Anal. Chern. 1980,
52,925-928
Determination of Oxides of Nitrogen (NO,) by Chemiluminescent Analysis R. A. Jenkins’ and
925
in Cigarette Smoke
B. E. Gill
Analytical Chemistry Division, Oak Ridge National Laboratory, P. 0. Box X , Oak Ridge, Tennessee 37830
The successful application of a commercial chemiluminescent NO, analyzer to the determination of oxides of nitrogen in cigarette smoke is reported. Individual puffs of the smoke vapor phase are rapidly diluted in an air stream before introduction into the analyzer. This acts to both reduce quenching of the chemiluminescent response by CO, and to prevent side reactions of the NO, with vapor phase organic constituents. Sweeping the dilute smoke through a reduced silver-Ion exchange resin bed removed a substantial positive Interference from hydrogen cyanide. A range of deliveries of 3-47 pmol of NO, per cigarette was observed for nine types of experlmental cigarettes. Statistically significant dilferences between NO, and NO levels (NO, NO = NO2) In smoke were observed in only one type of cigarette, presumably due to large cigarette-to-cigarette variability in constituent deliveries.
-
Oxides of nitrogen (NO,) in cigarette smoke have been implicated in the genesis of chronic obstructive pulmonary disease ( I ) and as precursors for carcinogenic N-nitrosamines (2). Because of this, the determination of oxides of nitrogen (NO,) as nitric oxide (NO) and/or nitrogen dioxide (NOz)in tobacco smoke has been the subject of several investigations. A number of these studies have been reviewed by Freeman and Juhos ( $ 3 ) . Since the development of commercial chemiluminescence instrumentation for the analysis of NO, in ambient air or automobile exhaust ( 4 ) ,there have been several attempts to apply this approach t o the determination of NO and/or NOz in cigarette smoke. Neurath and Dunger ( 5 ) and Freeman and Juhos (3)reported a number of positive interferences from volatile N-containing compounds in the vapor phase of smoke. Klimisch and Kircheim (6) have published the most extensive investigation of the chemiluminescent analysis of NO, in cigarette smoke. They reported substantial levels of quenching by COz in smoke. In addition, during puff-by-puff analysis of the cigarette smoke, the system throughput rate was sufficiently low to permit “aging” of the smoke, which could lead t o artifactually high levels of NOz being observed (7). I n this paper, we report the successful application of a commercial chemiluminescent NO/NO, analyzer t o the quantitative determination of oxides of nitrogen in cigarette smoke. A modified large-animal tobacco smoke inhalation exposure system (commercially available) has been employed to deliver fresh cigarette smoke for puff-by-puff analysis. Rapid dilution of the smoke mitigates chemiluminescence quenching by carbon dioxide, and a selective trapping material removes compounds which give rise t o artificially high NO, values. EXPERIMENTAL Apparatus. The experimental arrangement is portrayed in Figure 1. h i ADL/II Smoke Exposure System (Arthur D. Little, Inc., Cambridge, Mass.), designed to generate and deliver standard puffs of cigarette smoke twice a minute to a large bore stand tube for exposure of large animals t o cigarette smoke, was used to generate cigarette smoke. The ADL/II was modified to smoke 0003-2700/80/0352-0925$01 .OO/O
at 1 puff per min by removal of a cam on the machine timing gear. Thus, cigarettes were smoked under standard conditions (8) of one 35-mL putf of 2.0-s duration per min. Standing pressure waves from the pump in the NO/NO, analyzer caused the inhalation valve on the ADL/II to leak smoke. To prevent this, and to reduce internal dead volume, a small piece of Teflon tubing was inserted into the machine exhaust port. This effectively closed off the inhalation valve from the rest of the machine. The chemiluminescent analyzer was a commercially available unit (Beckman Model 951, Beckman Instruments, Fullerton, Calif.) with an optional sampling pump. The analyzer was used without any internal modifications. Because of the relatively high concentrations of NO, in cigarette smoke, pure oxygen, rather than ambient air, was supplied to the ozone generator in the analyzer. The output of the analyzer was registered