BS ISO 21160:2018 pdf download – Cigarettes — Determination of selected carbonyls in the mainstream smoke of cigarettes — Method using high performance liquid chromatography
5 Apparatus
The usual laboratory apparatus for use in preparation of samples, solutions and standards and, in particular, the following.
5.1 Equipment for conditioning of tobacco products.
5.2 Equipment for butt length marking.
5.3 Equipment for smoking of tobacco products, complying with ISO 3308.
5.4 Impingers for trapping mainstream smoke.
5.5 Erlenmeyer flasks, of capacities 150 ml, with ground glass stoppers, (or equivalent for combining impinger solutions).
5.6 Polyvinylchloride (PVC) tubing, suitable for connection of the trapping system.
5.7 Analytical balance, capable of measuring to four decimal places.
5.8 Amber glass volumetric flasks, of capacities 10 ml, 25 ml, 200 ml, 1 l and 2 l.
5.9 Glass micropipettes, of capacities 50 µl, 100 µl, 150 µl, 300 µl, 400 µl, 500 µl, 800 µl, 1 000 µl
and 2 000 µl.
5.10 Volumetric pipettes, of capacities 1 ml, 2 ml, 5 ml, 6 ml, 7 ml, 8 ml and 20 ml.
5.11 Glass graduated measuring cylinders, of capacities 25 ml, 50 ml and 100 ml.
5.12 Dispenser, capable of delivering 35 ml.
5.13 Hot plate/stirrer.
5.14 Syringe filter, 0,45 µm PVDF or equivalent.
5.15 Disposable syringes, 5 ml.
5.16 Disposable glass Pasteur pipettes.
5.17 Rubber bulbs.
5.18 Autosampler vials, caps and PTFE faced septa.
5.19 HPLC system, consisting of:
— tertiary gradient pump;
— auto-sampler with appropriate sampling loop;
— UV and/or DAD detector;
— data collection system;
— LC column: 250 mm × 4 mm, 100 Å, Reversed Phase (RP) 18e (5 µm), or equivalent;
— disposable guard column: 4 mm × 4 mm RP 18e (5 µm), or equivalent;
— vacuum filter;
— amber glass bottles 1 l and 4 l;
— desiccator;
— degasser (optional).
6 Reagents
6.1 Acetonitrile, MeCN, HPLC grade.
6.2 Isopropanol, IPA, HPLC grade.
6.3 Ethyl acetate, HPLC grade.
6.4 Tetrahydrofuran, THF, HPLC grade.
6.5 Ethanol, HPLC grade.
6.6 Phosphoric acid, 85 %.
6.7 Deionized water, resistivity > 18,0 MΩ.cm at 25 °C.
6.8 Formaldehyde-DNPH, min. 99 %.
6.9 Acetaldehyde-DNPH, min. 99 %.
6.10 Acetone-DNPH, min. 99 %.
6.11 Acrolein-DNPH, min. 99 %.
6.12 Propionaldehyde-DNPH, min. 98 %.
6.13 Crotonaldehyde-DNPH, min. 99 %.
6.14 2-Butanone-DNPH, min. 98 %; methyl ethyl ketone-DNPH derivative.
6.15 n-Butyraldehyde-DNPH, min. 99 %.
6.16 Tris-(hydroxymethyl)-aminomethane, ACS reagent grade 1) .
6.17 2,4-Dinitrophenylhydrazine (DNPH).
6.18 Helium, (UHP), if necessary for sparging of HPLC system mobile phase or equivalent degassing system.
7 Preparation
7.1 Preparation of glassware Glassware shall be cleaned and dried in such a manner as to ensure that contamination from glassware does not occur. All possible sources of contamination shall be removed from the work area (e.g. acetone solvent wash bottles).
7.2 Preparation of solutions
7.2.1 DNPH solution (using phosphoric acid) Add approximately 150 ml deionized water to a 200 ml volumetric flask, then carefully add 28 ml of 85 % phosphoric acid and mix the solution. Make up the solution to volume with deionized water. Weigh approximately 6,8 g (24,0 mmol should be achieved) of DNPH (approximately 30 % water) into a 2 l amber volumetric flask and add 1 l of acetonitrile. Dissolve DNPH by alternately gently swirling the flask. Make sure there are no crystals remaining.
WARNING — Do not sonicate as a precipitation of DNPH may occur. If using re-crystallized DNPH, weigh 4,8 g to achieve the same molarity (see Annex A). After the DNPH is dissolved, add 58 ml of the diluted phosphoric acid solution while gently mixing. Dilute to volume with deionized water. The colour of the solution will become bright orange upon addition of the deionized water. The addition of acid or water will cool the solution and may initiate the precipitation of the DNPH. Add the acid or water slowly. Gentle swirling may be required to maintain the solution at room temperature and to prevent the precipitation of DNPH. If crystals appear, do not sonicate. Store the solution in a 4 l amber bottle at room temperature in the dark to prevent or significantly reduce the chances of DNPH precipitation. This solution, if properly sealed, will remain stable for one week.
7.2.2 Tris-(hydroxymethyl)-aminomethane dilution solution, 80:20 (volume fraction), MeCN: 1 % aqueous solution. Dissolve 2,00 g of tris-(hydroxymethyl)-aminomethane in 200 ml of deionized water in a 1 l volumetric flask. Dilute to volume with acetonitrile. Store in a 1 l amber bottle with PTFE-lined cap or equivalent at ambient temperature.
7.3 Preparation of standards
7.3.1 HPLC calibration standards and working solutions The calibration should cover the concentration range of interest. Primary carbonyl standards Weigh the hydrazones as described in Annex B into individual 25 ml volumetric flasks and dissolve in acetonitrile. Record the concentrations of the free aldehyde equivalents in µg/ml. These solutions have been shown to be stable for up to one year when stored at approximately 4 °C. Stability and storage time should be checked by the laboratory.BS ISO 21160 pdf download.