Radicals from the Pyrolysis of Tobacco - American Chemical Society

Apr 27, 2005 - Biodynamics Institute and Department of Chemistry, Louisiana State University, ... different classes of radicals in tobacco smoke: thos...
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Radicals from the Pyrolysis of Tobacco Zofia Maskos, Lavrent Khachatryan, Rafael Cueto, William A. Pryor, and Barry Dellinger* Biodynamics Institute and Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 Received October 11, 2004. Revised Manuscript Received February 15, 2005

The fractional pyrolysis of Bright tobacco was studied in an inert nitrogen environment at atmospheric pressure over a temperature range of 200-700 °C, using a continuous-flow reactor system. The effect of pyrolysis conditions on the generation of free radicals and their distribution among total particulate matter (TPM), char residue, and the gas phase was characterized using electron paramagnetic resonance (EPR) and EPR spin-trapping techniques. It was found that the fractional pyrolysis of tobacco led to the generation of free radicals with g-factors of 2.00352.0040. They were formed during pyrolysis at temperatures of >380 °C and were present in both char and TPM. The concentration of these radicals in TPM varied from 0 spins/g at 240 °C to 11 × 1016 spins/g of TPM at 620 °C. The concentration of these radicals in the char residue increased as the pyrolysis temperature increased; a maximum concentration of 1.2 × 1019 spins/g of char at 480 °C was obtained, and then the value declined. The g-values of 2.0035-2.0040 are consistent with (i) surface-associated, carbon-centered radicals, where the unpaired electron is vicinal to an oxygen-containing functional group, or (ii) partially delocalized, polymeric, phenoxyl-type radicals. The analysis of the gas phase of tobacco smoke revealed the formation of alkoxyl radicals. The N-tert-butyl-R-phenylnitrone (PBN) adduct of the alkoxyl radicals is characterized by hyperfine coupling constants of aN ) 13.9 G and aH ) 2.0 G and a g-factor of 2.0064. The concentration of these radicals in the gas phase was insignificant ((5 ( 1) × 1014 spins/g of tobacco) and was not dependent on the pyrolysis temperature. After exposure to air, the concentration of TPM radicals increased, because of the production of “new” radicals, in addition to the “old” radicals, with the increasing time of storage, reaching a maximum value and then declining. The g-factor of these new radicals is 2.0053 ( 0.0003, and their TPM concentration achieved a maximum for a pyrolysis temperature of 280 °C. At this temperature, the concentration of these new radicals is 8.1 × 1015 spins/g of tobacco, which is 25 times greater than the concentration of radicals measured immediately after collection. The EPR spectra of the new radicals are consistent with oxygen-centered, semiquinone-type radicals. These results suggest that hydroquinone/ catechol-type species, which are free or chemically bound to TPM and do not produce EPR signals, are converted to semiquinone-type radicals by atmospheric oxidation.

Introduction Tobacco use is implicated in the pathology of several chronic conditions, including pulmonary cancer,1 cardiovascular disease,2 or peripheral vascular occlusive disease.3 Some studies suggest that reactive free-radical species produced by the oxidation or metabolism of smoke constituents have a major role in smoke toxicity. It has been reported that there are at least two different classes of radicals in tobacco smoke: those found in the total particulate matter (TPM)4,5 and those observed in the vapor phase.6,7 TPM is defined as the * Author to whom correspondence should be addressed. Telephone: 1-225-578-6759. Fax: 1-225-578-0276. E-mail address: [email protected]. (1) Pryor, W. A. Environ. Health Perspect. 1997, 105, 875-882. (2) Mehta, J. L.; Mehta, J. P. Cardiol. Rev. 1999, 7, 56-61. (3) Vallyathan, V.; Shi, X. Environ. Health Perspect. 1997, 105, 165177. (4) Pryor, W. A.; Hales, B. J.; Premovic, P. I.; Church, D. F. Science 1983, 220, 425-427. (5) Pryor, W. A.; Stone, K.; Zang, L.-Y.; Bermudez, E. Chem. Res. Toxicol. 1998, 11, 441-448.

portion of tobacco smoke that can be collected on a Cambridge filter,8 which collects aerosol particles >0.1 µm in diameter with an efficiency of 99.9%. The TPM contains relatively stable free radicals, which were first detected by electron paramagnetic resonance (EPR) in 1958.9 Additional studies4 on the TPM collected from smoked cigarettes resulted in the suggestion that the principal radical is a semiquinone radical that is associated with quinone and hydroquinone groups (Q/QH2) bound in a polymeric matrix. The radical is characterized by a broad EPR signal with a g-factor of 2.00352.0040 on TPM and in a solution of the benzene extract.4,10,11 (6) Pryor, W. A.; Tamura, A.; Church, D. F. J. Am. Chem. Soc. 1984, 106, 5073-5079. (7) Lachocki, T. M.; Church, D. F.; Pryor, W. A. Environ. Res. 1988, 45, 127-139. (8) Baker, R. R. Smoke Chemistry. In Tobacco. Production, Chemistry and Technology; Davis, D. L., Nielsen, M. T., Eds.; Blackwell Science: Oxford, 1999; pp 398-439. (9) Lyons, M. J.; Gibson, J. F.; Ingram, D. J. Nature 1958, 181, 1003-1004.