on a highspeed digital integrator. Permanent copy of the analyzer response profile was obtained from a conventional strip-c hart recorder. Reagents and Materials. All standard mixtures of NO andlor NOz or CO and COPin Nz were “Certified Standards” (Matheson, East Rutherford, N.J.). Pure gases were obtained from Linde (Union Carbide Corp., Linde Division. Indianapolis, Ind.). All cigarettes used in these studies were 85-inm, nonfilter, smoked to the puff nearest (but not beyond) a 23-ntm butt length. The Kentucky Reference 1R1 and 2R1 cigarettes were obtained from the Tobacco and Health Program, University of Kentucky (Lexington, Ky.). Other cigarettes were part ofthe Fourth Series of Experimental Cigarettes of the Smoking and Health Program of the National Cancer Institute (9). Selective T r a p p i n g Material. The material used to selectively remove HCN from dilute cigarette smoke was a modification of a previously described (10) stationary phase for the gas chromatographic determination of CO and COPin cigarette smoke. Large batches of the resin could be prepared reproducibly according to the following procedure: Seventy-five g of Amberlyst 15 cation-exchange resin (dry form, unsieved, Rohm & Haas, Philadelphia, Pa.) were slurried and washed with :!OOmL of 95% ethanol four times. Following equilibration in the H+ form by three 200-mL washings of 1 M HC1, the resin was packed into a 100-mL buret and washed with distilled water until the pH of the eluate was greater than 3 and no Cl eluted. The resin bed was washed with 0.1 N HN03-1.0 M AgN03 until breakthrough of the Ag+ occurred. The resin was then allowed to stand overnight in contact with the silver nitrate solution. Following washing with distilled water and air drying at room temperature the resin bed was placed in a tube furnace, and H2gas was directed through the resin at 20 mL m i d . The furnace temperature was raised to 225 “C. Heating for 16 h acted to reduce Ag+ to Ago. The resin bed was then purged with nitrogen and allowed to cool to room temperature. To make individual traps, .approximately 3 g of resin were packed into a glass tube 9 mm i.d. X 85 mm long, the ends of which were plugged with glass wool. Used resin could be reactivated by repeating the hot hydrogen treatment. Procedure. When the sample to be analyzed represented a fraction of the total sample flow into the analyzer, the apparent analyzer response was flow-rate dependent. Because the manner in which a puff of smoke was introduced into the 3ample stream altered its concentration profile within that stream, instrument standardization had to be performed in a manner analogous tc? the smoking process. The ADL/II periodically withdrew 35-mL portions of smoke from a lit cigarette and introduced them into the head of a Tygon stand tube (13 mm i.d. X 50 cm long). The end of the stand tube was enclosed with a one-way (in) flap valve Unspiron, C. R. Baird, Inc., Upland, Calif.). For standardization, 1980 American Chemical Society
926
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
S ‘4 CKI NC WOCPIkE
I
1
u
NO. NO2 IN ’J2
Figure 1. Experimental configuration for oxides of nitrogen (NO, NO,)
determinations in cigarette smoke the lit cigarette was replaced with a manifold containing continually replenished standard gases at atmospheric pressure (see Figure 1). The smoke (or standard gas) puffs were swept through a standard 44-mm Cambridge filter assembly (11) t o remove the particulate matter from the smoke aerosol, and then were swept into the analyzer at a flow of 2000 mL min-’. In situations in which an Ago-Amberlyst trap was required, the trap was installed immediately downstream of the Cambridge filter assembly. The nitrogen oxide content of smoke was determined by comparing the total integrated response when the cigarette was smoked with the accrued response t o a number of “puffs“ of the standard N O / N 0 2 mixture. Total nitrogen oxide (NO,) in smoke was measured with the Ago-Amberlyst trap in place in either the “NO Mode” or the “NO, Mode” of the analyzer. Nitric oxide (NO) content in smoke was measured without the trap in place when the analyzer was in the “NO Mode”. Nitrogen dioxide (NO,) content was taken to be the difference between the NO, and NO contents.
