Anal. Chem. 1998, 70, 2008-2012
Technical Notes
Detection and Separation of Gas-Phase Carbon-Centered Radicals from Cigarette Smoke and Diesel Exhaust Thomas M. Flicker and Sarah A. Green*
Chemistry Department, Michigan Technological University, Houghton, Michigan 49931-1295
Carbon-centered radicals were trapped from gas-phase cigarette smoke and diesel engine exhaust by reaction with a nitroxide, 3-amino-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3AP). The resulting mixture of stable, diamagnetic adducts was derivatized with naphthalenedicarboxaldehyde (NDA) to produce highly fluorescent products. Derivatives were separated by high-performance liquid chromatography (HPLC), which revealed distinctly different suites of radicals present in the two systems. Integration of HPLC peaks gave approximately 22 ( 7 nmol of radicals per cigarette and 3 ( 1 nmol of radicals per liter of diesel engine exhaust. An estimated 8-10 different carbon-centered radical species are present in each system. Gas-phase free radicals are important in a large variety of environmental,1,2 photochemical, and biological3,4 processes. In particular, combustion reactions are dominated by radical species, and radical reactions can continue within smoke plumes well beyond the point of combustion. These plume reactions may be important in determining the ultimate composition and toxicity of smoke from combustion sources. In real-world situations, radicals are typically present in complex matrixes at relatively low concentrations. Thus, their exact concentrations and roles within a given system can be difficult to study. Spin-trapping methods have been used to detect the presence of radicals in several heterogeneous systems, such as cigarette smoke,5 wood smoke,6 polystyrene processing-plant air,7 and polluted air,8 and in fumes from test burns of a variety of (1) Church, D. Anal. Chem. 1994, 66, 419A-427A. (2) Blough, N. V.; Zepp, R. G. In Active Oxygen in Chemistry; Foote, C. S., Valentine, J. S., Greenberg, A., Liebman, J. F., Eds.; Chapman and Hall: New York, 1995; pp 280-333. (3) Pryor, W. A. Free Radical Biol. Med. 1992, 13, 659-676. (4) Halliwell, B.; Guttrtidge, J. M. C. Free radicals in biology and medicine; Oxford University Press: Oxford, 1989; p 317. (5) Pryor, W. A.; Tamura, M.; Church, D. F. J. Am. Chem. Soc. 1984, 106, 5073. (6) Lachocki, T. M.; Church, D. F.; Pryor, W. A. Free Radical Biol. Med. 1987, 7, 17. (7) Westerberg, L.-M.; Pfaffli, P.; Sundholm, F. Am. Ind. Hyg. Assoc. 1982, 43, 544-546. (8) Dulseva, G.; Skubnevskaya, G.; Tikhonov, A. Y.; Mazhukin, D. G.; Volodarsky, L. B. J. Phys. Chem. 1996, 100, 17523-17527.
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materials.9,10 However, identification of specific radical species is difficult with standard spin-trapping techniques. Computer modeling of combustion has focused on the immediate area of the flame or engine; we are unaware of any modeling work on radical reactions within cooling smoke plumes. There are currently few techniques amenable to detailed investigations of reactions in smoke, as noted by Church, who, in a general review for Analytical Chemistry, stressed the need for robust radical traps that both form stable adducts and allow for the identification of the trapped species.1 Here we present a simple method that can answer both of these requirements for gas-phase carbon-centered radicals. The use of di-tert-alkyl nitroxide compounds for the detection of radicals has become well established in the past several years.11-15 Nitroxides react rapidly with carbon-centered radicals to form stable alkoxyamine adducts; solution bimolecular rate constants are 108-109 M-1 s-1.16 The resulting alkoxyamine adducts are sufficiently stable for storage, manipulation, and analysis by high-performance liquid chromatography (HPLC). Our method is based on that developed by Kieber and Blough, which has proven to be especially sensitive for the detection of photochemically generated radicals in aqueous solutions.12,13 An amino nitroxide, 3-amino-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical (3AP, I), is used to trap carbon-centered radicals (R•) (Scheme 1). The resulting stable alkoxyamine adducts (R-3AP, II) are derivatized with naphthalenedicarboxaldehyde (NDA, III) to produce highly fluorescent products (R-3AP-NDA, IV). (The NDA derivative of the parent 3AP (3AP-NDA) has extremely weak fluorescence due to efficient intramolecular quenching of (9) Lachocki, T. M.; Church, D. F.; Pryor, W. A. Environ. Res. 1988, 45, 127139. (10) Lowry, W. R.; Peterson, J.; Petty, C. S.; Badgett, J. L. J. Forensic Sci. 1985, 30, 73-85. (11) Johnson, C. G.; Caron, S.; Blough, N. V. Anal. Chem. 1996, 68, 867-872. (12) Kieber, D. J.; Blough, N. V. Anal. Chem. 1990, 62, 2275-2283. (13) Kieber, D. J.; Blough, N. V. Free Radical Res. Commun. 1990, 10, 109117. (14) Kieber, D. J.; Johnson, C. G.; Blough, N. V. Free Radical Res. Commun. 1992, 16, 35-39. (15) Blough, N. V.; Simpson, D. J. J. Am. Chem. Soc. 1988, 110, 1915-1917. (16) Athelstan, L.; Beckwith, J.; Bowry, V. W. J. Org. Chem. 1988, 53, 16321641. S0003-2700(97)00858-5 CCC: $15.00
