Energy & Fuels 2008, 22, 1675–1679
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Radicals from the Oxidative Pyrolysis of Tobacco Zofia Maskos and Barry Dellinger* Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana ReceiVed NoVember 7, 2007. ReVised Manuscript ReceiVed February 18, 2008
The fractional, oxidative pyrolysis of Bright tobacco was performed in an oxygen/nitrogen atmosphere containing from 0 to 20.4% of O2 over the temperature range of 200–510 °C in a specially constructed, hightemperature flow reactor system. The effect of the temperature and the concentration of oxygen in the reaction atmosphere on the generation of free radicals from Bright tobacco and two major components of Bright tobacco (polyphenols and cell wall biopolymers) were studied. Electron paramagnetic resonance (EPR) spectroscopy was used to analyze for free radicals in the initially produced total particular matter (TPM) and in the gas phase. Two types of radicals were observed in the initially produced TPM: type I radicals characterized by a five-line EPR spectrum with an apparent g-value of ∼2.0064 that was tentatively assigned to a tyrosyl radical formed at lower temperatures from the cell wall biopolymer fraction of tobacco, and type II radicals characterized by a single-line EPR spectrum with g-factor of 2.0035 assigned as a surface-associated, carbon-centered radical that is vicinal to an oxygen-containing functional group or an unpaired electron in the bulk of an oxygencontaining polymeric matrix. The yields of both types of primary radicals decreased with increasing oxygen concentration. This decrease in TPM radicals was accompanied by a significant increase in the formation of the gas-phase, alkoxyl radicals. The type I and type II primary TPM radicals formed during the oxidative pyrolysis were the same as those observed from pyrolysis of Bright tobacco. The oxidative pyrolysis of chlorogenic acid (a major component of the polyphenol fraction) formed radicals characterized by a singleline EPR spectrum with g-factor of ∼2.0053 and ∆Hp-p of 10–13 G. The structure of these radicals is assigned as oxygen-centered, semiquinone-type radicals that were formed from the thermal decomposition of chlorogenic acid. Their yield was not dependent on the oxygen concentration and appeared to be the same radicals previously reported in aged TPM.
Introduction From a thermal reaction perspective, the burning or “smoking” of a cigarette can be effectively divided into endothermic pyrolysis/distillation zones and exothermic oxidation/combustion zones. The oxidation/combustion zone that is in or near the burning tip is characterized by temperatures >600 °C and O2 at or very near 20.4%. Because of the high temperature and oxygen concentration, this zone produces few reaction byproduct other than carbon dioxide, water, and carbon monoxide.1–3 In contrast, the pyrolysis/distillation zones, which are located behind the burning tip, are oxygen deficient (380 °C. The structure of these radicals was assigned as surface-associated, carbon-centered radicals, where the unpaired electron is vicinal to an oxygencontaining functional group or a partially delocalized electron associated with the bulk of a phenoxyl-type polymeric matrix.6 In addition, we observed secondary radicals formed upon exposure of TPM to aging4,5,7 that were identified as oxygencentered, semiquinone-type radicals formed from atmospheric oxidation of hydroquinone/catechol-type species which were free or chemisorbed to TPM.7 Only the latter results, which reveal the formation of semiquinone-type radicals in aged TPM are in (5) Maskos, Z.; Khachatryan, L.; Dellinger, B. Precursors of radicals in tobacco smoke and the role of particulate matter in forming and stabilizing radicals. Energy Fuels 2005, 19, 2466–2473. (6) Maskos, Z.; Khachatryan, L.; Dellinger, B. Formation of the persistent primary radicals from the pyrolysis of tobacco. Energy Fuels 2008, 22, 1027–1033. (7) Maskos, Z.; Dellinger, B. Formation of the secondary radicals from the aging of tobacco smoke. Energy Fuels 2008, 22, 382–388. (8) Dellinger, B.; Lomnicki, S.; Khachatryan, L.; Maskos, Z.; Hall, R. W.; Adunkpe, J.; McFerrin, C.; Troung, H. Formation and stabilization of persistent free radicals. Proc. Combust. Inst. 2007, 31, 521–528.