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It has been demonstrated5,12 that semiquinone radicals produced during the autoxidation of aqueous extracts of cigarette particulate matter lead to the formation of reactive oxygen species (ROS): superoxide radicals, hydrogen peroxide, and hydroxyl radicals. The formation of these active oxygen species by the components of TPM from either mainstream or environmental tobacco smoke in the lung may have an important role in the onset of smoking-related disease. The gas-phase fraction of tobacco smoke contains short-lived radicals that can be observed effectively by EPR spin-trapping techniques. Using this technique, it has been demonstrated10,13 that alkyl and alkoxyl radicals are present in the gas phase and their concentration increases in the smoke during the first minutes of aging.14 In the previously published reports,1,4-7,10,12-15 measurements of radicals were mostly performed on gasphase and TPM samples collected from a burning cigarette. However, the burning zone of a cigarette can be effectively divided into two regimes: an exothermic combustion zone and an endothermic pyrolysis/distillation zone.8 The pyrolysis/distillation zone is an oxygendeficient zone where the temperature is in the range of 200-600 °C. The distillation of tobacco leads to the formation of many volatile and semivolatile products that undergo pyrolysis reactions, and a solid residue that remains after pyrolysis (i.e., char).16 The separation of the gas and particulate phases of the smoke aerosol can be affected by many factors, including moisture content, temperature, flow velocity, and specific chemical interaction between aerosol constituents and the filters. In the research reported in this manuscript, a primary emphasis was placed on the reaction temperature and reaction atmosphere dependence of nature and yields of free radicals formed during the pyrolysis of tobacco and the post-pyrolysis, atmospheric oxidation of TPM. The main objective for these experiments was to characterize and compare the radicals generated in the char residues, the TPM, and the gas phase. In this manuscript, we report the fractional pyrolysis of Bright tobacco in an inert environment at atmospheric pressure over a temperature range of 200-650 °C, using a continuous-flow reactor system. The radicals were characterized by EPR and EPR spin-trapping techniques. The formation of different types of radicals during the fractional pyrolysis provides the experimental basis for the development of a predictive model for the generation of free radicals. Experimental Procedure Materials. Bright (flue-cured) tobacco was provided by Philips Morris USA (Richmond, VA) and was used without (10) Church, D. F.; Pryor, W. A. Environ. Health Perspect. 1985, 64, 111-26. (11) Sealy, R. C.; Felix, C. C.; Hyde, J. S.; Swartz, H. M. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic Press: New York, 1980; Vol. 4, pp 209-259. (12) Zang, L.-Y.; Stone, K.; Pryor, W. A. Free Radical Biol. Med. 1995, 19 (2), 161-167. (13) Pryor, W. A.; Prier, D. G.; Church, D. F. Environ. Health Perspect. 1983, 47, 345-355. (14) Cueto, R.; Pryor, W. A. Vib. Spectrosc. 1994, 7 (1), 97-111. (15) Pryor, W. A.; Terauchi, K.; Davis, W. H. Environ. Health Perspect. 1976, 16, 161-175. (16) Sharma, R. K.; Wooten, J. B.; Baliga, V. L.; Martoglio-Smith, P. A.; Hajaligol, M. R. J. Agric. Food Chem. 2002, 50 (4), 771-783.

Maskos et al. further treatment. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (free radical, 98%), N-tert-R-phenylnitrone (PBN) (98%), and 2,2,6,6tetramethyl-1-piperidynyloxy (TEMPO) (sublimed, 99%) were obtained from Aldrich Chemical Co. The EPR tubes were standard X-band Suprasil high-purity quartz tubes (2.3 and 3 mm inner diameter (ID)) and were supplied by the Wilmad/ Labglas Co. Pyrolysis. Tobacco samples were pyrolyzed in a commercial Pyroprobe 1000 (CDC Analytical, Inc.), using a continuousflow reactor system. A pyroprobe can be successfully used to study small quantities of tobacco and guarantees a rapid ramp with a duration of 10-15 s and good reproducibility. Tobacco (80 ( 2 mg) was placed in a quartz reaction tube (ID ) 6 mm), and the assemblage was inserted inside the pyroprobe coil. Both ends of the tube were fitted with quartz wool plugs. The pyrolysis was induced by rapidly heating the reaction tube at a rate of 100 °C/s. The temperature in the center of tobacco bed was measured using a retractable chromel-alumel (type-K) thermocouple. The reaction time was varied over a range of 30-90 s. Reaction time is defined as the length of time the tobacco is exposed to heating by the pyroprobe, including the time required to ramp the temperature to the final temperature. Nitrogen that contained no more than 10 ppm of oxygen was used as a carrier gas. The pyrolysis gas was introduced at a flow rate of 100-200 cm3/min, which corresponded to a residence time of 0.05-0.1 s at 20 °C. The residence time (τ) was calculated using the equation

τ)

(

T0 v W Texposure

)

(1)

where v is the volume of the tobacco bed, W the flow rate, T0 the ambient temperature, and Texposure the temperature in the middle of the tobacco bed during the reaction. The flow rate was controlled by a mass controller (McMillan Co., Model 80D). Fractional Pyrolysis. To separate radicals formed at different temperatures, a fractional method of TPM accumulation was applied. The tobacco was heated for a total reaction time of 30 s (or 60 or 90 s, depending on the conditions) at various temperatures and the TPM was collected on the filter. After a single fractional collection, the entire pyroprobe was flushed for 5 min with a carrier gas, which allowed the tobacco bed to cool to 0.8 µm in diameter. The amount of TPM fraction collected on the 0.1 µm Cambridge filter was undetectable. After drawing a smoke sample, the cellulose filter was cut into 2-mm-wide strips and inserted into an EPR tube, and the tube was filled with argon, using three 5-min vacuum-argon cycles. The samples were stored in a freezer at -20 °C. To further minimize the oxidation of TPM before the first EPR measurement, cellulose filters with collected TPM were stored in liquid nitrogen until it was analyzed by EPR. In some experiments, the condensable products on the cellulose filter were extracted with a mixture that contained 80% ethanol and 20% H2O, and the extract was analyzed via gas chromatography/mass spectroscopy (GC/MS) (using a Hewlett-Packard model 5971A mass selective detector and model 5890 Series II gas chromatograph) (Agilent Technology).

