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!$li“1917370www.din.deDDIN ISO 20369Material used for producing wrappings for cigarette filters, cigarettesand other tobacco products Determination of citrate content (ISO 20369:2009),English translation of DIN ISO 20369:2012-10Material zur Herstellung von Umhllungen fr Zigarettenf
3、ilter, Zigaretten und andereTabakerzeugnisse Bestimmung des Citratgehalts (ISO 20369:2009),Englische bersetzung von DIN ISO 20369:2012-10Matriaux utiliss pour la fabrication des enveloppes pour les filtres de cigarette, pour lescigarettes et pour les autres produits du tabac Dosage du citrate (ISO 2
4、0369:2009),Traduction anglaise de DIN ISO 20369:2012-10SupersedesDIN 10259:1998-06www.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 citrate determination .6 5.2.1 General 6 5.2.2 Reagent mixture 1 6 5.2.3 Reagent mixture 2 7 6 Apparatus .7 7 Procedure .7 7.1 Sample prepara
6、tion .7 7.2 Determination .7 8 Calculation 8 9 Precision .9 10 Test report 9 Bibliography . 11 2 DIN ISO 20369:2012-10 National foreword This standard has been prepared by Technical Committee ISO/TC 126 “Tobacco and tobacco products” (Secretariat: DIN, Germany). The responsible German body involved
7、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 20369:2009 has been adopted in this standard without any modification. Attentio
8、n 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 Standards referred to in this document are as follows: I
9、SO 3696 DIN ISO 3696 ISO 187 DIN EN 20187 ISO 287 DIN EN ISO 287 Amendments This standard differs from DIN 10259:1998-06 as follows: a) a warning note has been included; b) normative references have been updated; c) the absorption wavelength has been specified to be 340 nm; d) an example of a suitab
10、le 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) the citrate content is to be reported as a percentage by mass of anhydrous citric acid instead of citric acid mono
11、hydrate; g) the determination of the moisture 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 10259: 1998-06 3 DIN
12、 ISO 20369:2012-10 National Annex NA (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 sampl
13、es DIN EN ISO 287, Paper and board Determination of moisture content of a lot Oven-drying method 4 DIN ISO 20369:2012-10 Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of citrate content WARNING The use of this International Standard
14、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 safety and health practices and determine t
15、he applicability of regulatory limitations prior to use. 1 Scope This International Standard specifies a method for the determination of the citrate content of material used to produce wrappings for cigarette filters, cigarettes and other tobacco products. 2 Normative references The following refere
16、nced 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 pulps Standard atmosphere for conditioning
17、 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 and definitions For the purposes of this do
18、cument, the following terms and definitions apply. 3.1 citrate content materials for producing wrappings for cigarette filters, cigarettes and other tobacco products anhydrous citric acid content determined by the enzymatic method NOTE Citrate is generally added to wrapping materials, in particular
19、cigarette paper, as trisodium citrate and tripotassium citrate or mixtures thereof to influence the burning rate of the cigarette and, consequently, the puff number. 5 DIN ISO 20369:2012-10 4 Principle The citrate content is determined by an enzymatic method in which (citrate) citric acid, catalyzed
20、 by the enzyme citrate lyase (CL), is first converted to oxaloacetate and acetate in accordance with the following reaction: citrate CLoxaloacetate + acetate (1) In the presence of the enzymes L-malate dehydrogenase (L-MDH) and L-lactate dehydrogenase (L-LDH), oxaloacetate and its decarboxylation pr
21、oduct, pyruvate, are converted by reduced nicotinamide adenine dinucleotide (NADH) to L-malate and L-lactate, respectively, according to the following reactions: oxaloacetate + NADH + H+L-MDHL-malate + NAD+(2) pyruvate + NADH + H+L-LDHL-lactate + NAD+(3The amount of oxidized NADH is proportional to
22、the amount of citrate. The residual NADH is determined from its absorbance at 340 nm by spectrophotometry. 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 citrate determi
23、nation 5.2.1 General Commercially available test kits shall be used that generally contain two reagent mixtures Roche-Biopharm 10.139.076.035, or equivalent1). Optionally, the determination may be performed using individual reagents. In that case, the procedure is to be found in the literature or co
24、mmercial information documents. 5.2.2 Reagent mixture 1 Reagent mixture 1 shall be diluted with water in accordance with the manufacturers instructions to produce solution 1. The ready-to-use solution 1, which is buffered to a pH of 7,8 using glycylglycine buffer, contains the following: L-malate de
25、hydrogenase (L-MDH), about 12 IU2)/ml; L-lactate dehydrogenase (L-LDH), about 23 IU/ml; 1) Roche-Biopharm 10.139.076.035 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 en
26、dorsement by ISO of this product. 2) IU (international unit) is the amount of enzyme (activity) that catalyses the conversion of 1 mol of substrate per minute under standard conditions. 6 DIN ISO 20369:2012-10 reduced nicotinamide adenine dinucleotide (NADH), 0,5 mg/ml; stabilizers. Solution 1 may b