© 1998 American Chemical Society Published on Web 03/28/1998
Scheme 1
Figure 1. Apparatus used to trap carbon-centered radicals from the gas phase with 3AP.
chemically produced radicals are trapped by 3AP to form stable alkoxyamines. For the solution-phase standard, 3AP (1 mM) and acetone (100 mM) in standard borate buffer were irradiated in a 1-cm quartz cell with a 150-W xenon lamp for 30 min. Prior to irradiation, each sample was deoxygenated by bubbling with argon (99.999% purity, Interstate Welding) for 5 min. During irradiation, the cell headspace was slowly purged with argon to exclude oxygen. A 10-cm water filter was used between the lamp and irradiation cell to absorb IR radiation. A gas-phase standard was made by similar means. Approximately 5 mg of 3AP in methanol was added to a 5-cm irradiation tube (with quartz windows). This solution was rolled over the glass walls of the tube until dry. Acetone-saturated argon
was then passed through the tube and irradiated as above for 30 min. Upon completion, 3AP and 3AP adducts were washed from the cell with 10 mL of standard borate buffer. Trap Loading. Fifteen grams of clean glass beads, approximately 0.6 cm diameter, was loaded into a 100-mL roundbottom flask. A 1-mL aliquot of 35 mM 3AP in acetone was added to the beads and dried by rotory evaporation at 25 °C. The coated beads (0.05%, w/w) were stored in a desiccator at -10 °C until needed. For sampling, the 3AP-coated glass beads were loaded into a standard 15-cm distillation column (with a small ball of wire as a plug to hold beads in place, Figure 1), and cigarette smoke or diesel exhaust was flushed through the column. Cigarette Smoke Sampling. The puff protocol18 was used to sample cigarette smoke. Using a 50-mL syringe, 35-mL puffs of 2-s duration were drawn from the cigarettes. Teflon tubing (5 cm × 1 cm i.d.) was used to hold the cigarette and to attach the syringe to the sampling column. Puffs were expelled into the column over a 2-s duration; the complete cycle was repeated every 8-10 s. One sample consisted of five commercially available Marlboro filtered cigarettes smoked sequentially. Smoke was filtered only by the manufacturer’s cigarette filter before it reached the 3AP-coated beads. Diesel Exhaust Sampling. A 1995 Cummins M330E diesel research engine, with an exhaust gas recirculator (EGR) in use, operated at mode 9 (75% load), produced the exhaust samples. The stainless steel sampling port (1 cm i.d.) was located in the main exhaust line approximately 1 m from the engine. A short length of Teflon tubing connected the sampling port to the sampling apparatus as described previously. An estimated 5% of the total exhaust flow was sampled. During exhaust sampling, tap water was circulated through the water-cooling jacket of the column. Sampling flow rate was maintained at 1 L/min for 15 min. Control Samples. Two types of controls were generated for each radical source sampled. With the first method, a blank was made by performing all the usual steps except the final derivatization with NDA. For the second style of blank, the sampling procedure was performed exactly as usual (including the derivatization step) but with no 3AP on the beads. Derivatization Procedure. After sampling, the beads were transferred to a 50-mL Erlenmeyer flask, and 10 mL of standard borate buffer was added to dissolve the 3AP and adducts. Next, 2200 µL of water, 500 µL of 3AP sample solution, 100 µL of sodium cyanide solution, and 200 µL of NDA solution were added sequentially to a glass vial and allowed to react for 30 min. When the solution was filtered with a 0.2-µm syringe filter, the 3APNDA product precipitated onto the filter. Sample was washed from the filter and into a HPLC vial with 1 mL of 80% methanol in
(17) Green, S. A.; Simpson, D. J.; Zhou, G.; Ho, P. S.; Blough, N. V. J. Am. Chem. Soc. 1990, 112, 7337-7346.
