In the Laboratory
Chromatography, Absorption, and Fluorescence: A New Instrumental Analysis Experiment on the Measurement of Polycyclic Aromatic Hydrocarbons in Cigarette Smoke Lisa M. Wingen, Jason C. Low, and Barbara J. Finlayson-Pitts* Department of Chemistry, University of California, Irvine, Irvine, CA 92697-2025
The importance of including environmental issues in the traditional chemistry curriculum is growing as public concern for these issues heightens. In particular, the approval by the American Chemical Society of an undergraduate chemistry degree option in environmental chemistry has generated a need for environmentally focused experiments in undergraduate laboratories (1). We describe here an experiment that can be used to illustrate the basic principles of high-performance liquid chromatography (HPLC) and is especially suitable for a junior/ senior level undergraduate instrumental analysis laboratory. The experiment clearly demonstrates differences between absorption and fluorescence as detection methods and compares the sensitivity and selectivity of these methods with respect to a variety of polycyclic aromatic hydrocarbons (PAH). A great deal of research has been devoted to PAH. PAH are described structurally as molecules of two or more fused benzene or cyclopentadiene rings, and their derivatives. Benzo[a]pyrene (Fig. 1) was the first pure compound to be isolated and synthesized, and was subsequently found to be strongly carcinogenic in animals in 1933 (see ref 2 and references therein). Since then, many other PAH have been identified in ambient particles throughout the world. Many are carcinogenic to humans and other animals; and many are mutagens, able to attack and mutate DNA, or promutagens, which must first be metabolized to produce mutagenic compounds in vivo. Hence measurement of the sources and concentrations of PAH is of considerable importance. PAH are formed in the burning of tobacco as well as during incomplete combustion of organic matter in wood and fossil fuels (2, 3). It is not surprising, then, that PAH are ubiquitous in the atmosphere. Once formed, PAH of 5 or 6 rings tend to condense onto existing atmospheric particles, while smaller PAH exist in the gas phase. Whether gases or particles, PAH are respirable, and in the case of submicron particles, able to reach the deep lung.
destructive, they can be placed in series (as they are in this experiment) to obtain a wealth of information for each compound. The combination of these two detectors illustrates the wavelength dependence of absorption and fluorescence as well as the relative sensitivities of the two detection techniques. It was the application of fluorescence that allowed the sensitive detection of PAH in a variety of environmental samples beginning a number of years ago (e.g., see refs 5–8). The experiment described here illustrates this as well as the basics of HPLC in the context of an important environmental problem, environmental tobacco smoke, ETS. Experimental Procedure The HPLC separations and analyses are performed on a Hewlett Packard 1100 series system consisting of a vacuum degasser, a quaternary pump, a 5-µL injection loop, and a diode array detector (DAD). A fluorescence detector (HP1046A) is connected in series with the DAD to the output of a Phenomenex Envirosep-PP column (125 × 3.20 mm, polymeric bonded silica) preceded by a guard column (Phenomenex Envirosep-PP, 30 × 3.20 mm). The solvent
Purpose of This Experiment The experiment described here illustrates the exceptional reproducibility, sensitivity, and selectivity of high-performance liquid chromatography (HPLC) with fluorescence and absorption detectors for analyzing PAH in the complex mixture found in cigarette smoke. These qualities make HPLC a widely used analytical technique, and thus essential to include in the traditional chemistry curriculum (4). Moreover, the experiment demonstrates the application of this methodology to solving environmental problems. Several detection methods used with HPLC have the ability to increase the student’s analytical skills. Because both UV–visible absorption and fluorescence detectors are non*Corresponding author.
Figure 1. Structures and names of the 16 PAH in the EPA standard. The numbers are assigned in order of elution to identify individual PAH in the chromatograms.