Radicals from the Pyrolysis of Tobacco Analysis of Metals. Qualitative and quantitative analysis of metals in TPM collected during pyrolysis on the filter was performed via high-resolution inductively coupled plasmamass spectrometry (ICP-MS), using an Element-2 system (Thermo Finnigan, Bremen, Germany). Spin-Trapping Technique. In the spin-trapping experiments, the tobacco smoke (gases and ultrafine particles that were not collected by the cellulose and Cambridge filters) was passed through a bubbler with 1.5 mL of a spin-trapping solution that contained 0.1 M N-tert-butyl-R-phenyl-nitrone (PBN) in benzene. The total path length between the tobacco bed and the spin-trap solution was 45 cm. After 5 min, the volume of the spin-trapping solution was readjusted to 1.5 mL, and a 0.4 mL aliquot of the solution was placed into an EPR tube. The sample was then degassed using a freeze-pumpthaw technique and filled with argon. All samples were stored at -196 °C until analysis via EPR. Electron Paramagnetic Resonance. All EPR spectra were recorded at room temperature on a Bruker model EMX10/2.7 EPR spectrometer (Bruker Instruments, Billerica, MA) with dual cavities, X-band, and microwave frequency (9.72 GHz). The typical parameters were as follows: (i): center field, 3464 G; sweep width, 100 G; power, 2 mW; receiver gain, 3.56 × 104; modulation amplitude, 4 G; and time constant, 1.28 ms for the TPM and char residues; and (ii) center field, 3455 G; sweep width, 100 G; power, 20 mW; receiver gain, 3.56 × 104; modulation amplitude, 0.5 G; and time constant, 1.28 ms for spin-trap adducts. Values of the g-factor were calculated using Bruker’s WINEPR program. The concentration of free radicals in the TPM samples were calculated using the double integration method of the first derivative signal and comparison with a sample of DPPH. The concentration of radicals in the gas phase were determined by comparison with a known concentration of a TEMPO standard in benzene.

Results and Discussion The EPR spectrum of raw Bright tobacco exhibited a single line with a g-factor of 2.0064 and a peak-to-peak line width (∆Hp-p) of the EPR first derivative of 8.8 G. The concentration of free radicals was 1.6 × 1016 spins/g. The high g-factor for raw tobacco is typical for plant tissue. Using the raw Bright tobacco samples, a series of preliminary experiments was performed, at which the flow velocity of the carrier gas was changed over a range of 15-500 cm3/min and the reaction heating time was varied over a range of 30-90 s. The results revealed (data not shown) that the concentration of radicals calculated per gram of TPM collected at 425 °C increased as the residence time τ increased. On the other hand, an optimal flow velocity was needed to elute reaction products and collect them on a filter. A range of flow of 100-200 cm3/min was chosen that corresponded to a residence time (eq 1) of τ ) 0.05-0.1 s. The concentration of free radicals in TPM remained unchanged when the tobacco loading layer was heated for >30 s. Radicals in Char Residue. The char represents the solid residue that remains after pyrolysis. The samples of char residues were obtained in separate pyrolysis experiments performed at a total reaction time of 30 s and a gas-phase residence time of τ ) 0.05 s. The temperature in the center of the tobacco bed was varied over a range of 200-600 °C. The EPR measurements were performed on undegassed char samples taken directly from the pyroprobe without further treatment. The yield of char was 35% of the initial tobacco weight at 400 °C and then decreased to 25% at temperatures

