Determination of Oxides of Nitrogen in Mainstream Tobacco Smoke

Health Canada
T-110 December 31, 1999

Table of Contents

  1. Scope of Applications
  2. Normative References
  3. Definitions
  4. Method Summary
  5. Apparatus and Equipment
  6. Reagents and Supplies
  7. Preparation of Glassware
  8. Sampling
  9. Tobaco Product Preparation
  10. Smoking Machine Preparation
  11. Instrument Analysis
  12. Smoking Procedures
  13. Calculations
  14. Quality Control
  15. Modifications for Intense Smoking
  16. References
  17. Appendices

1 Scope of Applications

  1. This method describes the determination of Nitric Oxide (NO) and Total Oxides of Nitrogen (NOx) in the gas phase of mainstream tobacco smoke and their quantification by a dual channel chemiluminescence analyzer. This is applicable to the NO and NOx generated in the gas phase of mainstream tobacco smoke from cigarettes on a puff by puff basis.

2 Normative References

  1. Health Canada Test Method T-115 Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 1999-12-31.

3 Definitions

  1. Refer to T-115 for definitions of terms used in this document.

4 Method Summary

  1. One cigarette is smoked on a standard single port smoking machine and the unfiltered mainstream vapour phase is exhausted into an evacuated smoke mixing chamber (SMC) puff by puff.
  2. The gas is mixed, and at precise and consistent intervals, an aliquot of each puff is routed by vacuum through a filter to a dual channel chemiluminescence nitrogen oxides analyzer where the gas stream is split immediately into two channels.
    1. In channel A, the sample stream is reacted with ozone and the resultant chemiluminescent emission is directly proportional to the NO concentration in the sample.
    2. In channel B, the sample stream is chemically reduced first by a catalytic converter and then mixed with ozone in the reaction cell where the resultant chemiluminescent emission is due to NOx or NO + NO2.
    3. The NO2 concentration is then derived electronically by subtracting the NO signal from the NOx signal.
  3. Selective photomultiplier detection monitors the reaction cell gas stream and the NO and NOx found in the vapour phase of mainstream tobacco smoke are quantified by external standard calibration procedures.

    Note: The testing and evaluation of certain products against this test method may require the use of materials and or equipment that could potentially be hazardous and this document does not purport to address all the safety aspects associated with its use. Anyone using this test method has the responsibility to consult with the appropriate authorities and to establish health and safety practices in conjunction with any existing applicable regulatory requirements prior to its use.

5 Apparatus and Equipment

  1. Equipment needed to perform conditioning as specified in T-115.
  2. Equipment needed to perform marking for butt length as specified in T-115.
  3. Equipment needed to perform smoking of tobacco products as specified in T- 115.
  4. Dual Channel Chart Recorder.
  5. Dual Channel Chemiluminescence Nitrogen Oxides Analyzer.
  6. V/F Integrator.
  7. Digital Multimeter.
  8. Bubblemeter.
  9. Round Bottomed Three Neck boiling flasks - 1 L (SMC for Burly type tobacco).
  10. Round Bottomed Three Neck boiling flask - 5 L (SMC for Flue cured type tobacco).
  11. Round Bottomed Three Neck boiling flask - 12 L (SMC for cigars).
  12. Round Bottomed boiling flask -1 L (PEC).
  13. Flow Meter.
  14. Mechanical Stirrer.
  15. Teflon coated magnetic stirrers, football shape.
  16. Gas Regulator CGA 660 - Inlet 0-3000, Delivery 0-100.
  17. Gas Regulator CGA 580 - Inlet 0-4000, Delivery 0-100.
  18. Lighter.
  19. Vacuum Pump (Make-up).
  20. Rubber Stoppers.
  21. Gas Flow meter (FM3).
  22. Gas Flow meter (FM4).
  23. Glass fibre filter holders (X2) 44 mm [threaded, screw cartridge] see diagram 2.
  24. Glass tubing ¼" OD.
  25. Nalgene Tubing 1/4" ID × 3/8" OD.
  26. Teflon (TFE) tubing 1/4" OD (6.35 mm) × 5.8 mm ID.
  27. Inert Valves (X2) with three port housing and plug with 90o right angled flow (STC3 & STC4).
  28. Balance capable of measuring to four decimal places.
  29. Exhaust Canopy.
  30. Vacuum Pumps (Exhaust) (X2).
  31. Stop watch.
  32. Barometer.