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consists of water, acetonitrile, and tetrahydrofuran in a starting ratio of 50:40:10, used at a total flow rate of 1.0 mL per minute. A linear solvent gradient starts 4 minutes after the injection and linearly adjusts to the final ratio of 90:10 acetonitrile:tetrahydrofuran at 18 minutes. All the analyses are carried out at room temperature. A stock solution containing a mixture of 16 PAH is obtained by diluting 60 µL of the standard PAH sample (Supelco, EPA 610 PAH mix) with 1 mL of a 1:1 (v/v) methylene chloride:methanol (HPLC grade) solvent mixture. Calibration curves are produced from 50-µL aliquots of the stock solution diluted with the 1:1 CH2Cl2:CH3OH solvent in ratios of 1:1, 1:2, 1:3, and 1:5, giving four standards. The names and structures of the 16 PAH are shown in Figure 1, with the order of elution from this column shown in parentheses. Figure 2 illustrates the apparatus used to extract the PAH from cigarette smoke. Precautions should be taken to clear the cigarette smoke sampling site of flammable material and solvents. An unfiltered cigarette is lit and allowed to burn until 20–30% of it has been consumed. The smoke residue collects on the fritted disk of the funnel (Hirsch, 50 mL, medium porosity) and is extracted using 1 mL of the CH2Cl2:CH3OH solvent mixture. This solution is filtered through a 0.2-µm Acrodisc filter (Gelman Sciences) to remove particles, and the filtrate is evaporated to dryness using a flow of inert gas. The evaporated sample is then dissolved in 50 µL of the solvent mixture. It should be noted that this procedure results in the loss of the more volatile PAH (pyrene and smaller PAH) (2). Absorption, excitation, and emission wavelengths can be selected to optimize the detector sensitivity for specific compounds. The five wavelength settings chosen for the absorption measurements are 241, 252, 269, 289, and 297 nm, which correspond to characteristic peak absorption wavelengths for pyrene, phenanthrene and anthracene, chrysene, benzo[a]anthracene, and benzo[a]pyrene, respectively (9). Three combinations of excitation and emission wavelengths are chosen to optimize the fluorescence detection for phenanthrene (245 nm excitation, 359 nm emission), chrysene (263, 371 nm), and benzo[a]anthracene (288, 405 nm). These wavelength combinations were chosen using the excitation and fluorescence data of Karcher et al. (9). A listing of optimized excitation and fluorescence wavelengths for a variety of PAH is also given by Mahanama et al. (3).
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Results and Discussion
Analysis of Standard Mixture of PAH Initially, an injection of pure solvent is performed to familiarize the student with the injection procedure, as well as to flush the column. Between sample injections, the injection loop should be flushed to remove traces of previous samples. Injections of the PAH stock solution are then per-
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Figure 2. Apparatus used to collect the cigarette smoke.
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Figure 3. Typical fluorescence chromatograms using different combinations of excitation and emission wavelengths optimized for the detection of specific PAH: (a) (245, 359 nm) combination for phenanthrene, (b) (263, 371 nm) for chrysene, and (c) (288, 405 nm) for benzo[a]anthracene.
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In the Laboratory
formed sequentially using the three different wavelength combinations for fluorescence detection, resulting in the typical chromatograms shown in Figure 3. Identification of each PAH in the standard mixture is obtained by comparing the pattern of retention times of the experimental chromatograms to those reported by the supplier. The results also demonstrate the wavelength dependence of
Figure 4. Typical chromatograms for the standard PAH mixture at five absorption wavelengths: (a) 241 nm for optimal detection of pyrene, (b) 252 nm for phenanthrene, (c) 269 nm for chrysene, (d) 289 nm for benzo[a]anthracene, and (e) 297 nm for benzo[a]pyrene.
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Analysis of PAH in Cigarette Smoke A typical chromatogram of the cigarette extract using fluorescence detection with the (288, 405 nm) wavelength combination is shown in Figure 6. Peaks due to individual PAH in the fluorescence spectrum of the cigarette sample are superimposed on a broad background, which is typical of cigarette smoke (3, 10). Phenanthrene (peak #5) does not show a large peak because it is sufficiently volatile that it exists primarily in the gas phase under these conditions (2). In
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fluorescence. For example, as seen in Figure 3a, phenanthrene (peak #5) has a strong fluorescence signal optimized at the wavelength combination (245, 359 nm), but a much weaker signal at the wavelength combination (288, 405 nm) chosen to optimize benzo[a]anthracene detection (Fig. 3c). Using the (263, 371 nm) combination chosen to optimize the detection of chrysene, peak #10 due to chrysene is strong (Fig. 3b) while that due to phenanthrene (#5) is significantly smaller than in Fig. 3a. Finally, using the (288, 405 nm) combination optimized for benzo[a]anthracene, a strong peak (#9) (Fig. 3c) is detected for this compound but the peaks corresponding to phenanthrene (#5) and chrysene (#10) are small. One also can see similar trends in detection sensitivities using absorption spectra (Fig. 4a–e). The phenanthrene peak (#5) is strong near its characteristic absorbance peak of 252 nm, but gets progressively weaker at longer absorption wavelengths. In contrast, the benzo[a]anthracene peaks (#9) become progressively stronger as the absorption wavelength settings increase from 241 nm towards the characteristic peak absorption at 289 nm. Detection of chrysene (#10) is optimized at 269 nm. Each of the four standards is analyzed using absorption at five wavelengths and fluorescence with three wavelength combinations. Calibrations are created using either the integrated area of the peaks (as in the present experiment) or peak heights. Figure 5 shows typical calibration curves for chrysene from the PAH standard mixture using both absorption at 269 nm (Fig. 5a) and fluorescence with excitation at 288 nm and emission at 405 nm (Fig. 5b). Both are linear over the range 0 to 30 ng. Time permitting, calibration curves may be constructed using the other two wavelength combinations, (263, 371 nm) and (245, 359 nm).