-
RESULTS AND DISCUSSION Interferences. Quenching of the chemiluminescent reaction by collisional deexcitation of the NO2* has been reported (12,13)for a number of compounds, including carbon dioxide, water vapor, and hydrogen. Since these compounds are present in relatively large quantities in the vapor phase of cigarette smoke (14), it was necessary to determine the extent to which the response of the analyzer system would be affected by their presence. In the instrumental configuration used, puffs of smoke were rapidly diluted by air in the stand tube. Thus, assessment of the effect of carbon dioxide on system response under actual smoking conditions was made by comparing two sets of analyzer responses: the response of the analyzer to standard gas puffs diluted with 10% COS; and the response of the analyzer to standard gas puffs diluted with 10% NZ. The comparison indicated that the CO, had only a minimal (- 1 % ) effect on response beyond t h a t due to dilution. This contrasts to the 5-7% quenching reported by others (6). Rapid dilution of the simulated smoke apparently reduced the effective peak concentrations of C 0 2 within the analyzer reaction chamber below the point at which significant levels of quenching occur. Similar studies with carbon monozide, methane, hydrogen, water vapor, and argon indicated that these common smoke constituents did not affect analyzer response significantly. Collection of tobacco smoke vapor phase in Saran gas sampling bags, followed by continuous analysis of either NO or NO,., indicated a rapid decrease in the NO/NO, content of t h e smoke with time. By the time the last puff of smoke was collected, average NO, levels in the bag had decreased to 30-50% of the value determined when the smoke was analyzed on a puff-by-puff basis. Similar phenomena have been observed by other workers ( 6 , 1 5 ) ,and it has been sug-
-
-
gested that this decrease is due to reactions of the NO or NO, with organic constituents in the vapor phase of smoke. Nitro-alkanes and methyl nitrite have been found to be important products of these types of reactions in cigarette smoke which had been allowed to stand more than a few seconds (26, 1 7 ) . In order to verify that such mechanisms were plausible under collection conditions used in this work, methanol vapors were reacted with N O / N 0 2 mixtures in Saran bags containing varying amounts of ambient air. Following an initial induction period, NO and NO, levels in the bags decreased, albeit not as rapidly as in whole cigarette smoke. Gas chromatographic visualization of the contents of these model atmospheres by the procedure of Horton and Guerin (18) confirmed the presence of methyl nitrite as a significant reaction product. Because of the chemical complexity of the vapor phase of cigarette smoke, possible positive interferences with the chemiluminescent reaction were of concern. Many low molecular weight organic species may chemiluminesce under conditions which are present in the analyzer reaction chamber (19). However, most species emit photons of much shorter wavelength than the 600-nm low wavelength cut-off of the optical filter used in the analyzer system. Nevertheless, several species, including nicotine, ammonia, methane, methanol, acetaldehyde, acrolein, benzene, hydrogen sulfide, and pyridine, were investigated and found to elicit no positive response. Halocarbons have been reported to give rise to anomalously high NO, responses in some chemiluminescent analyzers with heated carbon-based converters (20). Methylene chloride exhibited no such effect on the configuration described in this work. Compounds which might have been expected to decompose and form nitrogen oxides, such as nitromethane or nitrobenzene, did not produce a positive response. A substantial positive response from hydrogen cyanide was observed when the system was operated in the NO, mode. Since significant quantities of HCN are produced during the smoking of a cigarette ( 1 4 ) [typically 1WOO pg per cigarette, approximately half of which is present in the gas phase ( 2 1 ) ] , the phenomenon was investigated further. Generation of small quantities (-250 pg) of gaseous HCN produced a response equivalent to 0.7-1.0 mol of NO,. (This is about 10% of the total NO, content of a Kentucky Reference 1R1 cigarette). The mechanism of this interference is unclear. Since the interference was not present in the NO mode of the analyzer, chemiluminescence of the HCN itself was not responsible. Preliminary evidence suggested that the HCN promotes desorption of trace amounts of NO or NO, from the converter assembly. We are currently investigating this hypothesis with mass spectrometry. Because of the magnitude of the interference, selective removal of the HCN from the diluted cigarette smoke vapor phase was investigated. Partial success using silver oxide led us to investigate the silver loaded, reduced Amberlyst 15 ion-exchange resin described above. The material proved to be very successful in quantitatively removing HCN from dilute smoke streams. Its effectiveness was confirmed by analyzing smoke streams for HCN by conventional procedures (22) following passage through the trap. One-thousand pg quantities of gaseous HCN in nitrogen elicited no discernible analyzer response if the analyte stream was first passed through an Ago-Amberlyst trap. The effect of the trapping material on oxides of nitrogen was very interesting. Response of the analyzer to puffs of mixed NO/NO, standards with the traps in-line was identical in both the NO and NO, modes. This indicated that the trapping material quantitatively reduced NO, to NO, essentially duplicating the function of the NOz-to-NO converter assembly in the analyzer. Thus, to determine NO, content (NO + NO,) in smoke, the analyzer was operated in the NO
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, M.4Y 1980
Table I. Comparison of Analytical Methods NO, in Cigarette Smoke, Kentucky Refererice 1R1 (in pmoles)
NO MOUE
