Anal. Chem. 1996, 68, 1226-1232
Liquid Chromatographic Determination of Nitro-Substituted Polynuclear Aromatic Hydrocarbons by Sequential Electrochemical and Fluorescence Detection Mitsunori Murayama† and Purnendu K. Dasgupta*
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
Nitro-substituted polynuclear aromatic hydrocarbons occur in ambient suspended particulate matter and are of special concern because they act as direct mutagens. Determination of these compounds in a complex matrix such as particulate matter present in diesel engine exhaust is complicatedseven mass spectrometry requires initial cleanup and separation steps. We propose a sensitive liquid chromatographic method with an unique selectivity: an electrochemical detector operating in the reductive mode is followed by a fluorescence detector. When the NO2 group is reduced to the NH2 group, there is a major increase in fluorescence; nitro-PAH compounds are essentially nonfluorescent. A difference fluorescence signal or a difference chromatofluorogram is generated by subtracting the chromatogram obtained with the electrochemical detector off from that obtained with the electrochemical detector on. Applications to diesel engine exhaust samples are demonstrated. Nearly two decades ago, Jager1 and Pitts et al.2 independently discovered that atmospheric nitrogen oxides are capable of readily nitrating polynuclear aromatic hydrocarbons (PAHs) present in ambient particulate matter to form nitro-PAHs (NPAHs). NPAHs are of particular interest because of their genotoxicity. These compounds have measurable solubilities in the aqueous phase;3 this lability aids their biological reactivity. In the Ames test, the parent PAH compounds are mutagenic only after metabolic activation,4,5 whereas the NPAH compounds are direct mutagens. Several studies have been carried out regarding the occurrence and concentrations of various NPAH species in ambient air and particulate matter; they occur significantly in both phases.6 Indeed, the available evidence strongly implicates NPAHs for the mutagenicity exhibited by extracts of ambient particulate material.7 Theoretical efforts to model the formation and decay of NPAHs † Permanent address: National Institute of Hygienic Sciences, Ministry of Health and Welfare, 1-18-1 Kamiyoga, Setagaya, Tokyo, Japan. (1) Jager, J. J. Chromatogr. 1978, 152, 575-578. (2) Pitts, J. N., Jr.; Van Cauwenberghe, K. A.; Grosjean, D.; Schmid, J. P.; Fitz, D. R.; Belser, W. L.; Knudson, G. B.; Hynds, P. M. Science 1978, 202, 515519. (3) Yu, G.; Xu, X. Chemosphere 1992, 24, 1699. (4) Talcott, R.; Wei, E. J. Natl. Cancer Inst. 1977, 58, 449-451. (5) Sasaki, J.; Arey, J.; Harger, W. P. Environ. Sci. Technol. 1995, 29, 13241335. (6) Wilson, N. K.; McCurrdy, T. R.; Chuang, J. C. Atmos. Environ. 1995, 29, 2575-2584. (7) Gundel, L. A.; Daisey, J. M.; de Carvalho, L. R. F.; Kado, N. Y.; Schuetzle, D. Environ. Sci. Technol. 1993, 27, 2112-2119.
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in the atmosphere have also been undertaken.8 Patterns and sources of PAHs and their derivatives in indoor air are also now attracting attention.9 Particulate material from diesel-fired engine exhaust has long been identified as a prime source of atmospheric NPAHs. However, 2-nitrofluoranthene and 2-nitropyrene, the two most abundant NPAH species in ambient particulate matter, do not typically occur in diesel exhaust samples.5 To determine individual NPAH compounds unambiguously in a matrix as complex as ambient atmospheric aerosol, the final analytical step must be carried out by mass spectrometry, preferably with certified reference materials run in parallel through the entire procedure.10,11 Much initial cleanup, typically by a liquid chromatographic technique, is usually required. MacCrehan et al.12 provided a survey of the analytical practice as reported in the literature. Although this article appeared in 1988, there have been few changes in the general approach since. Preliminary work can be extensive: if detailed fractionation is desired, a sequence might involve, for example, Soxhlet extraction of a filter sample, followed by gel filtration chromatography, normal phase liquid chromatography, and final analysis by