In the Laboratory
Fluorescence, Absorption, and Excitation Spectra of Polycyclic Aromatic Hydrocarbons as a Tool for Quantitative Analysis
W
A. M. Rivera-Figueroa, K. A. Ramazan, and B. J. Finlayson-Pitts* Department of Chemistry, University of California, Irvine, CA 92697-2025; *
[email protected] With the introduction of an undergraduate chemistry degree option in environmental chemistry, there is a need for laboratory experiments that illustrate fundamental chemical principles in the context of current environmental problems (1). One area of environmental concern is polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous in air, soils, and water as a result of both direct and indirect emissions (2–7). PAHs are discharged into our environment as the byproducts of the combustion of fossil fuels used for transportation and generation of electricity. Other sources of PAHs include industrial processes, biomass burning, waste incineration, oil spills, and cigarette smoke (2–9). While low molecular weight PAHs are gases, most PAHs have low vapor pressures and hence are adsorbed on airborne particles. These organic com-
benzo[k]fluoranthene MW = 252.32 g/mol abs = 307 nm fluor = 403 nm
benzo[a]anthracene MW = 228.29 g/mol abs = 288 nm fluor = 387 nm
benzo[a]pyrene MW = 252.32 g/mol abs = 297 nm fluor = 405 nm
chrysene MW = 228.29 g/mol abs = 268 nm fluor = 382 nm
Methods
phenanthrene MW = 178.23 g/mol abs. = 251 nm fluor = 366 nm
Figure 1. PAHs analyzed in this experiment with their structures, molecular weights, and absorption and fluorescence maxima.
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pounds have also been found in aquatic environments (6). Many PAHs have been identified as mutagens in bacterial and human cell assays, and some are human carcinogens (2). The U.S. Environmental Protection Agency (EPA) has identified 16 PAHs as priority pollutants (10, 11). Therefore, understanding the properties of PAHs, particularly as related to their qualitative and quantitative analysis, is important and highly relevant to the ACS-approved undergraduate degree option in environmental chemistry. PAHs generally absorb light in the 200–400 nm range and also strongly fluoresce. UV–vis absorption and fluorescence spectroscopic techniques have sensitivities for PAHs on the order of 0.1–1 µg兾L (8) and hence are widely used for analysis of PAHs. These properties of PAHs make them excellent candidates for illustrating basic concepts in instrumental analysis as well as physical chemistry undergraduate laboratories. The experiment reported here illustrates the relationship between the observed absorption, fluorescence, and excitation spectra for five different PAHs and the associated molecular transitions, as well as heavy atom quenching of fluorescence. It illustrates the close coupling between the magnitude of the molar absorption coefficient, ε, and the analytical sensitivity for a particular PAH by UV absorption spectrometry, in addition to the basis for the improved analytical sensitivities using fluorescence compared to absorption spectroscopy for some PAH. In our instrumental analysis laboratory, this experiment precedes the analysis of PAH in cigarette smoke using high-performance liquid chromatography (HPLC) with UV absorption and fluorescence detection (9), as it provides important insights into the HPLC detection methods and choice of analytical wavelengths. The structures, names, molecular weights, and wavelengths of absorption and fluorescence maxima for each of the PAHs studied are shown in Figure 1 and on page 245.
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The five PAH that were used in this study are benzo[a]anthracene, benzo[k]fluoranthene, benzo[a]pyrene, chrysene, and phenanthrene. Stock solutions of 200 mg L᎑1 were provided to the students. These solutions can be prepared by dissolving each solid PAH in dichloromethane. Alternatively, they can be directly purchased as solutions of this concentration in dichloromethane. The students prepared five solutions for each PAH by diluting the stock solutions with heptane to obtain concentrations ranging from 0.2 to 1 mg L᎑1.