10.1021/ef7006694 CCC: $40.75 2008 American Chemical Society Published on Web 03/15/2008
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agreement with previously reports8–17 that the burning of tobacco produces hydroquinone- and catechol-type molecular species that can induce oxidative stress and DNA damage through the formation of semiquinone-type free radicals. Similar experiments reported recently18,19 showed the presence of hydroquinone, catechol, and their methyl-substituted derivatives in the particulate phase of cigarette smoke. The formation of semiquinone radicals via oxidation of the dihydroxybenzenes was found to be dependent on the reduction potential of the corresponding quinine/semiquinone redox couple. However, the capacity to generate semiquinone radicals was found to be insufficient to explain the variance in the cytotoxicity among the dihydroxybnzenes and the authors18,19 suggested that other mechanisms of toxicity were involved. In the effort to better identify the radicals in cigarette smoke formed under the full range of smoking conditions and assess how they affect the toxicity of cigarette smoke, we report the effect of varying oxygen concentration on the yield and nature of PFRs associated with TPM as well as gas-phase free radicals. In addition, we report the formation of PFRs formed from the oxidative pyrolysis of (i) methanol/water extracts of Bright tobacco that contain mostly polyphenols and (ii) extraction residue that includes mostly cell wall biopolymers. Experimental Section Materials. Samples of Bright (flue-cured) tobacco, before and after methanol/water extraction, were provided by Philip Morris U.S.A. (Richmond, VA) and used without further treatment. The methanol/water extraction was carried out as follows: the sample of Bright tobacco was extracted with methanol (tobacco to methanol ratio of 1: 30) in 11 cycles in a Soxhlet apparatus, and the residue was further extracted with water (tobacco to water ratio of 1: 400). The remaining residue was dried at room temperature for 48 h. 2,2-Diphenyl-1-picrylhydrazyl (DPPH; free radical, 98%), N-tertR-phenylnitrone (PBN; 98%), 2,2,6,6-tetramethyl-1-piperidynyloxy (9) Stone, K.; Bermudez, E.; Zang, L. Y.; Carter, K. M. The ESR properties, DNA nicking, and DNA association of aged solutions of catechol versus aqueous extracts of tar from cigarette smoke. Arch. Biochem. Biophys. 1995, 319 (1), 196–203. (10) Levay, G.; Ye, Q.; Bodell, W. J. Formation of DNA adducts and oxidative base damage by copper mediated oxidation of dopamine and 6-hydroxydopamine. Exp. Neurol. 1997, 146, 570–574. (11) Pryor, W. A.; Stone, K.; Zang, L.-Y.; Bermudez, E. Fractionation of aqueous cigarette tar extracts: Fractions that contain the tar radicals cause DNA damage. Chem. Res. Toxicol. 1998, 11 (5), 441–448. (12) Barclay, L. R. C.; Cromwell, G. R. ; Hilborn, J. W. Photochemistry of a model lignin compound. Spin trapping of primary products and properties of an oligomer. Can. J. Chem. 1994, 72, 35–41. (13) Schweigert, N.; Acero, J. L.; von Gunten, U.; Canonica, S.; Zehnder, A. J. B.; Egge, R. I. L. DNA degradation by the minute of copper and catechol is caused by DNA-copper-oxo complexes, probably DNA-Cu(I) OOH. EnViron. Mol. Mutagen. 2000, 36, 5–12. (14) Pryor, W. A.; Hales, B. J.; Premovic, P. I.; Church, D. F. The radicals in cigarette tar: Their nature and suggested physiological implications. Science 1983, 220, 425–427. (15) Borish, E. T.; C, J. P.; Church, D. F.; Deutsch, W. A.; Pryor, W. A. Cigarette tar causes single-strand breaks in DNA. Biochem. Biophys. Res. Commun. 1985, 133, 780–786. (16) Leanderson, P. T. C. Cigarette smoke-induced DNA damage: role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine. Chem.-Biol. Interact. 1990, 75, 71–81. (17) Pryor, W. A. Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity. EnViron. Health Perspect. 1997, 105 (Suppl. 4), 875–882. (18) Chouchane, S.; Wooten, J. B.; Tewes, F. J.; Wittig, A.; Muller, B. P.; Veltel, D.; Diekmann, J.; Involvement of semiquinone radicals in the in Vitro cytotoxicity of cigarette mainstream smoke, Chem. Res. Toxicol. 2006, 19, 1602–1610. (19) Wooten, J. B., Chouchane, S. McGrath, T. E., Tobacco smoke constituents affecting oxidative stress. In Cigarette Smoke and OxidatiVe Stress; Halliwell, B. B., Poulsen, H. E., Eds.; Springer: New York, 2006; pp 5–46.
