1、October 2012 Translation by DIN-Sprachendienst.English price group 8No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS
2、 65.160; 85.080.99!$wv“1918483www.din.deDDIN ISO 20370Material used for producing wrappings for cigarette filters, cigarettesand other tobacco products Determination of acetate content (ISO 20370:2009),English translation of DIN ISO 20370:2012-10Material zur Herstellung von Umhllungen fr Zigarettenf
3、ilter, Zigaretten und andereTabakerzeugnisse Bestimmung des Acetatgehalts (ISO 20370:2009),Englische bersetzung von DIN ISO 20370:2012-10Matriaux utiliss pour la fabrication des enveloppes pour les filtres de cigarette, pour lescigarettes et pour les autres produits du tabac Dosage de lactate (ISO 2
4、0370:2009),Traduction anglaise de DIN ISO 20370:2012-10SupersedesDIN 10370:1998-06www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.1109.12 A comma is used as the decimal marker. Contents Page National foreword .3 National Annex NA (
5、informative) Bibliography 4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle 6 5 Reagents .6 5.1 General 6 5.2 Test kit for enzymatic acetate determination6 5.2.1 General 6 5.2.2 Reagent mixture 1 6 5.2.3 Reagent mixture 2 7 5.2.4 Reagent mixture 3 7 5.2.5 Reagent mixture 4
6、7 6 Apparatus .7 7 Procedure .8 7.1 Sample preparation .8 7.2 Determination .8 8 Calculation 9 9 Precision 10 10 Test report . 10 Bibliography . 11 2 DIN ISO 20370:2012-10 National foreword This standard has been prepared by Technical Committee ISO/TC 126 “Tobacco and tobacco products” (Secretariat:
7、 DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-04-01 AA Tabak und Tabakerzeugnisse. The text of ISO 20370:2009 has been adopte
8、d in this standard without any modification. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN shall not be held responsible for identifying any or all such patent rights. The DIN Standards corresponding to the International Sta
9、ndards referred to in this document are as follows: ISO 3696 DIN ISO 3696 ISO 187 DIN EN 20187 ISO 287 DIN EN ISO 287 Amendments This standard differs from DIN 10370:1998-06 as follows: a) a warning note has been included; b) normative references have been updated; c) the absorption wavelength has b
10、een specified to be 340 nm; d) an example of a suitable product for reagent mixtures and filter paper has been included; e) conditioning is now to be in accordance with ISO 187 instead of DIN EN 20187 (the procedure remains unchanged); f) Equation (6) has been modified; g) the determination of the m
11、oisture content is now to be in accordance with ISO 287 instead of DIN EN 20287 (the procedure remains unchanged); h) the standard has been editorially revised and brought in line with the current rules of presentation. Previous editions DIN 10370: 1998-06 3 DIN ISO 20370:2012-10 National Annex NA (
12、informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN EN 20187, Paper, board and pulps Standard atmosphere for conditioning and testing and procedure for monitoring the atmosphere and conditioning of samples DIN EN ISO 287, Paper and board Det
13、ermination of moisture content of a lot Oven-drying method 4 DIN ISO 20370:2012-10 Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of acetate content WARNING The use of this International Standard can involve hazardous materials, opera
14、tions 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 safety and health practices and determine the applicability of regulatory limitat
15、ions 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 Normative references The following referenced documents are indispensable for t
16、he 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 pulps Standard atmosphere for conditioning and testing and procedure for monitor
17、ing 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 and definitions For the purposes of this document, the following terms and defini
18、tions 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 added to wrapping materials, in particular cigarette paper, as sodium acetate and
19、 potassium acetate to influence the burning rate of the cigarette and, consequently, the puff number. 5 DIN ISO 20370:2012-10 4 Principle The acetate content is determined by an enzymatic method in which acetate (acetic acid), catalyzed by the enzyme acetyl coenzyme A synthase (ACS) with adenosine 5
20、-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: acetate + ATP + CoA ACSacetyl-CoA + AMP + pyrophosphate (1) Catalyzed by citrate synthase (CS), acetyl coenzyme A reacts wit
21、h 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 formed from malate and nicotinamide-adenine dinucleotide (NAD+) by catalysis using L-malate dehydrogenase (L-MDH) in accorda