Absorption Experiments For the absorption experiments, a few milliliters of each calibration solution were transferred into a quartz cuvette (1 cm), and spectra were collected from 200 to 600 nm using
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In the Laboratory
the UV–vis spectrophotometer (Jasco V–530). For each PAH, the wavelength of maximum absorbance in the wavelength region was identified, and the net absorbance determined. Plots of net absorbance as a function of the concentration (c) were generated for each PAH at its maximum absorbance, and values of the molar absorption coefficient (ε) were calculated from the slopes for each of the five PAHs using the Beer–Lambert law (A = εlc).
Fluorescence Experiments A spectrofluorometer (Jasco FP–750) was used to measure the fluorescence of each PAH. The wavelengths of maximum absorbance measured for each compound were set as the excitation wavelengths. Each of the 1 mg L᎑1 PAH solutions was diluted to four solutions in the range of 0.05–0.2 mg L᎑1. A few milliliters of each solution were transferred into a quartz cuvette with four polished sides. The fluorescence intensity at the peak of the fluorescence spectrum was plotted for each PAH as a function of concentration. Compounds containing heavy atoms are known to be particularly efficient in quenching fluorescence and are also present in the environment. Quenching of fluorescence was investigated using 1-bromoheptane and 1,7-dibromoheptane. The three processes occurring in the presence of a quencher are, absorption fluorescence quenching
A + hν1 A*
A* + Q
kf
kQ
A*
(1)
A + hν2
(2)
A + Q
(3)
where A is a molecule in its ground state, A* is a molecule in an excited state, and Q is any molecule that acts as a quencher. It can be shown that for this system, the Stern–Volmer relationship applies (12), kQ 1 1 1+ = [Q ] If Ia kf
(4)
where If is the intensity of the fluorescence, Ia the intensity of absorption, kQ the rate constant for quenching by Q at a concentration of [Q], and kf is the rate constant for fluorescence. The slope of a plot of 1兾If versus [Q] is [kQ兾(Iakf)] and the intercept is 1兾Ia. The ratio of the slope to the intercept then gives kQ兾kf, the ratio of the quenching rate constant to the fluorescence rate constant. An equivalent formulation (13, 14) of the Stern–Volmer relationship is given as, Φo F = o = 1 + K sv [Q ] Φ F
(5)
where Φo and Φ are the fluorescence quantum yields in the absence and presence of quencher at concentration [Q], respectively; Fo and F are the fluorescence intensities in the absence of quencher and in the presence of quencher at concentration [Q], respectively; and KSV = kQ兾kf is the Stern– Volmer quenching constant. Two sets of six solutions of benzo[a]anthracene were prepared using 2 mL of the 1 mg L᎑1 solution and varying the quantities of solvent and one of the quenchers, as shown in www.JCE.DivCHED.org
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Table 1. Preparation of Solutions of Benzo[a]anthracene and Quencher Heptane/ mL
1-Bromoheptane or 1,7-Dibromoheptaneb/ mL
2.0
3.0
0.0
2.0
2.8
0.2
2.0
2.6
0.4
2.0
2.4
0.6
2.0
2.2
0.8
2.0
2.0
1.0
Benzo[a]anthracenea/ mL
᎑1
a
Solution concentration is 1 mg L .
b
These compounds are quenchers.
Table 1. The fluorescence spectrum of each solution was recorded and the results were plotted in the form of the Stern– Volmer relationship, eq 4. The ratio kQ兾kf was calculated from the slope and intercept of the line for the 1-bromoheptane quenching of the PAH. This procedure was repeated using 1,7-dibromoheptane.