capillary gas chromatography/mass spectrometry.13 It would clearly be desirable to have a faster and less capital intensive analytical method, at least for initial screening purposes, to determine NPAHs in atmospheric samples and in other samples, such as foodstuff cooked in open flames. Liquid chromatography with electrochemical (EC) detection (differential pulse12 or conventional amperometry in the reductive mode12,14 ) has been shown to be applicable for the sensitive detection of NPAHs. In complex samples, however, many other species can respond under the same detection conditions, and additional selectivity is necessary. Krause and Wang15 have suggested that the prereduction of nitroaromatic species with subsequent oxidative determination to be advantageous, but the method has not actually been used for NPAH compounds. While most PAH compounds are markedly fluorescent, the strong electron-withdrawing effect of the nitro group renders (8) Kamens, R. M.; Zhi-hua, F.; Yilin, Y. Chemosphere 1994, 28, 1623. (9) Mitra, S.; Ray, B. Atmos. Environ. 1995, 29, 3345-3350. (10) Nishioka, M. G.; Howard, C. C.; Contos, D. A.; Ball. L. M.; Lewtas, J. Environ. Sci. Technol. 1988, 22, 908-915. (11) Lindsey, A. S.; Belliardo, J. J.; Wagstaffe, J. J. Fresenius Z. Anal. Chem. 1989, 333, 599-606. (12) MacCrehan, W. A.; May, W. E.; Yang, S. D.; Benner, B. A., Jr. Anal. Chem. 1990, 60, 194-199. (13) Niles, R.; Tan, Y. L. Anal. Chim. Acta 1989, 221, 53-63. (14) Ang, K. P.; Tay, B. T.; Gunasingham, H. Int. J. Environ. Stud. 1987, 29, 163-170. (15) Krause, R. T.; Wang, Y. J. Chromatogr. 1988, 459, 151-162. 0003-2700/96/0368-1226$12.00/0
© 1996 American Chemical Society
Figure 1. Experimental arrangement schematic.
NPAHs nonfluorescent. If the NO2 functionality is reduced, however, to the NHOH or NH2 functionality, the resulting compounds will be strongly fluorescent. Thus, detection of NPAHs in liquid chromatography has been accomplished after reduction by Zn10 or Pt/Rh on Al2O314 or electrochemical means.16 After reduction to the amine, detection can be accomplished by fluorometry;10,14,17 additional selectivity in this case can be gained by acquiring a full fluorescence spectrum of the eluite.18 The feasibility of obtaining very high resolution fluorescence spectra of these compounds at low temperature has been recently demonstrated.19 Chemiluminescence detection can also be performed;16 early reports indicate that it is more sensitive than fluorescence detection.20 By combining reductive electrochemical detection with fluorometry, we suggest here a new, uniquely selective approach to the problem of sensitive detection of NPAHs. A coulometric detector is operated in the reductive mode, followed by fluorometric detection. Once the time delay between the two detectors is accounted for, an eluite peak is uniquely assigned to a NPAH species if it meets both of the following criteria: (a) both detectors respond to the eluite and (b) the fluorescence detector signal disappears if the coulometric detector is turned off (no reduction of NO2 group, thus no fluorescence). EXPERIMENTAL SECTION Reagents and Test Analytes. NPAH test substances used in this study included 1-nitronaphthalene (1-NN), 2-nitronaphthalene (2-NN), 2-nitro-1-naphthol (2-NNOH), 1-amino-4-nitronaphthalene (1-A-4-NN), 9-nitroanthracene (9-NA), 2-nitrofluorene (2NF), 9-hydroxy-3-nitrofluorene (9-OH-3-NF), 1-amino-7-nitrofluorene (1-A-7-NF), 2-nitro-9-fluorenone (2-N-9-F), and 1-nitropyrene (1NP) (all from Aldrich). Methanol (HPLC grade, Mallinckrodt), analytical reagent grade H2SO4, and distilled deionized water was used for the preparation of the chromatographic eluent (85:15 MeOH-1.0 mM H2SO4). Other chemicals/solvents used were of analytical reagent grade. (16) Imaizumi, N.; Hayakawa, K.; Suzuki, Y.; Miyazaki, M. Biomed. Chromatogr. 1990, 4, 108-112 (17) Hayakawa, K.; Terai, N.; Suzuki, Y.; Dinning, P. G.; Yamada, M.; Miyazaki, M. Biomed. Chromatogr. 1993, 7, 262-266. (18) Tejada, S. B.; Zweidinger, R. B.; Sigsby, J E., Jr. Anal. Chem. 1986, 58, 1827-1834. (19) Matsuzawa, S.; Garrigues, P.; Budzinski, H.; Bellocq, J.; Shimizu, Y. Anal. Chim. Acta 1995, 312, 165-177. (20) Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56, 10961102.