27、e stored for two weeks at +4 C or for four weeks at 20 C. 5.2.3 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 about 40 IU/ml of citrate lyase. Solution 2 may be stored for
28、two weeks at +4 C or for four weeks at 20 C. The activity of the enzyme system shall be (100 5) %. 5.3 Citric acid monohydrate. 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 pa
29、per, 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 pipettes might be used as well 2. 6.5 Piston-operated pipette, of nominal capacity 20 l. 6.6 Double-beam spectrophotometer, suitable for
30、 a wavelength of 340 nm. 6.7 Glass or plastic cuvets, 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
31、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 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
32、 temperature of between 20 C and 25 C. The following pipetting procedure (see Table 1) has proved satisfactory for the blank solution (water) and the test solution (sample extract as prepared in 7.1). 3) Whatman No. 40 is an example of a suitable product available commercially. This information is g
33、iven for the convenience of the users of this International Standard and does not constitute an endorsement by ISO of this product. 7 DIN ISO 20369:2012-10 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
34、 water as reference. The total volume, V, of test solution in the cuvet shall be 3,02 ml. To calibrate the method, replace the sample extract by standard solutions of citric acid monohydrate (5.3) having mass concentrations of 50 mg/l, 25 mg/l and 12,5 mg/l and proceed as described in this clause. T
35、able 1 Pipetting procedure Pipette into cuvets Blank cuvet ml Test cuvet ml Solution 1 according to 5.2.2 Water Sample extract 1,00 2,00 1,00 1,80 0,20 Mix, read off the absorbance of the solutions (A1) after about 5 min, and start the second reaction by addition of: Solution 2 according to 5.2.3 0,
36、02 0,02 Mix, and on completion of the reaction (after about 5 min), read off the absorbance of the solutions (A2). 8 Calculation In the reactions on which this determination is based, there is a linear proportionality between the amount of NADH consumed and, consequently, the absorbance difference,
37、A and the concentration by mass of citric acid (see Clause 4). Calculate the absorbance difference using the following equation: 1 2 sample 1 2 blank() ()AAA AA= (4) Occasionally a negative value is obtained for the absorbance difference of the blank solution, (A1 A2)blank. In such cases, Equation (
38、4) is still used, and |(A1 A2)blank| is added to (A1 A2)sample. To obtain reliable results, the absorbance difference of the sample extract should be at least 0,1. If the absorbance difference of the sample extract is higher than 0,850, the concentration by mass of citric acid in the sample extract
39、is too high. In this case, the sample extract should be diluted until the concentration by mass of citric acid in the cuvet is less than 80 g. Calculate the citric acid mass content, C, in grams per litre of the sample extract, using the following equation: CP1000VMFAV= (5) where V is the total volu
40、me of test solution in the cuvet, in millilitres (generally 3,02 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
41、me of sample solution used for the preparation of the test solution, in millilitres; 8 DIN ISO 20369:2012-10 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 citrate content, C, given as a concentration by mass in
42、 grams per litre of sample extract, as anhydrous citric acid (M = 192,1 g/mol) using the following equation: C0,461 A = (6) Calculate the content of anhydrous citric acid as a mass fraction, C, in the wrapping material, as a percentage by mass, using the following equation: CCP100 %= (7) where Pis t
43、he mass concentration 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 and the content of anhydrous citric acid, C, is given by the following equation: CC10 %= (8) Report the citrate content (mas
44、s fraction) as a percentage by mass of anhydrous citric 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, using three cig
45、arette paper samples with citrate levels between 0,6 % and 2,5 %. Nine 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 % citric acid monohydrate C1 8 0,68 0,025 0,038 C
46、2 6 2,44 0,046 0,060 C3 8 0,60 0,012 0,022 10 Test report The test report shall include the following: a) all information required for the complete identification of the sample (type of sample, its origin and its designation); b) reference to this International Standard; c) date of sampling and meth
47、od; 9 DIN ISO 20369:2012-10 d) details of conditioning; e) date of sample delivery; f) date of analysis; g) moisture content of wrapping material, determined in accordance with ISO 287; h) the analysis results and the units in which they are reported; i) any special features observed during the anal
48、ysis; j) any working conditions that are not specified in this method or are considered as optional, and that can have affected the results. NOTE If the wrapping material samples are taken from cigarettes, the results can be influenced by external parameters, such as additives of the tobacco blend. 10 DIN ISO 20369:2012-10 Bibliography 1 CORESTA Recommended Method No 34, Determination of Citrate in Cigarette Paper, January 1993 2 DIN 12699, Laboratory