Maskos and Dellinger (TEMPO; sublimed, 99%), and silica gel (Devisil, grade 633, 200–425 mesh) were obtained from Aldrich Chemical Co., and chlorogenic acid was obtained from Sigma Chemical Co. The EPR tubes were standard X-band Suprasil high-purity quartz tubes (3 and 4 mm inner diameter (ID)) and were supplied by the Wilmad/ Labglas Co. Fractional Oxidative Pyrolysis. Tobacco samples were pyrolyzed in a commercial Pyroprobe 1000 (CDC Analytical, Inc.) using a continuous flow-reactor system. The experimental details are given elsewhere.4 The samples (80 ( 2 mg) were placed in a quartz tube (i.d. ) 6 mm) and inserted inside the pyroprobe coil. The tobacco samples were diluted with silica in a ratio of 1:1. Both sides of the tube insert were plugged with quartz wool to prevent sample loss. Prior to the experiments, samples were flushed with nitrogen or argon for 24 h to remove traces of oxygen. 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 (K type) thermocouple (o.d. ) 0.25 mm). The temperature gradient along the layer of tobacco in the reaction tube did not exceed (5 °C. Argon or nitrogen containing no more than 5 ppm of oxygen was used as a carrier gas in the pyrolysis experiments. For the oxidative pyrolysis, nitrogen was mixed with air to obtain from 0.01 to 20.4% of oxygen in the reaction gas mixture. The reaction carrier gas was introduced at a flow rate of 200–330 cm3/min, which corresponded to a gas residence time of 0.05 s. The flow rate was controlled by a mass controller (McMillan Co., Model 80D). The sample was heated for a reaction time of 60 s at each temperature, and TPM was collected on the filter. The filter with TPM was flushed for 5 min with the carrier gas, then removed and replaced with a new filter. The entire experiment was performed with the same bed of tobacco and the temperature was varied from 200 to 510 °C in 30–60 °C increments. The smoke from the heated tobacco was passed through a cellulose filter (Osmonic Inc.) supported by conventional glass fiber Cambridge filter, and the TPM was collected and weighed. The cellulose filter had an active surface area of 0.5 cm2 and was efficient for particles larger than 0.8 µm in diameter. After collecting the smoke sample, the cellulose filter was cut into 2 mm wide strips, and the strips were inserted directly into an EPR tube. The sample was stored at -196 °C until it was analyzed using EPR at room temperature. In some experiments, the condensable products on the cellulose filter were extracted with methanol, and the extract was analyzed via gas chromatography/mass spectroscopy (GC/MS) using a model 6890N series gas chromatograph coupled to a 5973 mass selective detector (Agilent). A 30 m, 0.32 mm i.d. capillary column (HP-5) with a film thickness of 0.25 µm was used for separation. Spin-Trapping. 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-tertbutyl-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 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 degassed using a freeze–pump–thaw technique and filled with argon. All samples were stored at –196 °C until EPR analyses were performed. Electron Paramagnetic Resonance. All EPR spectra were recorded at room temperature on a Bruker EMX-10/2.7 EPR spectrometer (Bruker Instruments, Billerica MA) with double rectangular resonator ER 4105DR, 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 (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, 128 ms for spin-trap adduct. Values of g-factors were calculated using Bruker’s WINEPR program. The calculated g-values were checked using a g-factor
Radicals from the OxidatiVe Pyrolysis of Tobacco
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Figure 1. The EPR spectra of the initially produced TPM from pyrolysis (N2) and from oxidative pyrolysis (0.78% O2 in N2).
of standard DPPH sample. The concentrations of free radicals in the TPM were calculated using double integration of the first derivative signal and comparison with a known concentration of a DPPH standard. The concentration of gas-phase radicals was determined by comparison with a known concentration of a TEMPO standard in benzene.
Results and Discussion Primary Radicals in TPM. Figure 1 depicts the EPR spectra of TPM generated during the oxidative pyrolysis of Bright tobacco at 280 and 450 °C. The properties of these EPR spectra, the g-value, and the resolution strongly depended on the pyrolysis temperature. The 280 °C/N2 TPM spectrum exhibited five lines, with an apparent g-factor of 2.0060 and total spectral width of 42 G. When the temperature was increased from 280 to 450 °C, the resolved spectrum was gradually converted to the single line with a g-value of 2.0036 and ∆Hp-p of 8.5 G. The fractions of TPM collected at higher pyrolysis temperatures were extracted with benzene and filtered. The EPR spectrum of these radicals in benzene exhibited a single, symmetrical Lorentzian line with a g-value of 2.0035 and ∆Hp-p of 6.6 G. These spectra were almost identical to those of radicals previously reported14 in TPM from the burning of 1R1 research cigarettes. Our previously reported results of the EPR microwave power dependence6 implied that these two fractions contained different types of radicals: type I that consisted of a five-line spectrum, with an apparent g-factor ∼ 2.0064 and overall width of ∼ 42 G that can be observed in TPM produced from the pyrolysis of tobacco at temperatures e380 °C, and type II radicals with a characteristic EPR spectrum consisting of singlet with g ∼ 2.0035 that was observable for pyrolysis temperatures of >380 °C. Under oxidative pyrolysis conditions (0.78% of O2/N2), similar spectra were obtained but the radical concentrations were lower and resolution was reduced for the type I radicals. Figure 2 compares the temperature dependence of the radical concentration under pyrolysis and oxidative pyrolysis conditions. The radical concentration increased from ∼8.0 × 1014 spins/g of tobacco at 240 °C to ∼3.5–4.0 × 1015 spins/g of tobacco at 450-510 °C when the experiments were performed in an inert
Figure 2. The effect of temperature on the concentration of primary radicals formed during the fractional pyrolysis of Bright tobacco in N2 (O) and in 0.78% of O2 in N2 (b).