method
value
chemiluminescence nitrate electrodea colorimetricb (Saltzman)
11.0 i: 0.4 6.9 i 0.6 6.7 i 0.5
According t o the procedure in Ref. 25. to the procedure in Ref. 9. --
/I 2
3
4 5 TIME imin)
927
6
-
Flgure 2. Response of system to puffs of cigarette smoke in NO and NO, modes. Cigarette: Kentucky Reference 1 R 1
mode with a trap in the analysis stream. For NO determinations, the trap was removed. While it was determined that each 3-g trap would exhibit no detectable loss of effectiveness until after the passage of smoke from three cigarettes, a procedure was adopted by which a trap would be exposed to the smoke of only two cigarettes before the resin was rejuvenated by the process described above. Batches of the resin have remained effective after four rejuvenations. Response Profiles. Responses to cigarette smoke of the system in both operating modes according to the configuration in Figure 1 (without t h e Ago-Amberlyst trap) are compared in Figure 2. There are only minor differences in the response profiles of the two modes, with the smoke completely clearing the analyzer system within 10 s following the initiation of response. Insertion a t the Ago-Amberlyst trap had no discernible effect on the response profiles of either mode. That peak NO, concentrations in the analyzer average around 200 ppm is indicative of -31 dilution of the smoke in the analysis stream (see below). Rapid dilution and clearance from the analyzer act to reduce aging of the smoke and prevent conversion of NO to NOz (7). T h e traces in Figure 2 are fairly representative of most of the cigarettes analyzed. Unlike constituents such as CO, tar,or nicotine (23),the per puff NO or NO, content of the smoke was highly variable and did not steadily increase as the cigarette was consumed. Comparison with Other Methods. Values for the total nitrogen oxide content (NO,) of Kentucky Reference 1R1 cigarette smoke determined by two methods (24, 25) in use in our laboratory are compared in Table I with the values determined by the chemiluminescent procedure. The data obtained by the wet chemical methods were in good agreement, but were -40% lower than the values obtained by chemiluminescence. Both of the wet chemical procedures involve introduction of the smoke puffs into a partially evacuated flask containing trapping solution. Following smoking, the flasks are brought to atmospheric pressure with ambient air. Ideally, over a 45-min period, oxygen in the air reacts with unreacted NO to form NO2, which is more effectively trapped in the solution. Studies with these procedures using puffs of mixed NO/NOz gas standards (-700 ppm
According
_ .
NO, in Nz) indicated that they are only partially effective in recovering oxides of nitrogen. Both the nitrate electrode and the Saltzman procedure recovered only about 6c-70% of the added NO/N02. Even after the specified reaction period, about 25% of the added NO/NOz remained in the headspace above the trapping solution, as determined by the chemiluminescent procedure. Some of the NO/NOz may be lost t o reaction flask walls. This was suggested in light of the 90% recoveries achieved after the NO/NOz standards were introduced into partially evacuated flasks containing no trapping solution, allowed to react with ambient air, and then let stand for 45 min. While this trapping inefficiency may not account for all of the observed differences between the wet chemical and chemiluminescent procedures, the long reaction time may permit a fraction of the oxides of nitrogen in cigarette smoke to react with organic vapor phase constituents, as described above. These two factors may explain the large differences between NO, values determined by the two types of analytical procedures. Loss of Nitrogen Dioxide (NO2)in the Smoking Machine. Careful comparisons of the N0,:NO ratios of standard gases before and after passage through the modified ADL/II smoking machine suggested that a significant portion of the NOz was being lost inside the machine. Presumably, this was due to reaction of the NOz with the machine components or with smoke particulate matter residues on the inner working surfaces. To determine if this phenomenon was peculiar t o the ADL/II, several analytical smoking machines were surveyed after modification to reduce their internal dead volume. All machines investigated exhibited significant losses of NOz. The ADL/II was found to behave most reproducibly. Over a wide range of NOz concentrations (-3 ppm 150 ppm), recoveries of NOz from “puffs” of standard gas averaged 67%. There was no evidence to suggest any loss of NO upon passage through the smoking machine. While the survey of smoking machines was not exhaustive, the high reactivity of NOz will probably prevent its quantitative passage through any smoking machine capable of routine use. Thus, the NOz delivery of a cigarette must be considered as that which emanates from an analytical smoking machine, rather than that which is generated by the cigarette being smoked under standard conditions. The latter is probably 50% greater than the former. Oxides of Nitrogen Contents of Cigarette Smokes. Using the procedures described above, the NO and NO, deliveries of selected experimental cigarettes were determined. The results of these determinations are listed in Table 11. (In most cases, each NO or NO, determination was a n average from four randomly selected cigarettes.) Generally, relative standard deviations for the NO and NO, values were 6% or less, which were similar in magnitude to those of other smoke constituents (9). Only one of the cigarettes surveyed, the Code 37. delivered a n amount of NO, greater than NO which was statistically significant a t the 90 % confidence level. T h e difference between the two values, 2.3 pmol, was taken t o be the NOz delivery of the cigarette. This amounted to 6.7% of the total nitrogen oxides present. For the other cigarette types surveyed, the NO, and NO deliveries were not statistically distinguishable.