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of 400-600 °C (data not shown). This behavior is consistent with the results of other reported detailed studies,16 in which the yield of char was 30% at 450 °C before leveling off at ∼22% at higher temperatures. An increase in char yield can be only observed when the volatile products are not removed quickly from the reaction zone.17 Under severe pyrolysis conditions, the secondary reactions of the volatile products can lead to the formation of polycyclic aromatic hydrocarbons (PAHs).18 Based on the char yield dependence on temperature, we believe that such reactions, which occur mainly because of the inability of the volatile products to escape rapidly from the thick substrate sample,19 were absent under the pyrolysis conditions used in our experiments. However, there is the second route for the formation of aromatic compounds.20 The primary char that is formed in the temperature range of 300-400 °C can undergo a chemical transformation at temperatures of >400 °C to produce aromatic hydrocarbons and PAHs.21 This pathway of PAH formation does not represent a significant loss of weight, compared to the first pathway.20 The concentration of radicals, the peak-to-peak line width (∆Hp-p), and g-factor of char residues are shown in panels A, B, and C, respectively, in Figure 1, each as a function of the pyrolysis temperature. The EPR spectra of the char consist of a single symmetric resonance line, with a line shape intermediate between Gaussian and Lorentzian for temperatures of 500 °C. These results are consistent with those previously reported in the literature.22 The concentration of free radicals found in the raw tobacco (1.6 × 1016 spins/g of tobacco) increased as the pyrolysis temperature increased, up to a concentration of ca. 1.2 × 1019, and then sharply declined, resulting in a maximum at 480 °C (see Figure 1A). The appearance of this maximum is related to the shape and line width of EPR signals. As can be seen in Figure 1B, the line width (∆Hp-p is 8.8 G for the raw Bright tobacco) slowly decreases as the pyrolysis temperature increases up to 350 °C and then decreases sharply in the region from 350 °C to 450 °C. Above 500 °C, the line width reaches the value of ∼3.5 G, which varies only slightly at higher temperatures. Similar observations have been22 reported at 450 °C and above, in which the EPR resonance line for a char formed during pyrolysis of cellulose begins to narrow. This was attributed to electron-spin-exchange interactions, because of the high concentration of delocalized unpaired π-electrons of polyaromatic structures in the char.23 Figure 1C demonstrates that the g-factor of char residue decreases from 2.0045 to 2.0034 as the pyrolysis (17) Mok, W. S.; Antal, M. J.; Szabo, P.; Varhegyi, G.; Zelei, B. Ind. Eng. Chem. Res. 1992, 31, 1162-1166. (18) Marsh, N. D.; Ledesma, E. B.; Sandrowitz, A. K.; Warnat, M. J. Energy Fuels 2004, 18, 209-217. (19) Shafizadeh, F. J. Anal. Appl. Pyrol. 1982, 3 (4), 283-305. (20) Hajaligol, M. R.; Waymack, B.; Kellogg, D. Fuel 2001, 80, 17991807. (21) Sharma, R. K.; Wooten, J. B.; Baliga, V. L.; Hajaligol, M. R. Fuel 2001, 80 (12), 1825-1836. (22) Wind, R. A.; Li, L.; Maciel, G. E.; Wooten, J. B. Appl. Magn. Reson. 1993, 5 (2), 161-176. (23) Singer, L. S. Review of Electron Spin Resonance on Carbonaceous Materials. In Proceedings of the Conference on Carbon, 5th; Macmillan: New York, 1962; pp 37-64.

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Figure 1. Effect of temperature on the formation of radicals in char residue during the pyrolysis of tobacco: (A) the concentration of radicals, (B) the line width of electron paramagnetic resonance (EPR) signals (∆Hp-p), and (C) the g-factor. Reaction conditions are as follows: reaction time, 30 s; and residence time, 0.05 s.

temperature is increased. The decrease of the g-factor is attributed to the thermal decomposition of less-stable, oxygen-centered radicals with increasing temperature. The loss of oxygen and other heteroatom linkages between aromatic units at high temperatures causes a gradual increase in the ordering of the polyaromatic skeleton and, hence, a shift of the g-factor toward the free electron value (2.0023). 13C cross-polarization magic angle spinning (CPMAS) NMR analysis of char16 (generated during the pyrolysis of tobacco or other biomassderived materials) has shown that the aromatic resonances increased monotonically with temperature, whereas the phenolic resonances achieved a maximum at 300 °C and gradually decreased at higher temperatures. Radicals in TPM. In preliminary experiments, the concentration of free radicals in TPM generated during the nonfractional pyrolysis increased significantly as the temperature increased from 300 °C to 600 °C (data not shown). The EPR spectra of the TPM generated in that set of experiments seemed to be essentially identical and consisted of a single line with no indication of fine

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Figure 2. Effect of temperature on the formation of radicals in the total particulate matter (TPM) collected during the fractional pyrolysis of tobacco: (A) the yield of TPM, (B) the concentration of radicals in TPM, and (C) the g-factor of the EPR signal. Pyrolysis conditions are as follows: reaction time, (b) 30 s, (O) 60 s, and (2) 90 s; and residence time, 0.05 s. Storage conditions are as follows: EPR tubes with the collected TPM were filled with argon, using three 5-min vacuum-argon cycles, and stored in a freezer at -20 °C until analysis via EPR.

structure. The line width varied in the range of 7.47.9 G, and g-factor varied in the range of 2.0038-2.0043. These results revealed that this type of experiment did not differentiate radicals collected during pyrolysis that was performed at different temperatures. Therefore, to separate radicals formed at lower temperature from those formed at higher temperature, a modified method of TPM collection method was used in subsequent experiments. In this method, which is called fractional pyrolysis, the tobacco was heated for 30 s (and, for additional experiments, reaction times of 60 and 90 s) at 240 °C and the TPM was collected on the filter. The filter with the TPM was flushed for 5 min with the carrier gas, replaced by a new filter, and repeated at temperatures from 240 °C to 620 °C, in 3060 ° C increments. The entire experimental sequence was performed using the same bed of tobacco. The results obtained in this manner are presented in Figure 2. The yield of TPM collected during the pyrolysis of tobacco increased as the temperature increased, reaching a maximal value at temperatures of 250-350 °C (see Figure 2A) and then sharply declining. Simultaneously, the concentration of the free radicals per gram of TPM increased as the temperatures increased, especially dramatically at temperatures >400 °C (see

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Figure 3. Effect of storage time on the relative intensity of the EPR signal for TPM collected during the pyrolysis of tobacco and then exposed to air and light in open EPR tubes. Pyrolysis conditions are as follows: temperature, 360 °C; reaction time, 60 s; and residence time, 0.05 s. Storage conditions are as follows: cellulose filters with collected TPM were stored directly in liquid nitrogen until analysis via EPR.