(18) Pryor, W. A.; Terauchi, K.; Davis, W. H. Environ. Health Perspect. 1976, 16, 161-175.
fluorescence by nitroxide free radicals.17) The adducts are separated by HPLC and detected fluorometrically.12 We have adapted this originally solution-based method to the trapping of carbon-centered radicals from the gas phase by adsorbing 3AP molecules onto a solid support, over which the sample gas is passed. Additionally, we have replaced the fluorophore used in previous work (fluorescamine) with NDA. This has greatly improved the peak resolution and limits of detection. EXPERIMENTAL METHODS Chemicals. Acetone and sodium borate were purchased from Fisher Scientific. NDA, sodium cyanide, 3AP, cyclopentylamine (CPA), and HPLC grade methanol were purchased from Acros. All chemicals were of the highest purity available and were used without further purification. Marlboro brand cigarettes were purchased from a local vendor. Solutions of NDA in methanol (5.0 mM) were prepared weekly and stored in the freezer at -5 °C. Stock sodium cyanide solutions (10.0 mM) were prepared weekly and stored at room temperature. Stock sodium borate buffer (25 mM, pH 9.2) was prepared by dissolving the appropriate amount of sodium borate in water and adjusting the pH with 1 M NaOH. Water used for all solutions was from a Millipore Milli-Q system. Generation of Standard Radicals. With near-ultraviolet light, Norrish type I photolysis of acetone proceeds through reaction 1 to produce methyl and acetyl radicals. The photo-
CH3C(O)CH3 + hv f •CH3 + •C(O)CH3
(1)
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water. Experiments performed on standards show that this filtering sequence effectively collects and concentrates the 3APNDA species. In addition to protecting the HPLC (by filtering the sample), this procedure provided a better sample matrix for injection and removed any particle-bound 3AP adducts. High-Performance Liquid Chromatography. The HPLC was a Hewlett-Packard Series 1100 with a binary pump, autosampler, vacuum degasser, thermostated column compartment, diode array detector, and HP 1046A programmable fluorescence detector. A Supelco 2.1-mm × 250-mm column with 5-µm reversephase (C18) packing was used for all separations. Consistent with published parameters for NDA adducts, the fluorescence detector was set at 420 (excitation) and 480 nm (emission). Allowable sensitivities for the fluorescence photomultiplier tube (PMT) were between 1 and 18; samples were run at PMT ) 13, 14, or 15. Separations (of 3AP-NDA and adducts) were carried out at 30 °C, with a flow rate of 0.3 mL/min. The mobile-phase composition for the gradient was (A) water and (B) methanol. The selected gradient consisted of 30% A/70% B, ramped to 20% A/80% B in 20 min. The run was stopped at 30 min. CPA-NDA standards were run under the same conditions, except that the mobile phase was isocratic at 20% A/80% B. CPANDA injection volumes ranged from 5 to 50 µL, and runs were complete in less than 7 min. Cyclopentylamine-NDA Standard. CyclopentylamineNDA (CPA-NDA) was used for quantification approximations because it was easy to make and structurally similar to 3APNDA. CPA was derivatized by the same methods described above for 3AP. A stock solution of 1 µM CPA-NDA was used for HPLC quantification purposes at two different PMT gain settings. Calibration Curves. Calibration curves for two PMT sensitivities were generated with the CPA-NDA. Nine standard injections were run on the HPLC and yielded linear calibration curves (one at each PMT setting) with excellent correlation coefficients (r2 ) 0.999). The nine calibration injections ranged from 5 to 50 pmol of CPA-NDA. The fluorescence yield of CPA-NDA was found to be only slightly dependent on solvent, increasing