--
928
A N A L Y T I C A L CHEMISTRY, VOL. 52, NO. 6, M A Y 1980
Table 11. Oxides of Nitrogen (NO, NO,) Deliveries' of Selected Experimental Cigarettesb cigarette code 06 12 20 23 26 32 37 1R1 2R1
composition of filler reconstituted tobacco sheet - 10%cellulose same as Code 06, with waxy substances removed bright leaf, with full return of stems extracted standard experimental blend with nicotine added burley leaf, with full return of stems standard experimental blend reconstituted tobacco sheet made from burley leaf, with additives Kentucky Reference Blend remake of 1 R 1
a Deliveries in pmoles per cigarette, mean Cigarettes; see Ref. 9.
+_
one standard deviation.
Table 111. Comparison of NO, Deliveries as Determined by Saltzman' and Chemiluminescent Proceduresb (NO,, mean
* one standard deviation)
cigarette code 06 12 20 23 26 32 37 1R1
2R1 a
F'rom Ref. 9.
NO
NO, via Saltzman, pmoles per cigarette
NO, via chemiluminescence, pmoles per cigarette
1 . 2 f 0.1 3.4: 0.2 1 . 3 i 0.2 10.4: 0.6 30.4: 2.3 9.8 t 0.5 1 9 . 6 r 0.2 6.7 + 0.5 7.0 * 0 . 3
3.2 i8.2 i 3.4 2 17.8 i47.0 = 15.8 t 34.3 * 11.0 i 13.3 i
0.2 0.3 0.1 0.6
1.4 0.7 1.0 0.4 0.8
FromTable 11.
In Table 111, NO, deliveries determined by the modified Saltzman procedure (9) are compared with those obtained by chemiluminescence. I n all cases, the chemiluminescent determined values were substantially greater, ranging from 50% for the higher NO, delivery cigarettes to 170% and greater for the low delivery cigarettes. Differences in the levels obtained by the two methods are due to those factors discussed above. However, these data suggest that the values obtained by the chemiluminescent procedure would serve to rank cigarettes according to relative NO, deliveries in much the same manner as the more classical analytical methods. CONCLUSIONS T h e chemiluminescent analysis of oxides of nitrogen in cigarette smoke provides for a more accurate assessment of NO or NO, deliveries than the more classical, wet chemical procedures. Rapid dilution of single puffs of smoke reduces quenching of the chemiluminescent response and discourages side reactions of the NO or NOz with organic gas phase constituents in the smoke. Drawing the diluted, filtered smoke through a selective trapping material removed a significant interference from hydrogen cyanide which would otherwise be registered as NOz. In addition, use of the selective trapping material permitted the more interference-prone N 0 2 - t o - N 0 converter assembly in the analyzer to be by-passed. Presumably, the fact that only one of the cigarette types surveyed exhibited statistically different levels of NO and NO, does not imply that freshly generated cigarette smokes contain no nitrogen dioxide whatsoever. Rather, the data indicate that t h e relative fraction of NO, that the NOz represents is less than t h a t which can be distinguished given cigarette-to-cig-
3.2 8.0 3.4
NO,
0.1 0.2 i 0.1 18.1 t 0.4
3.4 i17.8i 0.6
47.8 * 1 . 7 15.9 t 0.4 32.0 k 1.7
47.0 i 1.4 15.8 i- 0.7 34.3 i 1.0
10.6 * 0.5 13.0 * 0.6
11.0 + 0 . 4 13.3 i 0.8
i-
3.2
i
i
8.2
i-
0.2 0.3 0.1