atmosphere. The presence of even a minute quantity of oxygen in the reaction atmosphere decreased the concentration of radicals (especially g380 °C), namely, the concentration of the radicals was reduced from 4 × 1015 spins/g at 450 °C, but only from 1 × 1015 spins/g at 280 °C. Figure 3A depicts the variations in the concentration of primary radicals in the fractions of 280 and 450 °C fractions versus the concentration of oxygen in the reaction atmosphere. As the concentration of oxygen increased from 0 to 5.2%, the concentration of radicals decreased from ∼1 × 1015 to 0.2 × 1015 spins/g of tobacco for the 280 °C fraction and from ∼4 × 1015 spins/g of tobacco to nearly undetected for the 450 °C fraction (Figure 3A). Simultaneously, the g-values of type I radicals decreased slightly from 2.0060 to 2.0053 but increased significantly for type II radicals from 2.0036 to ∼2.0052 (Figure 3B). The g-factor of 2.0034–2.0039 is characteristic of carboncentered radicals with a nearby oxygen heteroatom which is known to increase spin–orbit coupling that results in increased g-factors over that of purely carbon-centered radicals.11,20 The increase of the g-value from ∼2.0036 to ∼2.0052 with the increasing concentration of oxygen can be attributed to either (i) the oxidation of some carbon-centered radicals to oxygencentered radicals or (ii) the carbon-centered radicals were (20) Graf, F.; Loth, K.; Gunthard, H.-H. Chlorine hyperfine splittings and spin density distribution of peroxy radicals. An ESR and quantum chemical study. HelV. Chim. Acta 1977, 60 (76), 710–720.
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Figure 5. Formation of primary radicals during the oxidative pyrolysis of methanol/H2O extracted Bright tobacco residue (cell wall biopolymer fraction) and chlorogenic acid performed in N2 (white) and 0.78% O2/ N2 (black). Figure 3. The effect of oxygen concentration (A) on the concentration and (B) on the g-factor of primary radicals formed during the fractional pyrolysis of Bright tobacco for the fraction of 280 and 450 °C.
Figure 4. Effect of temperature and oxygen concentration on the formation of gas-phase radicals (Insert: EPR signal of the spin adduct trapped in 0.1 m PBN in benzene during the oxidative pyrolysis of Bright tobacco).
destroyed and the lower concentration of oxygen-centered radicals dominated the EPR spectrum. Radicals in Gas-Phase Smoke. The analysis of the gas-phase component of tobacco smoke revealed the formation of alkoxyl radicals. The N-tert-butyl-R-phenylnitrone (PBN) adduct of 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 (insert in Figure 4).4 A total of 7 acyl and 11 alkylaminocarbonyl radicals have recently been identified in the fresh whole smoke from the combustion of 2R4F cigarette.21 Because these radicals would be expected to react rapidly with oxygen before they reached the spin trap solution (the rate constant for the reaction of simple alkyl radicals with oxygen is very fast, approximately 109 L/mol × s),22 the carbon-centered radicals were not observed as a spin-adduct in our system. The small quantities of alkyl (21) Bartalis, J.; Chan, W. G.; Wooten, J. B. A new look at radicals in cigarette smoke. Anal. Chem. 2007, 79, 5103–5106. (22) Johnson, W. R. Incorporation of atmospheric oxygen into components of cigarette smoke. Chem. Ind. (London) 1975, 521.
radical or cyclohexadienyl radicals as spin-trap adducts have also been observed previously.23 Figure 4 depicts the yield of gas-phase radicals versus temperature and oxygen concentration. The concentration of radicals from pyrolysis (0% O2) was