by 2% as the proportion of methanol in a methanol/water solution increased from 70 to 80%. Since 3AP-NDA adducts eluted over this mobile-phase range, the approximate concentrations reported have a minimum uncertainty of 2%. RESULTS AND DISCUSSION Comparison of Solution and Gas-Phase Trapping. Chromatograms illustrating the separation of trapped acetyl and methyl adducts from the photolysis of acetone are shown in Figure 2. Samples irradiated with no 3AP present yielded no fluorescent peaks at standard conditions (not pictured). Irradiated 3AP samples, free of acetone and derivatized, yielded one early fluorescent peak and several small bumps in the chromatogram (Figure 2a). The result of solution-phase radical trapping of acetone photolysis products, using a procedure similar to that described by Kieber and Blough,12 is shown in Figure 2b. Gasphase trapping, as outlined above, provided a chromatogram with nearly identical peaks and retention times (Figure 2c). Adducts were assigned by comparisons to previous work.11-13 The observed peaks correspond to adducts of •CH3 (D) and •COCH3 (B). Peaks A and C are unknown components that grow in after 2010 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
Figure 2. Fluorescence chromatograms for the comparison of solution and gas-phase trapping with the described 3AP-NDA method. (a) Blank run (PMT ) 15) of irradiated sample solution (standard procedure) with no acetone. (b) 3AP and acetone irradiated in solution (PMT ) 13). (c) 3AP on solid-phase support with irradiated acetone-saturated argon gas blown over trap (PMT ) 13). Peaks D correspond to the adduct •CH3, peaks B to •C(O)CH3, and peaks A and C to unidentified products.
irradiation. Absorbance spectra of peaks B and D matched those of 3AP-NDA derivatives, as expected. The •COCH3/•CH3 peak ratios were different for the liquid- and gas-phase generation schemes. The decrease in the amount of •CH3 trapped from the gas phase may indicate that it has a shorter lifetime than •COCH3 in the presence of acetone and trace oxygen, or that it is less efficiently trapped from the gas phase. These chromatograms clearly indicate that the 3AP-NDA method, established for solution-phase sampling, can also be applied to gas-phase radical trapping. Cigarette Smoke. To test this method in a real-world scenario, we examined tobacco smoke, a system that is known to contain gas-phase carbon-centered radicals.1 Cigarette smoke was sampled using the sampling apparatus and puffing method described above. The first control, run (and derivatized) with no 3AP on the beads, produced three early fluorescent peaks and some noise (Figure 3a). The second control, with 3AP but omitting the final derivatization step, showed a minimal number of fluorescent peaks (Figure 3b). Peaks in the blanks correspond either to fluorescent compounds trapped from the smoke (a and b) or to primary amines in the smoke which are derivatized by NDA (a). Figure 3c shows the result for sampling cigarette smoke using the method described. Fluorescent peaks which correspond to a variety of carbon-centered radicals were observed in the chromatogram. The cigarette smoke chromatogram (Figure 3c) was integrated from 2 to 25 min, and peak areas from the blank (a) were
Figure 4. Fluorescence chromatogram from sampling 15 min of diesel engine exhaust with standard procedure (PMT ) 14). Figure 3. Fluorescence chromatograms from sampling cigarette smoke from five commercial cigarettes. (a) Blank generated by sampling smoke over clean glass beads (no 3AP) and derivatizing with NDA as usual (PMT ) 14). (b) Blank sample with 3AP-coated beads, but omitting derivatization step (PMT ) 14). (c) Cigarette smoke sample trapped with 3AP and derivatized with NDA (PMT ) 14).