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Figure 5. Calibration curves for chrysene using (a) absorption at 269 nm and (b) fluorescence with the (288, 405 nm) wavelength combination.
Figure 6. Typical chromatogram of a cigarette smoke sample using fluorescence with 288 nm excitation.
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Figure 7. Typical chromatogram of a cigarette smoke sample using absorption at 289 nm.
addition, the wavelength combination is not optimized for this compound. However, fluorescence signals for chrysene (#10) and benzo[a]anthracene (#9) are sufficiently large that these compounds can be quantitatively analyzed. For the particular sample shown in Figure 6, the peaks on top of the broad background correspond to 20.4 ± 1.7 (2σ) ng chrysene and 6.00 ± 0.55 (2σ) ng of benzo[a]anthracene. Given that these amounts were obtained from the smoke from burning 23% of a cigarette, dissolving the evaporated sample in 50 µL of solvent, and injecting through a 5-µL loop, the sample contained 887 ± 74 (2σ) ng of chrysene per cigarette and 261 ± 24 (2σ) ng of benzo[a]anthracene per cigarette. The average total mass of the cigarette was 0.807 g, giving 1100 ± 92 (2σ) ng of chrysene and 324 ± 30 (2σ) ng of benzo[a]anthracene per gram of tobacco burned. On the other hand, signals using the photodiode array detector for absorption measurements (Fig. 7) are much smaller and would be difficult to use to quantify the PAH with high accuracy. This provides an excellent illustration of the differences in detection sensitivity for PAH provided by these two detection methods. However, this chromatogram can be used for qualitative analysis, as is shown by the identification of chrysene (peak #10), in Figure 7. Other Applications Tobacco smoke is a quick and convenient sample for demonstrating the utility of HPLC in the analysis of PAH. The results reported here can be compared with those from other studies of cigarette smoke reported in the literature. For example, environmental tobacco smoke particles from Kentucky Reference 1R4F cigarettes were reported to contain 412 ± 36 ng of chrysene and 152 ± 23 ng of benzo[a]anthracene per cigarette (10), values similar to those reported here for an unfiltered commercial cigarette. In another study, side-stream smoke collected from a Kentucky Reference cigarette 1R4F on filter pads and analyzed by HPLC gave 219 ng of benzo[a]anthracene per cigarette (11). Chrysene in cigarette smoke condensate, which was isolated by cold trap and liquid–liquid extractions followed by several chromatographic separations, was reported to be 70 ng per cigarette (12), a factor of 10 less than that found in this experiment. 1602
Other samples such as diesel exhaust particles (13), tap water, river water, and ambient air samples (14, 15), biological samples (16 ), extracts from charcoal grilling of meat (17), and the residue of coal samples (18) can be used to demonstrate the same concepts. However, such samples are not as readily obtained as cigarette smoke and the extraction processes for these samples are much more complex and time-consuming. In addition, the much smaller concentrations found in many of these samples make such sources less suitable for use in an undergraduate laboratory. For comparison, GC and GC-MS analysis of PAH extracted from coal fly ash samples collected from the walls of a coal-burning steam engine and a coal-burning gas production plant (19) showed benzo[a]anthracene in the range of 12– 31 µg/g and chrysene from 13 to 41 µg/g. Similarly, PAH in the smoke and drippings from charcoal grilling of meat range from tens of micrograms per kilogram of meat for the 3-to4-ringed PAH to below 1 µg/kg meat for the 5- and 6-ringed PAH (17 ). Thus, the cigarette sample in the experiment described in this paper contains concentrations of PAH one to two orders of