6 Reagents and Supplies

Note: All reagents shall be, at the least, recognized as analytical grade in quality.

  1. Compressed Primary Standard Gas Mixture - 990 ppm NO balance nitrogen (certified).
  2. Compressed Primary Standard Gas Mixture - 508 ppm NO balance nitrogen (certified).
  3. Compressed Primary Standard Gas Mixture - 260 ppm NO balance nitrogen (certified).
  4. Compressed Primary Standard Gas Mixture - 160 ppm NO balance nitrogen (certified).
  5. Compressed Primary Standard Gas Mixture - 70 ppm NO balance nitrogen (certified).
  6. Compressed Primary Standard Gas Mixture - 35 ppm NO balance nitrogen (certified).
  7. UHP Nitrogen (Zero Gas).
  8. Methanol.
  9. Reagent Alcohol HPLC Grade.
  10. Glass fibre filter discs (pads), 44 mm in diameter, with no more than 5% acrylic type binder.
  11. Chart Paper.

7 Preparation of Glassware

  1. All round bottomed glassware is rinsed three times with ethanol and once with methanol and then dried in an oven at 200 °C.
  2. All Teflon three-way stopcocks are disassembled and rinsed with ethanol to remove the tar and once with methanol and allowed to air dry.
  3. The piston assembly of the single port smoking machine is disassembled and the piston is cleaned and rinsed with methanol and then allowed to air dry. The piston is then reassembled, free of any lubricants.
  4. The solenoid valve assembly is disassembled and cleaned with methanol.

8 Sampling

  1. The sampling of tobacco products for the purpose of testing shall be as those specified in T-115.

9 Tobaco Product Preparation

  1. Product shall be conditioned as specified in T-115.
  2. Cigarettes shall be marked for butt length as specified in T-115.
  3. Cigarettes to be smoked under intense smoking conditions shall be prepared as specified in T-115.

10 Smoking Machine Preparation

  1. Ambient Conditions
    1. The ambient conditions for calibration shall be between 21 and 23 °C.
  2. Machine Conditions
    1. The machine conditions shall be as those specified in T-115 with the following modifications.
      1. Allow the smoking machine to warm up on the automatic cycling for several minutes and ensure that the piston is oil-free.
      2. Because the analysis is dynamic and ongoing, it is important to ensure that the mainstream tobacco smoke generated is characteristic of the test sample. Also, because the sample stream is unfiltered; a pad in the mainstream path is not used. Therefore puff count information is the only criterion that can then be used to characterize the vapour phase samples and monitor the smoking process.
    2. Fit a conditioned pad into each of the exhaust and flow meter filter pad holders with the rough side towards the incoming smoke.
    3. The NOx Analyzer inlet pad is changed every second observation.
  3. Set-up of Mainstream Flow Detection Equipment
    1. The NO/NOx Analyzer is pumped down (left on) overnight, then the ozone generator and molycon catalytic converter are activated.
    2. The mainstream NO/NOx equipment and tubing are assembled as per Diagram 1 in the Appendix.
    3. The FM1 and filter are connected inline to the Make-up air Pump on the downstream side of the NO/NOx Analyzer inlet. The vacuum flow is calibrated to 0.9 L/minute (SS ball) and is meant to augment the total flow to the NO/NOx Analyzer (total flow 1.5 L/minute).
    4. The reaction vessel (SMC) is assembled upstream of the NO/NOx Analyzer inlet as per the following configuration:
      1. A glass tube is inserted into the left-hand neck of the SMC through a rubber stopper. This is the exhaust port through which the SMC is evacuated after every puff. The glass tube is connected to a Teflon stopcock (STC3) which is also connected to an inline filter and then to an unrestricted flow Vacuum pump (which is exhausted to the outside). STC3 rotates, and directs the vacuum stream either to the SMC or the room air (when a puff has occurred).
      2. Two glass inlet lines are inserted through a rubber stopper in the centre neck of the SMC. One tube is a pressure equalization device whereas the second tube is connected directly to the outlet port of the single port smoking machine.
      3. A glass tube is inserted through a rubber stopper into the right hand neck of the SMC. This is connected to a Teflon stopcock (STC4) which is connected directly to a T at the inlet port of the NO/NOx Analyzer. The STC4 is rotated with every puff at precise intervals to direct the vacuum from the NO/NOx Analyzer either to the SMC or the room air.
    5. Insert a magnetic stirrer bar into the SMC and mount the SMC directly over the mechanical stirrer.
    6. Electronic output from the NO/NOx Analyzer is routed to the V/F Integrator which is connected to the digital multimeter.
    7. An output signal is also directed to the two-pen chart recorder where one pen records the NO output and the other record the NOx output.
    8. Prior to smoking, record:
      1. V/F Integrator Span setting at the beginning and end of the experiment.
      2. NO/NOx span pots settings at the calibration gas settings.
      3. The average channel counts from the calibration step.
      4. Instrument status checks and flow rates.