From NCI Fourth Series of Experimental
arette variability. T h a t only two thirds of the NOz entering a smoking machine can be recovered from the exit, probably contributes to the statistical "nonexistence" of NO2 in the smoke. ACKNOWLEDGMENT We thank R. B. Quincy for his assistance in the development of the silver-loaded ion-exchange resin selective trapping material. LITERATURE CITED "Criteria for a Recommended Standard , . . Occupational Exposure to Oxides of Nitrogen (Nitrogen Dioxide and Nitric Oxide)", DHEW Publication NO. (NIOSH) 76-149; pp, 20-85. Schmeltz, I.; Hoffmann, D. Chem. Rev. 1977, 77, 295-311. Freeman, G.; Juhos, L. T. "Trace Substances and Tobacco Smoke in Interaction with Nitrogen Oxides. Biological Effects", EPA-600 1-76-021. April 1976. Stevens, R. K.; Hodgeson. J. A. Anal. Chem. 1973, 4 5 , 443A-449A. Neurath, G. B.; Dunger, M. Proc. IARC 1974, 9 , 177-179. Klimisch, H. J.; Kircheim, E. 2.Lebensm. Unters.-Forsch. 1977, 163. 48. Sloan, C. H.; Kiefer, J. E. Tob. Sci. 1969, 13, 180-182. Pillsbury, H. C.; Bright, C. C.; O'Connor, K. J.; Irish, F. W. J . Assoc. Off. Anal. Chern. 1969, 52, 458-462. Griest. W. H.: Guerin, M. R.; Quincy, R. B.; Jenkins, R. A,; Kubota, H. "Chemical Characterization of Experimental Cigarettes and Cigarette Smoke Condensates in the Fourth Cigarette Experiment," in "Report No. 4, Toward Less Hazardous Cigarettes-The Fourth Set of Experimental Cigarettes", Gori, G. B., Ed.; DHEW Publication, in press. Horton, A. D.; Guerin, M. R. J . Assoc. Off. Anal. Chem. 1974. 57, 1-7. Cobgill, E. C; Harlow, E. S. J. Assoc. Off. Anal. Chem. Wartman, W. 6.; 1959, 3 1 , 1705-1709. Manhews, R. D.; Sawyer, R. F.; Schefer, R. W. Environ. Sci. Technol. 1977, 7 7 . 1092-1096. Siewert, R. M. Combust. Flame 1975, 25, 273-276. Wynder, E. L.; Hoffmann, D. "Tobacco and Tobacco Smoke", Academic Press: New York. 1967; Chapter 8. Vilcins, G.; Lephardt, J. 0. Chem. Ind. (London) 1975, 974-975. Philippe, R. J.; Hackney, E. J. Tob. Sci. 1959, 3 , 139-143. Rathkamp, G.; Hoffmann, D. Beitr. Tabakforsch. 1970, 5, 302-306. Horton, A. D.; Guerin, M. R. Tob. Scl. 1974, 18, 19-22. Shlyapintokh, V. Ya. "Chemiluminescent Techniques in Chemical Reactions"; Consultants Bureau: New York, 1968. Joshi, S. B.; Bufalini, J. J. Environ. Sci. Technol. 1978, 72, 597-599. Guerin, M. R.; Shuns, W. D. "Tobacco Smoke Analysis Program Progress Report for the Period January 1, 1970 to September 1, 1970", ORNL4642. Available from NTIS. Collins, P. F.: Sarji, N. M.; Williams, J. F. Tob. Sci. 1970, 1 4 , 12-15. Williams, T. B.; Belk, C. W.. 11, Beitr. Tabakforsch. 1972, 6 . 210-215. Saltzman, 8 . E. Anal. Chem. 1954, 26, 1949-1955. Sioan, C. H.; Morie, G. P. Tob. Sci. 1974, 18, 98-99.
RECEIVED for review September 13, 1979. Accepted January 31,1980. This work was presented in part at the 32nd Tobacco Chemists' Research Conference, Montreal, Quebec, October 30-November 1, 1978. This research was sponsored by the National Cancer Institute under Interagency Agreement NIH(NC1) 40-485-74 under Union Carbide Corporation contract W-7405-eng-26 with the U.S. Department of Energy.