magnitude higher than those from charcoalgrilled meat and one to two orders of magnitude lower than those from fly ash samples. It should be noted that while this laboratory focused on the application of HPLC to the detection and measurement of PAH, other techniques, such as GC-MS, could also be applied to the extract (13, 17, 19–22). Gaseous emissions from cigarettes, such as HCHO, can also be analyzed by GC and HPLC (23). Conclusions The experiment described here is appropriate for a junior/ senior-level laboratory on instrumental analysis. It illustrates the application of HPLC to the analysis of complex mixtures in the context of an important environmental problem, PAH in tobacco smoke. The use of absorption and fluorescence as detection methods is illustrated and the large difference in their sensitivities for this analytical problem is demonstrated. Hence this experiment should be especially useful for introduction into instrumental analysis laboratories used in the undergraduate degree option in environmental chemistry. Acknowledgments We would like to thank Meitai Shu for experimental assistance. We gratefully acknowledge the Camille and Henry Dreyfus Foundation and the University of California, Irvine, for financial support and A. R. Chamberlin, R. J. Cicerone, J. C. Hemminger and J. N. Pitts, Jr. for helpful discussions. Literature Cited 1. Hewitt, S. A. Environ. Sci. Technol. 1995, 29, A130–A132. 2. Finlayson-Pitts, B. J.; Pitts, J. N., Jr. Atmospheric Chemistry: Fundamentals and Experimental Techniques; Wiley: New York, 1986. 3. Mahanama, K. R. R.; Gundel, L. A.; Daisey, J. M. Int. J. Environ. Anal. Chem. 1994, 56, 289–309. 4. Willis, W. V. Laboratory Experiments in Liquid Chromatography; CRC: Boca Raton, FL, 1991. 5. Wheals, B. B.; Vaughan, C. G.; Whitehouse, M. J. J. Chromatogr. 1975, 106, 109–118. 6. Wise, S. A.; Chesler, S. N.; Hertz, H. S.; Hilpert, L. R.; May, W. E. Anal. Chem. 1977, 49, 2306–2310.
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In the Laboratory 7. Smillie, R. D.; Wang, D. T.; Meresz, O. J. Environ. Sci. Health 1978, A13, 47–49. 8. Ogan, K.; Katz, E.; Slavin, W. Anal. Chem. 1979, 51, 1315–1320. 9. Karcher, W.; Fordham, R. J.; Dubois, J. J.; Glaude, P. G. J. M.; Ligthart, J. A. M. Spectral Atlas of Polycyclic Aromatic Compounds Including Data on Occurrence and Biological Activity; D. Reidel: Dordrecht, The Netherlands, 1985. 10. Gundel, L. A.; Mahanama, K. R. R.; Daisey, J. M. Environ. Sci. Technol. 1995, 29, 1607–1614. 11. Risner, C. H. Beitrage Tabakforsch. Int. 1991, 15, 11–17. 12. Schmid, E. R.; Bachlechner, G.; Varmuza, K.; Klus, H. Fres. Z. Anal. Chem. 1985, 322, 213–219. 13. Pointet, K.; Renou-Gonnord, M.-F.; Milliet, A.; Jaudon, P. Bull. Soc. Chim. Fr. 1997, 134, 133–140. 14. Núñez, M. D.; Centrich, F. Anal. Chim. Acta 1990, 234, 269–273. 15. Kicinski, H. G.; Adamek, S.; Kettrup, A. Chromatographia 1989, 28, 203–208.
16. Thompson, D.; Jolley, D.; Maher, W. Michrochem. J. 1993, 47, 351–362. 17. Dyremark, A.; Westerholm, R.; Överik, E.; Gustavsson, J. Atmos. Environ. 1995, 29, 1553–1558. 18. López García, A.; Blanco González, E.; García Alonso, J. I.; SanzMedel, A. Chromatographia 1992, 33, 225–230. 19. Gohda, H.; Hatano, H.; Hanai, T.; Miyaji, K.; Takahashi, N.; Sun, Z.; Dong, Z.; Yu, H.; Cao, T.; Albrecht, I. D.; Naikwadi, K. P.; Karasek, F. W. Chemosphere 1993, 27, 9–15. 20. Simonsick, W. J., Jr.; Hites, R. A. Anal. Chem. 1984, 56, 2749–2754. 21. Lee, H. K. J. Chromatogr. A 1995, 710, 79–92. 22. Gere, D. R.; Knipe, C. R.; Castelli, P.; Hedrick, J.; Randall Frank, L. G.; Schulenberg-Schell, H.; Schuster, R.; Doherty, L.; Orolin, J.; Lee, H. B. J. Chromatogr. Sci. 1993, 31, 246–258. 23. Wong, J. W.; Ngim, K. K.; Shibamoto, T.; Mabury, S. A.; Eiserich, J. P.; Yeo, H. C. H. J. Chem. Educ. 1997, 74, 1100– 1103.
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