11 Instrument Analysis

  1. Performance Test of the NO/NOx Analyzer
    1. The NO/NOx Analyzer electronic output to the two-pen chart recorder and to the V/F Integrator is synchronised in the range of 10mV (ppm)
    2. Various NO/NOx Analyzer instrument tests are performed prior to any smoking or calibration.
    3. Test T1, T2, and T3 outputs and measure on the analogue scale. This should remain constant day to day.
    4. Switch the Range PPM to the Optic and Electric test modes. Allow to stabilize at least 15-20 minutes before reading. These outputs should be consistent day to day but some variation is experienced.
    5. Switch the Range PPM to 10 for the rest op the experiment.
  2. Zero the NO/NOx Analyzer with Nitrogen
    1. Connect the Nitrogen to FM3 to deliver 1.8 L/minute to the inlet port of the pressure equalisation chamber (PEC).
    2. There are two outlets from the PEC, one to the exhaust and the other is connected to STC4 which is connected to the SMC (Smoke mixing chamber) inlet.
    3. Set the Nitrogen regulator to deliver 10 psi.
    4. STC4 is directed to deliver flow to the NO/NOx Analyzer.
    5. Allow to equilibrate 30 minutes.
    6. Adjust Zero pots on NO/NOx Analyzer to give an average of 0.000 over 60 seconds on the digital multimeter.
  3. Confirm the mainstream NO/NOx configuration with Nitrogen (zero gas)
    1. Connect the Nitrogen to FM3 to deliver 1.8 L/minute to the inlet port of the pressure equalisation chamber (PEC).
    2. There are two outlets from the PEC, one to the exhaust and the other is connected to STC4 which is connected to the SMC (Smoke mixing chamber) inlet.
    3. Set the Nitrogen regulator to deliver 10psi.
    4. SMC exhaust is directed to room air.
    5. STC4 is directed to deliver flow to the NO/NOx Analyzer.
    6. Make certain the mixing bar inside the SMC is rotating.
    7. Allow to equilibrate 30 minutes.
    8. Read zero values over 60 seconds on the digital multimeter. Results should agree with 12.2.6.
  4. Span the NO/NOx Analyzer
    1. Connect the 9 ppm NO primary standard to FM3 to deliver 1.8 L/minute to the inlet port of the pressure equalization chamber (PEC).
    2. There are two outlets from the PEC, one to the exhaust and the other is connected to STC4 which is connected to the SMC (Smoke mixing chamber) inlet.
    3. Set the gas regulator to deliver 10psi.
    4. STC4 is directed to deliver flow to the NO/NOx Analyzer.
    5. Allow to equilibrate for 30 minutes.
    6. Adjust Span pots on NO/NOx Analyzer to give an average of 9.000 over two minutes on the digital multimeter. (Adjust the span pots to give a corresponding voltage to concentration i.e. NO=9.1 NOx=9.3).
    7. Record Span settings and adjust attenuation on plotter if necessary.
    8. Optional: The above procedure can be repeated with a 5 ppm NO standard gas or another standard NO gas that fall in the linear range.
  5. Confirm the mainstream NO/NOx configuration (Span)
    1. Connect the 9 ppm NO primary standard to FM3 to deliver 1.8 L/minute to the inlet port of the pressure equalization chamber (PEC).
    2. There are two outlets from the PEC, one to the exhaust and the other is connected to STC4 which is connected to the SMC (Smoke mixing chamber) inlet.
    3. Set the gas regulator to deliver 10psi.
    4. SMC exhaust is directed to room air.
    5. STC4 is directed to deliver flow to the NO/NOx Analyzer.
    6. Make certain the mixing bar inside the SMC is rotating.
    7. Allow to equilibrate for 30 minutes.
    8. Read the actual Span voltage over 60 seconds on the digital multimeter.
  6. Calibrate the NOx mainstream set-up
    1. Connect the 990ppm NO primary standard to FM3 to deliver 1.8 L/minute to the inlet port of the PEC.
    2. Set the gas regulator to deliver 10 psi.
    3. The outlet line from the PEC chamber is connected directly to the smoking (inlet) port of the single port smoking machine. The exhaust port of the smoking machine is connected directly the inlet port of the SMC.
    4. Sample the contents of the SMC as per the following schedule:
      1. Initiate automated smoking machine operation to take one 35 cc, two-second puff every 60 seconds.
      2. As the smoking machine piston is drawing the puff, rotate STC3 (SMC exhaust) to room air. STC4 (SMC sample line) is already directed to room air.
      3. The 35 cc puff is mixed in the SMC by the stirring bar for five seconds.
      4. At t=7 seconds STC4 is rotated to the sample line to the NO/NOx Analyzer
      5. The NOx Analyzer samples the SMC for 10 seconds.
      6. At t=17 seconds both STC3 and STC4 are rotated simultaneously. STC3 is positioned to allow vacuum purging of the SMC until the start of the next puff. STC4 is positioned to direct room air to the NO/NOx Analyzer inlet line.
    5. This sampling procedure is repeated as many times as necessary to effect the following outcome:
      1. First the NO/NOx span pots are adjusted to give the expected calibration voltage on the digital multimeter and the chart recorder.
      2. Use a 5 L smoke mixing chamber when testing Burly type tobacco and a 1L smoke mixing chamber when smoking Flue-cured tobacco.
      3. [990 × 0.035L] / 5L (SMC) = 6.93mV
      4. or
      5. [170 × 0.035L] / 1L (SMC) = 5.95mV
      6. Read baseline from V/F Integrator.
      7. Add baseline to expected voltages to give actual target values.
      8. Repeat sampling procedure and adjust the threshold pots on the V/F Integrator to give reproducible counts on each channel. Record and average the counts. This figure is inserted into the calculation equation at a latter stage.
      9. The NO/NOx Analyzer is now calibrated for operation.
      10. Read the 500 ppm NO balance nitrogen when using the 5 L flask and read the 70 ppm NO balance nitrogen gas mixture when using the 1 L flask for confirmation.
    6. Turn off gases and disconnect line from PEC to smoking machine. Be sure room and smoking machine is evacuated of calibration gas before starting smoking procedure.