Determination of Detection Limits The detection limits (3σ) were calculated for each PAH in the following manner. First, 20 measurements of the absorbance and fluorescence spectra of the heptane solvent without added PAH were made. It is the variability in this signal that determines the limit of detection when the PAH is present. The average of the 20 measurements gives an average “blank” signal, Sbl, and a standard deviation for this blank of sbl. The minimum detectable analytical signal, SM, is then taken as equal to the blank signal plus 3 times the standard deviation of the blank: (6) S M = S bl + 3s bl Calibration plots of signal (either absorbance or fluorescence) versus concentration are then determined and the slope of these plots, m, obtained. The limits of detection (LD) were obtained by applying (15):
LD =
SM − Sbl 3 s bl = m m
(7)
Hazards The analytes in this experiment are used as dilute (µM and lower) solutions in heptane. The National Fire Protection Association (NFPA) ratings for the PAHs and solvents are in the range of 1 to 2. However, many PAHs as well as dichloromethane are mutagenic or carcinogenic; for three of the compounds used here (benzo[a]anthracene, benzo[k]fluoranthene, and benzo[a]pyrene) there is sufficient evidence to indicate they are carcinogenic (2). The dilutions with heptane should be carried out using a hood and appropriate gloves, as should handling of the quenchers, 1bromoheptane and 1,7-dibromoheptane. The PAH stock solutions provided to the students can be purchased directly; if they are prepared by dissolving the PAH in dichloromethane, a glove box should be used for weighing the standards.
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In the Laboratory 100
0.6
90
excitation 0.5
Absorbance
0.4
70 60 50
0.3
40 0.2
30 20
0.1
absorption 0.0
250
Relative Intensity
80
fluorescence
PAH
350
400
450
Results and Discussion A typical set of absorption, fluorescence, and excitation spectra for benzo[a]anthracene is shown in Figure 2. Plots of absorbance and fluorescence as a function of concentration for this and the four additional PAHs, as well as the Stern– Volmer plots, are given in the Supplemental Material.W The molar absorptivities, limits of detection using absorption and fluorescence, and the Stern–Volmer constants (kQ兾kf) for 1bromoheptane and 1,7-dibromoheptane obtained from such plots are summarized in Tables 2–4. These data illustrate the close coupling between the magnitude of the molar absorption coefficient and the detection limit for that compound measured using absorption spectroscopy. They also illustrate the generally improved limits of detection using fluorescence compared to absorption, particularly for those PAHs that fluoresce strongly, such as benzo[k]fluoranthene and benzo[a]pyrene (Table 3). The limits of detection using fluorescence are in good agreement with the literature (8), except for phenanthrene where our higher limit of detection is due to increased noise in the detection region on our particular instrument. While both of the brominated heptanes quench the fluorescence, the 1,7-dibromoheptane shows a much larger quenching effect than 1-bromoheptane, as expected for a compound with a larger number of heavy atoms. This part of the experiment can be used in an instrumental analysis laboratory to illustrate the potential effects of the presence of unrecognized quenching compounds when using fluorescence analysis. This may be a problem particularly with the complex mixtures found in environmental samples, for example, where hundreds of unidentified compounds may be present. Once students have completed this experiment, they can use the results to choose the optimum absorption, excitation, and fluorescence wavelengths for analysis of these PAHs in cigarette smoke using HPLC with UV–fluorescence detection. In our instrumental laboratory, they perform this experiment in the lab session prior to the one in which they measure PAHs in cigarette smoke (9). •
Karchera
288
9.8 ± 0.4
8.9
297
6.0 ± 0.1
6.4
Benzo[k]fluoranthene
307
6.7 ± 0.1
6.8
Chrysene
268
14.3 ± 0.1
15.2
Phenanthrene
251
6.5 ± 0.1
6.4
a
Ref 16.
0 500
Figure 2: Comparison between the absorption, fluorescence, and excitation spectra of benzo[a]anthracene at a concentration of 1 mg L᎑1.
