procedure is to be found in the literature or commercial information documents. 5.2.2 Reagent mixture 1 The ready-to-use solution 1, which is buffered to a pH of 8,4 using triethanolamine buffer, contains the following: L-malic acid, about 4,2 mg/ml; magnesium chloride, about 2,1 mg/ml. Soluti
28、on 1 will be stable for one year at +4 C. 1) Roche-Biopharm 10.148.261.035 is an example of a suitable product available commercially. This information is given for the convenience of the users of this International Standard and does not constitute an endorsement by ISO of this product. BS ISO 20370
29、:2009ISO 20370:2009(E) ISO 2009 All rights reserved 35.2.3 Reagent mixture 2 Reagent mixture 2 shall be diluted with water in accordance with the manufacturers instructions to produce solution 2. The ready-to-use solution 2 contains the following: adenosine 5-triphosphate (ATP), about 25,0 mg/ml; co
30、enzyme A (CoA), about 2,6 mg/ml; nicotinamide adenine dinucleotide (NAD), about 12,3 mg/ml. Solution 2 will be stable for four weeks at +4 C. 5.2.4 Reagent mixture 3 The ready-to-use solution 3 contains the following: L-malate dehydrogenase (L-MDH), about 2 750 IU2)/ml; citrate synthase, about 675 I
31、U/ml. Solution 3 will be stable for one year at +4 C. The activity of the enzyme system shall be (100 5) %. 5.2.5 Reagent mixture 4 Reagent mixture 4 shall be diluted with water in accordance with the manufacturers instructions to produce solution 4. The ready-to-use solution 4 contains about 20 IU/
32、ml of acetyl coenzyme A synthase (ACS). Solution 4 will be stable for five days at +4 C. 5.3 Sodium acetate trihydrate. 6 Apparatus Usual laboratory apparatus and, in particular, the following items. 6.1 Conical flasks, of nominal capacity 250 ml. 6.2 Funnel, of diameter 80 mm. 6.3 Filter paper, of
33、diameter 125 mm Whatman No. 40, or equivalent3). 6.4 Pipettes, with graduations suitable for nominal capacities of 1 ml, 2 ml, 5 ml and 10 ml; enzyme assay pipettes might be used as well 2. 6.5 Piston-operated pipettes, of nominal capacities 10 l and 20 l. 6.6 Double-beam spectrophotometer, suitable
34、 for a wavelength of 340 nm. 2) IU (international unit) is the amount of enzyme (activity) that catalyses the conversion of 1 mol of substrate per minute under standard conditions. 3) Whatman No. 40 is an example of a suitable product available commercially. This information is given for the conveni
35、ence of the users of this International Standard and does not constitute an endorsement by ISO of this product. BS ISO 20370:2009ISO 20370:2009(E) 4 ISO 2009 All rights reserved6.7 Glass or plastic cuvets, of light path 10 mm and capacity 5 ml. 6.8 Ultrasonic bath or magnetic stirrer. 6.9 Analytical
36、 balance, suitable for measuring to the nearest 0,001 g. 7 Procedure 7.1 Sample preparation Extract approximately 1,0 g, to the nearest 0,001 g, of cut wrapping material previously conditioned as specified in ISO 187, in 100 ml of water in a 250 ml conical flask (6.1), by the aid of an ultrasonic ba
37、th or magnetic stirrer (6.8) for 30 min. Then filter the extract through a filter paper (6.3). 7.2 Determination Perform the determination at a constant temperature of between 20 C and 25 C. The following pipetting procedure (see Table 1) has proved satisfactory for the blank solution (water) and th
38、e test solution (sample extract as prepared in 7.1). The absorbance shall be determined using a double-beam spectrophotometer (6.6) at a wavelength of 340 nm with air (no cuvet in the beam path) or water as reference. The total volume, V, of the test solution in the cuvet shall be 3,23 ml. To calibr
39、ate the method, replace the sample extract by standard solutions of sodium acetate trihydrate (5.3) having mass concentrations of 300 mg/l, 100 mg/l and 50 mg/l and proceed as described in this clause. Table 1 Pipetting procedure Pipette into cuvets Blank cuvet ml Test cuvet ml Solution 1 according
40、to 5.2.2 Solution 2 according to 5.2.3 Water Sample extract 1,00 0,20 2,00 1,00 0,20 1,90 0,10 Mix, read off the absorbance of the solutions, A0, and then add the following. Solution 3 according to 5.2.4 0,01 0,01 Mix, and on completion of the reaction (after about 3 min), read off the absorbance of
41、 the solutions, A1. Then start the second reaction by adding the following. Solution 4 according to 5.2.5 0,02 0,02 Mix, and on completion of the reaction (after about 10 min to 15 min), read off the absorbances of the solutions, A2. If the reaction has not stopped after 15 min, read off the absorba
42、nce at 2 min intervals until the absorbance increases constantly for 2 min. If the absorbance increases at a constant rate, extrapolate it to the time when the solution was added. BS ISO 20370:2009ISO 20370:2009(E) ISO 2009 All rights reserved 58 Calculation In the reactions on which this determinat
43、ion is based, there is not a linear proportionality between the amount of NADH consumed and, consequently, the absorbance difference, A and the acetic acid concentration (see Clause 4). Calculate the absorbance difference using the following equation: 2210sample1 0 blank20sample 20blank20sample 20bl
44、ank()()() ()() ()AAAAAAA AAAA AA = (4) To obtain reliable results, the absorbance difference of the sample extract should be at least 0,1. The acetic acid concentration in the cuvet shall be between 1 g and 15 g, and the sample shall therefore be diluted beforehand so that the acetic acid content is
45、 0,01 g/l to 0,15 g/l. The absorbance difference should be between 0,2 and 0,4. Calculate the acetic acid mass concentration, A, in grams per litre of the sample extract, using the following equation: AP1000VMFAV= (5) where V is the total volume of test solution in the cuvet, in millilitres (general
46、ly 3,23 ml); M is the molar mass of the substance to be determined; F is the dilution factor of the sample solution; is the absorption coefficient of NADH at 340 nm: 6,30 lmmol1cm1, is the light path of the cuvet, in centimetres; VPis the volume of sample solution used for the preparation of the tes
47、t solution, in millilitres; A is the absorbance difference. If the volumes are the same as in 7.2 and it is unnecessary to dilute the sample extract, calculate the acetate content, A, given as a concentration by mass in grams per litre of sample extract, as anhydrous acetic acid (M = 60,05 g/mol) us
48、ing the following equation: A2,566 A = (6) Calculate the content of acetate as mass fraction, A, in the wrapping material given as a percentage by mass as anhydrous acetic acid, using the following equation: AAP100 %= (7) where Pis the mass concentration of the wrapping material sample, in grams per
49、 litre of sample extract. If 1 g of wrapping material is extracted with 100 ml of water, Pis equal to 10 g/l. In this case, the content of acetate, A, is given by the following equation: AA10 %= (8) BS ISO 20370:2009ISO 20370:2009(E) 6 ISO 2009 All rights reservedReport the acetate content (mass fraction) as a percentage by mass of anhydrous acetic acid in the wrapping material. 9 Precision Table 2 shows mean
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