12 Smoking Procedures

  1. Turn the Puff Counter switch to ON and switch to automation.
  2. Weigh each cigarette and insert into the single port smoking machine.
    1. Gently insert the cigarette into the labyrinth seal or dental dam in the cigarette holder to a depth greater than 9 mm. Withdraw the cigarette until the 9 mm mark is just visible.
    2. Light the cigarette and initiate the puff count according to the following schedule. Be sure to light the cigarette on the first puff.
      1. Normal lighting procedure is 15 second warm-up beginning at t-18 seconds followed by a five-second ignition. (three seconds prior to puff plus the two-second puff)
    3. Record the puff by puff NO and NOx counts.
  3. Record
    1. The barometric pressure and the room temperature. (Do not proceed if room temperature is less than 21°C).
    2. Volume of the SMC and the calibrated puff volumes of the single port smoking machine.
    3. V/F Integrator Span setting at the beginning and end of the experiment.
    4. NO/NOx span pots settings at the calibration gas settings.
    5. The average channel counts from the calibration step.
    6. Instrument status checks and flow rates.
  4. Position an exhaust canopy above the cigarette to draw off the sidestream smoke.
  5. The unfiltered smoke from each puff is injected directly into the SMC and mixed.
  6. Initiate the sampling procedure as per step 11.6.4 of the calibration procedure. It is very important that this aspect of the procedure be repeated as precisely and consistently as possible to achieve reproducible results.
  7. Record the counts of each channel for each puff on a run sheet and the number of puffs to the end of smoking.
  8. When the cigarette has been smoked to the predetermined butt length. Remove the butt with a pair of tweezers and extinguish in a jar of water. Then take two clearing puffs at the same intervals and record the counts.
  9. The mainstream smoke pattern should be similar to Figure 1.
  10. At the end of smoking, re-test the 990 ppm standard gas as per 11.6 (read only).