Journal of Chemical Education
This Work
Benzo[a]anthracene
Table 3. Limits of Detection Using Absorption and Fluorescence Spectroscopy
Wavelength / nm
244
nm
Benzo[a]pyrene
10 300
Table 2. Experimental Molar Absorptivities Compared to Literature Values 4 ᎑1 ᎑1 Wavelength/ ε (± 2σ)/(10 M cm )
Absorption
Fluorescence λ/ nm
Limits of Detection/ µg L᎑1
PAH
λ/ nm
Limits of Detection/ µg L᎑1
Benzo[a]anthracene
288
1.1
387
0.32
Benzo[a]pyrene
297
2.7
405
0.12
Benzo[k]fluoranthene
307
2.1
403
0.04
Chrysene
268
0.8
382
0.33
Phenanthrene
251
1.4
366
4.7
Table 4. Stern–Volmer Constants for the Quenching of Benzo[a]anthracene Constanta (± 2σ)/(L mol᎑1)
Quencher
a
1-Bromoheptane
0.53 ± 0.02
1,7-Dibromoheptane
2.88 ± 0.06
Stern–Volmer constant is equal to kQ/kf.
This experiment is carried out during one, seven-hour laboratory, and students typically use most of the allotted time; the major time factor is preparing the solutions. It can be adapted for shorter laboratory time slots by using fewer PAHs and studying only one of the quenchers. Alternatively, the students could be provided with solutions that have already been diluted to the desired concentrations. Acknowledgments We are grateful to the University of California at Irvine for support of this work. We also thank Bradley Fahlman and Meitai Shu for instrumental assistance. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Hewitt, S. A. Environ. Sci. Technol. 1995, 29, A130. 2. International Agency for Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Polynuclear Aromatic Compounds, Part I: Chemical,
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3. 4. 5.
6.
7.
8. 9.
Environmental and Experimental Data; WHO-IARC: Lyon, France, 1984; Vol. 32–35, 46. Mastral, A. M.; Callen, M. S. Environ. Sci. Technol., 2000, 34, 3051. Mastral, A. M.; Callen, M. S.; Garcia, T.; Lopez, J. M. Environ. Sci. Technol. 2001, 35, 2645. Dickhut, R. M.; Canuel, E. A.; Gustafson, K. E.; Liu, K.; Arzayus, K. M.; Walker, S. E.; Edgecombe, G.; Gaylor, M. O.; MacDonald, E. H. Environ. Sci. Technol. 2000, 34, 4635. Hillery, B. R.; Simcik, M. F.; Basu, I.; Hoff, R. M.; Strachan, W. M. J.; Burniston, D.; Chan, C. H.; Brice, K. A.; Sweet, C. W.; Hites, R. A. Environ. Sci. Technol. 1998, 32, 2216. Finlayson-Pitts, B. J.; Pitts J. N., Jr. Chemistry of the Upper and Lower Atmosphere: Theory, Experiments and Applications; Academic Press: San Diego, CA, 2000. Mahanama, K. R. R.; Gundel, L. A.; Daisey, J. M. Intern. J. Environ. Anal. Chem. 1994, 56, 289. Wingen, L. M.; Low, J. C.; Finlayson-Pitts, B. J. J. Chem. Educ. 1998, 75, 1599.
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10. US-EPA, Code of Federal Regulations, Title 40, Part 302, U.S. Environmental Protection Agency: Washington, DC, 2001. 11. Kelly, T. J.; Mukund, R.; Spicer, C. W.; Pollack, A. J. Environ. Sci. Technol. 1994, 28, 378. 12. Calvert, J. G.; Pitts, J. N., Jr. Photochemistry; John Wiley & Sons, Inc.: New York, 1966. 13. Eftink, M. R. Fluoresence Quenching. In Topics in Fluoresence Spectroscopy; Lakowicz, J. R., Ed.; Plenum Press: New York, 1991; Vol. 2, pp 53. 14. Szabo, A. G. Fluorescence Principles and Measurement. In Spectrophotometry and Spectrofluorimetry; Gore, M. G., Ed.; Oxford University Press: Oxford, U.K., 2000; p 33. 15. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed.; Saunders College Publishing: Chicago, IL, 1998. 16. Karcher, W.; Fordham, R. J.; Dubois, J. J.; Glaude, P. G. J. M.; Ligthart, J. A. M. Spectral Atlas of Polycyclic Hydrocarbons; D. Reidel Publishing Company: Dordrecht, The Netherlands, 1985.
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