22、nce 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 at 340 nm. Since the determination reaction is preceded by an indicator reaction, the amount of NADH formed is not linea
23、rly 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 of ISO 3696. 5.2 Test kit for enzymatic acetate determination 5.2.1 General Commercially available test kits shall be use
24、d 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, the procedure is to be found in the literature or commercial information documents. 5.2.2 Reagent mixture 1 The ready-
25、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. Solution 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
26、 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. 6 DIN ISO 20370:2012-10 5.2.3 Reagent mixture 2 Reagent mixture 2 shall be diluted with water in accordance with the manufacturers i
27、nstructions to produce solution 2. The ready-to-use solution 2 contains the following: adenosine 5-triphosphate (ATP), about 25,0 mg/ml; coenzyme 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 mixtu
28、re 3 The ready-to-use solution 3 contains the following: L-malate dehydrogenase (L-MDH), about 2 750 IU2)/ml; citrate synthase, about 675 IU/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 di
29、luted with water in accordance with the manufacturers instructions to produce solution 4. The ready-to-use solution 4 contains about 20 IU/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 an
30、d, 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 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 pipet
31、tes 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 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 s
32、tandard conditions. 3) 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 does not constitute an endorsement by ISO of this product. 7 DIN ISO 20370:2012-10 6.7 Glass or plastic cuve
33、ts, of light path 10 mm and capacity 5 ml. 6.8 Ultrasonic bath or magnetic stirrer. 6.9 Analytical 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 specif
34、ied in ISO 187, in 100 ml of water in a 250 ml conical flask (6.1), by the aid of an ultrasonic bath 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 followi
35、ng pipetting procedure (see Table 1) has proved satisfactory for the blank solution (water) and the 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 wate
36、r as reference. The total volume, V, of the test solution in the cuvet shall be 3,23 ml. To calibrate 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.
37、Table 1 Pipetting procedure Pipette into cuvets Blank cuvet ml Test cuvet ml Solution 1 according 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.
38、4 0,01 0,01 Mix, and on completion of the reaction (after about 3 min), read off the absorbance of 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 ab
39、sorbances of the solutions, A2. If the reaction has not stopped after 15 min, read off the absorbance 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. 8 DIN ISO 20370:2012-1
40、0 8 Calculation In the reactions on which this determination 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 e
41、quation: 2210sample1 0 blank20sample 20blank20sample 20blank()()() ()() ()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
42、 be diluted beforehand so that the acetic acid content is 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 volu
43、me of test solution in the cuvet, in millilitres (generally 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 volu
44、me of sample solution used for the preparation of the test 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 samp
45、le extract, as anhydrous acetic acid (M = 60,05 g/mol) using 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 co
46、ncentration of the wrapping material sample, in grams per 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) 9 DIN ISO 20370:2012-10 Report the acetate c
47、ontent (mass fraction) as a percentage by mass of anhydrous acetic acid in the wrapping material. 9 Precision Table 2 shows mean standard deviations of repeatability, sr, and reproducibility, sR, obtained from a collaborative study. The study, involving nine laboratories, was conducted in 2005, usin
48、g three cigarette paper samples with acetate levels between 0,4 % and 1,1 %. Eight laboratories reported results obtained by this method. Table 2 Data analysis of collaborative study Mean value mNsrsRSample n data considered Results expressed as mass fraction in % anhydrous acetic acid A1 8 0,52 0,025 0,031 A2 8 0,40 0,035 0,043 A3 8 1,11 0,037 0,057 10 Test report The test report shall include the following: a) all i