13 Calculations

  1. The total counts for each channel for each test brand are summarized and the mainstream NO and NOx deliveries in [µg/cigarette] are quantitated.
    NO µmole/cigarette = [TC(NO) / AC(NO)] × [ 990 / 22.4] × 0.035 × [273 / (273+ °C)] × [BP / SBP]
    NO µg/cigarette =[ µmole / cigarette] × [µg / µmole] and
    NOx µmole/cigarette = [TC(NOx) / AC(NOx)] × [ 990 / 22.4] × 0.035 × [273 / (273+ °C)] × [BP / SBP]
    NOx µg/cigarette =[ µmole / cigarette] × [µg / µmole]

where:

14 Quality Control

  1. Typical Graph
    1. see Figure No.1
  2. Recoveries and Levels of Contamination
    Each analytical run of test cigarettes should also include:
    1. A Laboratory Reagent Blank (LRB) to evaluate the extent of any interference due to glassware, reagents, and analyzer effects (see section 7.2). In this case it would be the Zero gas.
    2. A Laboratory Fortified Blank (LFB) to evaluate the extent of potential analyte loss. The 9ppm NO standard gas is the LFB.
    3. Test a reference sample such as 1R4F to determine the inter-experimental reproducibility of the entire method of analysis.
  3. Method Detection Limit (MDL) and Limit of Quantitation (LOQ)
    1. Method Detection Limit (MDL)
      1. The method detection limit is determined by analyzing the lowest level standard at least 10 times as an unknown over several days. The MDL is then calculated as three times the standard deviation of these determinations.
    2. Limit of Quantitation (LOQ)
      1. The limit of quantification is determined by analyzing the lowest level standard at least 10 times as an unknown over several days. The LOQ is then calculated as 10 times the standard deviation of these determinations.
  4. Stability of Reagents and Supplies
    1. All primary standard gas mixtures are maintained at room temperature (22 °C).
    2. All samples are analyzed on a puff by puff basis.

15 Modifications for Intense Smoking Conditions

  1. No modifications are necessary under intense smoking conditions.

16 References

  1. Rickert, W.S., Robinson, J.C., and Collishaw, N.E. Decay of Cigarette Smoke NOx an Ambient Air Under Controlled Conditions, Environmental International, 13, 1987, p. 399-407.
  2. Norman, V., Ibrig, A.M., Larson, T.M., and Moss, B.L. The Effect of Some Nitrogenous Blend Components on NO/NOx and HCN Levels in Mainstream and Sidestream Smoke, Beitrige zur Tabakforschung International, 12 , No. 2, 1983, p. 55-62.
  3. Williams, T.B. The Determination of Nitric Oxide in Gas Phase Cigarette Smoke by Non-Dispersive Infrared Analysis, Beitrige zur Tabakforschung International, 10, No. 2, 1980, p. 91-99.

17 Appendices

Figure 1. Graph of Vapour Phase NO and NOx in Mainstream Tobacco Smoke

The figure shows a graph of Vapour Phase NO and NOx Analyses in Mainstream Tobacco Smoke

Diagram 1: Set-up of mainstream NOx analysis

The figure shows a graphical representation of mainstream NOx smoking and analysis setup

Diagram 2: Glass fibre filter disc holder: Threaded screw cartridge

The figure shows a graphical drawing of a glass fibre filter disc holder with a threaded screw cartridge

Pad Holder Setup (Measurements are inner diameters only)

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