Figure 2B). Thus, the concentration of TPM radicals is the greatest for the smaller fractions of TPM obtained at the higher temperatures. The effect of temperature on the g-factor is depicted in Figure 2C. The g-factor decreased from a value of 2.0064 (found for TPM collected during pyrolysis at 240 °C) to 2.0043 (for TPMassociated radicals at 400 °C). The line width was 9 ( 0.5 G and did not vary with pyrolysis temperature. We also observed that, during the storage of TPM at room temperature in open EPR tubes, changes occurred in the concentrations of radicals in all of the pyrolysis fractions, and these changes were dependent significantly on the storage conditions (especially the storage time). Figure 3 presents the dependence of the relative concentration of radicals versus the storage time in the EPR tubes exposed to air and light for the 360 °C fraction, which is representative of the other fractions. The concentration of radicals increased as the storage time increased, reaching a maximum at 20 days (480 h) (see Figure 3) and then declining. Consequently, our next experiments were designed (i) to investigate if an oxidation occurs under exposure of TPM samples to air at room temperature and (ii) to compare the free radicals formed during the pyrolysis with those formed during post-pyrolysis exposure to air at room temperature. Pyrolysis Radicals. Fractional pyrolysis of the Bright tobacco was performed in the temperature range of 240-510 °C. To minimize any oxidation of the TPM before the first EPR measurements, the TPM samples were transferred to EPR tubes and stored in liquid nitrogen. After the first measurement (Figure 4A, open circles), all TPM samples were stored in open EPR tubes at room temperature. Periodically, the concentration of radicals was determined and the maximum values were chosen (Figure 4A, closed circles), to compare with the initially measured concentration of radicals. For a better presentation of the effect of atmospheric air on the TPM, the results are given as numbers of spins per gram of tobacco. Figure 4B depicts the g-factors before and after oxidation. Figure 5 presents EPR spectra before and after exposure to air for the most characteristic samples: TPM collected at 280 °C is shown in Figure 5A, and

Figure 4. Effect of storage on the concentration of radicals in TPM collected at different pyrolysis temperatures during the fractional pyrolysis of Bright tobacco samples: (A) the concentration of radicals (per gram of tobacco) after collection of TPM during pyrolysis (open circles) and after 480 h of storage at room temperature in open EPR tubes (closed circles); and (B) the g-factor of radicals after collection (open circles) and after exposure to air (closed circles). Pyrolysis conditions are as follows: reaction time, 60 s; residence time, 0.05 s. EPR tubes with the collected TPM were stored directly in liquid nitrogen until analysis via EPR.

TPM collected at 510 °C is shown in Figure 5B. For the samples collected at 380 °C, the g-factor decreased to 2.0037-2.0038 and the ∆Hp-p value decreased to 8.5 ( 0.2 G. The parameters of these spectra were compared to those obtained for the char residue, and the comparison indicates close similarity between the spectra found for char residue and those for TPM collected at the same temperatures. The sharp decrease of the g-factor and ∆Hp-p values in the temperature range of 350-450 °C are analogous in both the char residue and TPM samples. Clearly, this suggests structural similarities in the TPM and char radical. This suggests that they may both result from a common tobacco precursor or that burnout of the char releases the char radicals, which undergo additional reaction before being incorporated into the TPM. Further study of the thermal degradation of char radicals is needed to understand the contribution of these two pathways to TPM radicals. The initially formed radicals with a g-factor of 2.00352.0040 are consistent with two identifiable sources: (1) Carbon-centered radicals with the unpaired electron probably vicinal to an oxygen-containing functional group. [Note: Resonance structures are a concept from valence bond theory. A phenoxyl radical (or neutral

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Figure 5. Effect of storage on the EPR spectra of TPM radicals collected during the pyrolysis of Bright tobacco at (A) 280 °C and (B) 510 °C. Line 1 represents the spectra of TPM after collection, and line 2 represents the spectra of TPM after 480 h of storage at room temperature in open EPR tubes. Scheme 1: Resonance Structures for the Phenoxyl Radical

semiquinone radical, viz, a orthohydroxy or parahydroxy phenoxyl radical) can be represented as a superposition of four resonance structures: an oxygen-centered resonance structure (I) and three carbon-centered resonance structures (II, III, IV) (see Scheme 1). The contribution of these different resonance structures to the actual structure of the radical can be estimated from a molecular orbital theory viewpoint, on the basis of the localization of corresponding spin densities on radical active centers. As resonance structures, there are no formal reactions for their interconversion within the valence bond theory of chemical reactions. However, there may be conditions when the reactions occur on surfaces, in which these moieties are separately identifiable chemical species rather than resonance structures.] This explanation is consistent with the results of a previous study that used a nitroxide trap on solidstate supports,24 in which 8-10 different carboncentered radicals species were identified in cigarette smoke. It is also consistent with studies of 2-chlorophenols in which carbon-centered radicals were observed on a CuO/silica surface following the surface-mediated oxidation of 2-chlorophenol in the temperature range of 250-400 °C.25 (24) Flicker, T. M.; Green, S. A. Anal. Chem. 1998, 70, 2008-2012.