subtracted. Comparison with the CPA-NDA calibration curves gives a concentration of 11 ( 4 µM of trapped carbon-centered radicals in the 10 mL of solution rinsed from the glass beads. This corresponds to 22 ( 7 nmol of radicals trapped per cigarette, based on four independent trials of five cigarettes each. Pryor et al. reported that the yield of radicals from one 1R1 research cigarette, using the spin trap R-phenyl-N-tert-butylnitrone (PBN) in benzene, was 17 nmol per cigarette.5 PBN traps both alkyl and alkoxyl radicals; these two types were differentiated, although more specific identification was not possible with their method. In Pryor’s samples, approximately 30% of the radicals trapped were from carbon-centered species, giving 5 nmol of R• per cigarette. Our value is about 4-fold higher, 22 ( 7 nmol/ cigarette. Some of this difference may be due to variable amounts of radicals generated in different experiments, depending on the particular tobacco tested and the smoking method employed. However, it is possible that some radicals have been missed in earlier spin-trapping experiments because of degradation of the spin adducts before EPR analysis or phase-partitioning limitations. Given the uncertainties in quantification for both methods, we consider the values to be in reasonable agreement. Diesel Engine Exhaust. The method was also tested on diesel engine exhaust. Blank runs (with no 3AP) produced no detectable fluorescence over background, indicating that no
primary amines were trapped from the exhaust. Unlike tobacco smoke, nonderivatized diesel samples yielded no fluorescent peaks. A chromatogram of radical adducts produced from 15 min of exhaust sampling is shown in Figure 4. Again, it is clear that a variety of carbon-centered radicals were trapped from the exhaust. The chromatogram from diesel exhaust sampling (Figure 4) was integrated from 2.0 to 26.0 min. Calculations based on the CPA-NDA calibrations (PMT gain 14) gave 41 ( 13 nmol of carbon-centered radicals trapped from 15 min (15 L) of exhaust sampling, or 3 ( 1 nmol/L for three trials. We are unaware of previous measurements of radicals in gas-phase diesel exhaust for comparison. However, we note that the number of radicals detected in 8 L of exhaust was equal to about the amount from one cigarette. Diesel exhaust sampling (Figure 4) yielded a variety of fluorescent peaks. The most prominent feature on these chromatograms is the number of large, early-eluting peaks. We suggest that peaks eluting prior to the parent 3AP-NDA (6.1 min) are very polar or ionic radical species that were trapped from the smoke. Cigarette smoke sampling did not produce such large peaks early in the chromatogram. Adducts of NO2-R• species would be quite polar and may be responsible for early-eluting peaks. NO2-R• radicals are postulated to result from NO2 additions to alkenes. We are currently investigating the generation of standard NO2-R•-type radicals. We expect radical concentrations in diesel exhaust to be dependent on a variety of engine parameters, such as fuel composition, air/fuel mixture, engine load and type, and pollutioncontrol equipment. For example, several measurements performed without the EGR in place yielded radical concentrations about 10-fold higher than those reported here. Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
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Sample-to-sample variability in both cigarette and diesel experiments is most likely due to variation in combustion materials or parameters (temperature, humidity, burn rate); better control of these parameters is expected to improve our error estimates. For a fixed radical concentration, the factors controlling trapping efficiency have not yet been fully investigated. The maximum number of radicals that could be trapped in our system as presently configured is 40 µmol, assuming all 3AP on the beads is available for reaction. So, the number of trapping sites is not limiting; however, the flow characteristics of gas within the column could limit the number of sites encountered. In addition, we do not yet know the sticking coefficient for the reaction of radicals with adsorbed 3AP. Humidity and temperature of the gas stream could be critical in determining trapping efficiency, and efforts are underway to quantify these parameters. CONCLUSIONS Application of a known solution-phase radical trapping system to a solid-phase support for the trapping of gas-phase radicals has been demonstrated. A simple standard was used for comparison and showed that acetyl and methyl radicals were trapped directly from the gas phase. Samples from cigarette smoke yielded chromatograms with a large number of fluorescence peaks corresponding to trapped radical adducts. Radicals were found
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in tobacco smoke at somewhat higher concentrations than previously reported.5 Diesel exhaust was also found to contain an array of carbon-centered radicals. While the specific radicals have not yet been identified, it is clear that a minimum of 8-10 different radicals are present in each source examined and that a distinctly different suite is produced in each of the two systems studied thus far. The system described is very flexible and easily adapted to a variety of gas-phase radical sampling applications. Ultimately, application of HPLC-mass spectrometry will be employed to identify trapped radicals. In the future, we plan to extend this study to other combustion systems, such as gasoline engines and pyrolysis of synthetic and natural materials. ACKNOWLEDGMENT We thank Dr. J. Perlinger for generously allowing use of her HPLC, the Mechanical Engineering Department at MTU for access to the diesel engine, Ms. Linda Gratz for helpful discussions, and the Department of Chemistry at MTU for funding.
Received for review August 8, 1997. Accepted February 19, 1998. AC970858F