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(2) Radicals that are part of, or associated with, polymeric phenolic units. This explanation is supported by previous studies that reported the presence of hydroquinone-semiquinone-quinone polymeric systems in TPM and the saturation behavior of the EPR signal of TPM collected during the combustion of cigarettes and characterized by g ) 2.0035 and ∆Hp-p ) 6.6 G.4 Room-Temperature Oxidation Radicals. After exposure to air, the concentration of radicals increased over the entire range of studied temperatures (see Figure 4A, closed circles); however, the maximal increase was observed for TPM collected at 250-350 °C. At 280 °C, the concentration of these radicals was 8.1 × 1015 spins/g of tobacco, which is greater than the concentration of radicals measured immediately after collection, by a factor of 25. The g-factor also increased for all TPM samples (see Figure 4B, closed circles). TPM collected in the range of 230-280 °C did not contain radicals, but, after exposure to air, some radicals appeared, with a g-factor of 2.0053 ( 0.0003. For TPM collected at higher temperatures, the g-factors are 2.0041-2.0052, which are greater than the g-factors measured before exposure to air (see Figure 4B, open circles). This suggests that, during exposure to air, new radicals are formed. These radicals, with a g-factor of 2.0053 ( 0.0003, are consistent with oxygen-centered semiquinone-type radicals, rather than the carbon-centered (vicinal oxygen) or polymeric, delocalized radicals formed by direct pyrolysis of the tobacco. Similar phenomena were reported recently in the literature.26,27 Char/air interactions of cellulose, pectin, and tobacco were studied using EPR spectroscopy. These results suggested that, during the air exposure of char from tobacco and pectin, “new” radicals form at nonradical sites of the char surface, not through direct reactions of oxygen with “old” radicals generated by pyrolysis. It was suggested that the air exposure involves two different types of chemical processes: the production and annihilation of free radicals. However, the g-factors of these new radicals formed on the char surface (g ≈ 2.0040) differ significantly from those formed during the air exposure of TPM radicals in our experiments (g ≈ 2.0053). This suggests that different types of oxygen-centered radicals are present in the TPM and char. To better determine the precursors for the observed semiquinone radicals, the TPM sample obtained during pyrolysis at 250 °C was subjected to GC/MS analysis. The TPM was extracted from the filter with 1 mL of a mixture containing 80% ethanol and 20% H2O, and the extract was analyzed by GC/MS. The product assignments are made by matching the mass spectral data with a spectral library (Wiley). The analysis identified catechol (m/z ) 110, 81, 64, 31) and hydroquinone (m/z ) 110, 81, 55), as well as some other oxygenated and heteroatom compounds, such as phenol (m/z ) 96, 66, 55), glycerol (m/z ) 61, 43), 5-methyl-furfurol (m/z ) 110, 81, 53, 39), 2,3′-bipyridyl (m/z ) 156, 130), steric acid (m/z ) 284, 241, 185, 129, 97), and nicotine (m/z ) (25) Lomnicki, S.; Dellinger, B. J. Phys. Chem. A 2003, 107, 43874395. (26) Feng, J.-W.; Zheng, S.; Maciel, G. E. Energy Fuels 2004, 18, 560-568. (27) Feng, J.-W.; Zheng, S.; Maciel, G. E. Energy Fuels 2004, 18, 1049-1065.

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Figure 6. Possible pathways for the conversion of hydroquinone (I) to semiquinone radical (IV) and semiquinone radical anion (V) in protic solvent solution. In the upper pathway, a dissociation of both hydroxyl radical protons is required to form quinone dianion (III) before oxidation by O2 to form V. In the lower pathway, only one hydroxyl proton is dissociated24 before oxidation of the semiquinone monoanion (II) to form IV. V can also be oxidized by O2 to form the quinone (VI). Superoxide is formed in each step involving O2. Transition metals are required to induce similar reaction for catechol (ortho-dihydroxybenzene).25,26

162, 133, 84). These data suggest that hydroquinone and catechol in TPM are available precursors for the formation of semiquinone radicals after exposure to air. These results are in accordance with the observations of the model reactions of semiquinone radicals in solution that may have some pertinence to formation in the gas phase and the surface of particles.28 For example, it has been suggested, based on experimental studies, that para- and ortho-dihydroxy compounds, under continuous access to the molecular oxygen, are oxidized to semiquinone-type radicals in a diffusionlimited process, then subsequently oxidized to quinones.28 It has been proposed that both hydroxyl protons of dihydroxy organic compounds in protic media must be dissociated (pK1 ) 9.91 and pK2 ) 11.56 for hydroquinone) before one-electron oxidation occurs (cf. Figure 6). This theory suggests that semiquinone radicals should not be significantly formed at physiological pHs of ∼7.4. However, Pederson29 observed that radical formation from hydroquinone in solution is initiated at a pH slightly below 7.0, implying that the oxidation occurs after the loss of only one hydroxyl proton (cf. Figure 6). He concluded that the mono anion, which exists in small amounts at pH 7 in the presence of molecular oxygen, can be oxidized to the p-hydroxyphenoxyl radical (neutral semiquinone). This radical is unstable and immediately loses a proton to form a semiquinone radical anion (for the neutral radical, pKa ) 4.0). This can explain why some semiquinone radicals might occur in nature at or slightly above the physiological pH, under aerobic conditions. In contrast to hydroquinone, catechol is not oxidized under physiologi-

(28) Chetverikov, A. G.; Kalmanson, A. E.; Kharitonenkov, I. G.; Blumenfeld, L. A. Biofizika 1964, 9, 18-24. (29) Pedersen, J. A. Spectrochim. Acta, Part A 2002, 58 (6), 12571270.

Figure 7. EPR signal of the spin adduct trapped in 0.1 m PBN in benzene during the pyrolysis of 80 mg of tobacco (spectrum A), compared to those obtained during the experiment performed without tobacco (spectrum B) and during the oxidative pyrolysis of tobacco (spectrum C).

cal conditions,30,31 except in the presence of a heavy metal (e.g., copper, iron). The ionic chemistry discussed for reactions in solution is not obviously transferable to gas-phase and particle-surface chemistry. However, transition metals may promote the formation of semiquinone radicals, as has been shown for ortho-chlorinated phenols that are isoelectronic with catechol.32,33 To assess this possibility, a filter loaded with TPM was analyzed for heavy metals, using the high-resolution ICP-MS method. The cellulose filter (pore size of 0.8 µm) with 8.1 mg of TPM collected during the pyrolysis of 86 mg of tobacco at 320 °C was analyzed, and an empty filter after an experiment at 320 °C without tobacco was used as a blank sample. The following elements were detected: cadmium, 3 ng (0.04%); lead, 22.5 ng (0.28%), zinc, 82 ng (1.0%); cobalt, 1.2 ng (0.01%); copper, 53.5 ng (0.66%); chromium, 180 ng (2.22%); manganese, 12 ng (0.15%); nickel, 54 ng (0.67%); and iron, 520 ng (6.4%). Copper is known to catalyze the oxidation of hydroquinone and catechol34,35 and chlorinated phenols to chlorinated phenoxyl radi(30) Schweigert, N.; Zehnder, A. J. B.; Eggen, R. I. L. Environ. Microbiol. 2001, 3 (2), 81-91. (31) Irons, R. D.; Sawahata, R. Phenols, Catechols, and Quinones. In Bioactivation of Foreign Compounds; Anders, M. W., Ed.; Academic Press: San Diego, CA, 1985; pp 259-279. (32) Lomnicki, S.; Dellinger, B. Symp. (Int.) Combust., [Proc.] 2002, 29th (2), 2463-2468. (33) Taylor, P. H.; Sidhu, S. S.; Rubey, W. A.; Dellinger, B.; Wehrmeier, A.; Lenoir, D.; Schramm, K. W. Evidence for a Unified Pathway of Dioxin Formation from Aliphatic Hydrocarbons. Symp. (Int.) Combust., [Proc.] 1998, 27th, 1769-1775. (34) Hirakawa, K.; Oikawa, S.; Hiraku, Y.; Hirosawa, I.; Kawanishi, S. Chem. Res. Toxicol. 2002, 15, 76-82. (35) Li, Y.; Thrush, M. A. Arch. Biochem. Biophys. 1993, 300 (1), 346-355.

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Maskos et al.

Figure 8. Model of TPM and gas-phase radical production from the pyrolysis of macromolecular tobacco constituents I (e.g., lignin (Ia) and chlorogenic acid (Ib)). Pyrolysis forms gas-phase alkoxyl radicals (II) over the entire range of pyrolysis temperatures (240-510 °C). TPM-associated (IIIa) or polymeric (IIIb) carbon-centered radicals (g ) 2.0035-2.0040) are formed at temperatures of g380 °C. For temperatures in the range of 240-380 °C (extending to 510 °C), TPM-associated, molecular catechols/hydroquinone (IV) are formed from the pyrolysis of tobacco constituents. These species are oxidized in air at room temperature by transition metals in TPM to oxygen-centered semiquinone radicals (V) (g ) 2.0053).

cals.25,32,36 The same behavior is expected for iron, on the basis of its reactivity toward chlorophenols.25,32,36 The high concentration of iron, copper, and nickel (which are known to be catalytically active transition metals) suggest that they may be catalyzing the oxidation of catechol and catechol-type compounds, as well as hydroquinone-type compounds, to the semiquinone-type radicals on associated particles at, or near, room temperature. Gas-Phase Radicals. Figure 7 depicts the EPR spectra of PBN spin-trapped adducts formed by gasphase reactions of tobacco pyrolysis products after the TPM has been removed by passing the reactor effluent through a 0.8-µm cellulose filter and a Cambridge filter. The spectra are comprised of a triplet of doublets that result from the interaction of the unpaired electron with the nitroxyl nitrogen (14N, I ) 1) and β-hydrogen (1H, I ) 1/2). The hyperfine coupling constants measured from these spectra are as follows: aN ) 13.9 G and aH ) 2.0 G, with a g-factor of 2.0064 and a line width of 12.1 G. On the basis of these constants and spectral simulation experiments, the gas-phase radicals seem to be alkoxyl radicals. These hyperfine coupling constants agree very well with previous determinations of the parameters for the alkoxyl spin adduct of PBN in benzene.15,37 In some experiments, di-tert-butyl-nitroxide was detected as a result of the oxidation of PBN by NO238 or the thermal decomposition of PBN at >100 °C,39 which yielded an EPR spectrum that exhibited three lines, with aN ) 15.2 G.40 (36) Farquar, G.; Alderman, S.; Poliakoff, E.; Dellinger, B. Environ. Sci. Technol. 2003, 931-935. (37) Baum, S. L.; Anderson, I. G. M.; Baker, R. R.; Murphy, D. M.; Rowlands, C. C. Anal. Chim. Acta 2003, 481, 1-13.

The amplitude of spectrum A in Figure 7 was compared with the amplitudes of spectra obtained during control experiments performed without tobacco (spectrum B in Figure 7) and with that obtained during the combustion (oxidation) of tobacco in air (spectrum C in Figure 7). The integrated intensity of the spectrum allows quantification of the yields of trapped radicals. The concentration of the pyrolysis radicals in tobacco smoke after passing through a 0.8-µm filter and Cambridge filter was ∼(5 ( 1) × 1014 spins/g of tobacco, which is ∼100 times less than that generated during combustion, and only ∼10 times higher than that of the background blank. The concentration of gas-phase radicals formed during the pyrolysis of tobacco is relatively insignificant, compared to radicals formed during oxidation. Conclusions The radicals identified in this study and their assigned sources are presented in Figure 8. It has been found that the fractional pyrolysis of tobacco led to the formation of the following: (1) Alkoxyl radicals in the gas phase of tobacco smoke. The N-tert-butyl-R-phenylnitrone (PBN) adduct of the alkoxyl radicals is characterized by hyperfine coupling constants of aN ) 13.9 G and aH ) 2.0 G and a g-factor of 2.0064. The concentration of these radicals, formed (38) Jantzen, A. G.; Blackburn, B. J. J. Am. Chem. Soc. 1969, 91, 4481-4490. (39) Sommermeyer, K.; Seiffert, W.; Wilker, W. Tetrahedron Lett. 1974, 20, 1821-1824. (40) Forrester, A. R. Magnetic Properties of Free Radicals. In Landolt-Bornstein, New Series, Group II; Springer-Verlag: Berlin, 1979; Vol. 9, 192-1066.

Radicals from the Pyrolysis of Tobacco

under pyrolytic conditions, was insignificant ((5 ( 1) × 1014 spins/g of tobacco) and was not dependent on the pyrolysis temperature. (2) Radicals with g-factors of 2.0035-2.0040 in the total particulate matter (TPM) and char. They are formed from the direct pyrolytic degradation of tobacco constituents. The results indicate that, during the pyrolysis of tobacco, a threshold temperature of ∼380 °C exists above which some components of tobacco or primary char thermally decompose to form these radicals. The g-values of 2.0035-2.0040 are consistent with (i) carbon-centered semiquinone-type radicals, in which the unpaired electron is largely distributed on carbons with vicinal oxygen,41 or (ii) a delocalized electron distributed between oxygen and carbon centers within a polymeric, phenolic matrix.11 In either case, the g-value of 2.0035-2.0040 is between that for a pure carbon-centered organic radical (∼2.003) and a pure oxygen-centered radical (∼2.005). Radical type IIIa in Figure 8 corresponds to a surface-associated, physisorbed or possibly chemisorbed monomeric unit of the radical that is stabilized on the particle surface as a largely carbon-centered radical. Radical type IIIb in Figure 8 corresponds to a radical that is an intrinsic part of the TPM or a polymeric material associated with the TPM. This polymeric material contains both carbon and oxygen functionalities. The electron is delocalized throughout the polymer and exhibits a g-factor determined by the fraction of the unpaired electron density associated with the oxygen and carbon centers. A shift of the g-factor toward the free-electron value at higher temperatures is attributed to loss of oxygen and other heteroatomic functional groups and a gradual increase in the ordering of the polyaromatic skeleton. (3) “New” TPM-associated radicals after the exposure of TPM samples to air at room temperature. The concentration of the new radicals increased as the exposure time increased, reaching a maximum value (41) Barclay, L. R. C.; Cromwell, G. R.; Hilborn, J. W. Can. J. Chem. 1994, 72, 35-41.

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and then declining. The g-factor of these radicals is 2.0053 ( 0.0003, and their concentration reached a maximum for TPM collected during pyrolysis at 280 °C. At this temperature, the concentration of these “new” radicals is 8.1 × 1015 spins/g of tobacco, which is greater than the concentration of radicals measured immediately after collection, by a factor of 25. The g-factor of these radicals is consistent with an oxygen-centered semiquinone radical (cf. Species V in Figure 8). The concentration of the old radicals does not decrease; therefore, the likely source of these new radicals is molecular hydroquinone/catechol precursors detected in the TPM. These molecular precursors (species IV in Figure 8) may be free or chemically bound to particles and do not produce EPR signals, until they are converted to semiquinone-type radicals by atmospheric oxidation. They have been reported in the mainstream of tobacco smoke at concentrations of 100-360 µg/g of tobacco for catechol and 110-300 µg/g of tobacco for hydroquinone and can be selectively collected on cellulose acetate filters.42,43 Because significant quantities of catechol or hydroquinone have not been detected in the tobacco leaf, they are apparently formed during the thermal decomposition of some constituents of tobacco and transferred directly from tobacco/char residue by distillation. Based on the literature and elemental analysis of TPM, it is suggested than transition metals in TPM may be responsible for the formation and stabilization of the “new” semiquinone-type radicals via chemisorption and electron transfer between catechol and the transitions metals. Acknowledgment. Support from Philip Morris, USA (under Grant No. 14358) is gratefully acknowledged. EF040088S (42) Williamson, J. T.; Graham, J. F.; Allman, D. R. Beitr. Tabakforsch. 1965, 3, 233-242. (43) Williamson, J. T.; Allman, D. R. Beitr. Tabakforsch. 1965, 3, 590-596.