NPAH stock solutions (1-10 mM) were prepared in methanol. Working standards were prepared by dilution with the chromatographic eluent. All NPAH reagents, stock solutions, and working standards were stored refrigerated in the dark. Equipment. The overall experimental arrangement is shown in Figure 1. The majority of the instrumentation used in this work ranges in age from 10 to 15 years. Thus, the performance data reported in this paper should not be regarded as limits of the described technique. Liquid chromatography was carried out isocratically with a Beckman Model 110A pump, equipped with a Model 210 sample injection valve (100 µL loop) and a Spherisorb ODS II reversed-phase column (3 µm particles, 6 mm × 150 mm), preceded by a 6 mm × 20 mm guard column packed with the same material, at a flow rate of 0.5 mL/min. The EC detector was a Coulochem 5100A (ESA Inc., Bedford, MA), equipped with a Model 5010A analytical cell. This instrument uses a palladium-based modified H2/H+ electrode as the reference. The cited voltages are therefore ∼0.2 V greater those obtained against an Ag/AgCl electrode. The working electrode is porous graphite. EC detectors are particularly sensitive to flow pulsations. Because the flow noise from the pump used was relatively high, a 4.6 mm × 250 mm column, packed with 10 µm macroporous poly(styrenedivinylbenzene) resin (PRP-1, Hamilton Co.), was put in before the injector as a pulse dampener. The flow noise was reduced only partially; significant residual noise remained. This noise is the dominant factor in determining the limits of detection (LODs) cited in this paper. Reductive EC detection requires removal of oxygen from the eluent; a column packed with zinc particles and later Pt on Al2O3 has been recommended for this purpose in fluorometric detection;18,21 others have reported that both packing materials are incompatible with an EC detector downstream.15 The ESA detector has two independent potentiostats, one of which, intended for operation with a guard cell/electrode, can be operated with a current up to 1 mA. We incorporated such a guard cell, operated at -2.5 V (unless otherwise stated), after the pump and before the injection valve. With the guard cell in operation, the background current of the detector following the separation column decreased dramatically; oxygen removal by this method may not have been quantitative but was sufficient for satisfactory testing of the serial detection scheme. The fluorometric detector was Shimadzu Model RF-530. Fluorescence spectra were recorded on a RF-540 recording (21) MacCrehan, W. A.; May, W. E. Anal. Chem. 1984, 56, 625-628.
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Figure 2. Hydrodynamic voltammograms and resulting fluorescence signals for four selected NPAH compounds. Note different ordinate scaling for the fluorescence signals in the four panels.
spectrofluorophotometer. A filter fluorometer (Fluoromonitor III, Thermo Separation Systems) was also used in some experiments. Chromatographic data were recorded on a Model TY-2 dualchannel strip chart recorder (Knauer) or a PC-based system using AI-450 software (Dionex). Sampling and Processing of Diesel Engine Exhaust Samples. Exhaust from a diesel-powered electric generator was sampled on a 47 mm glass fiber filter (type GF/A, Whatman Inc.) at sampling rates mass flow controlled between 10 and 15 L/min under no-load and 50% of maximum permissible engine load conditions. The sample temperature was continuously measured with a thermocouple-based recording thermometer. The exhaust temperature at the sampling point ranged from 70 to 75 °C. Doubtless, the partition of volatile PAH/NPAH species to the particle phase would be much greater in an aged exhaust that is at ambient temperature than under our sampling conditions. However, the present purpose was not so much to quantitatively characterize diesel engine exhaust as to study the ability of the proposed analytical system to handle matrices associated with real samples. Therefore, no efforts were made to age and cool the exhaust before sampling. After sampling, the filter was extracted in a Soxhlet extractor for 24 h with 150 mL of CH2Cl2. The dichloromethane was then carefully evaporated under a controlled stream of pure N2, and the residue was sonicated with 10 mL of 85:15 MeOH-6.67 mM H2SO4. The supernatant was injected into the HPLC system, after further dilution with methanol (typically 20-fold). RESULTS AND DISCUSSION Choice of Fluorometric Detection Conditions. While the scheme does not preclude the use of a real-time spectrally imaging fluorescence detector or a rapid scanning instrument (either would 1228
Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
Table 1. Fluorescence Characteristics of Selected NPAH Compounds after Reductiona
NPAH compound (1) 1-nitronaphthalene (2) 2-nitronaphthalene (3) 2-nitro-1-naphthol (4) 1-amino-4-nitronaphthalene (5) 9-nitroanthracene (6) 2-nitrofluorene (7) 9-hydroxy-3-nitrofluorene (8) 1-amino-7-nitrofluorene (9) 2-nitro-9-fluorenone (10) 1-nitropyrene
maxima, nm excitation emission 288 (285) 280 (290) 287 (290) 323 (305) 262 (260) 265 (265) 302 (300) 269 (275) 262 (265) 275 (275)
442 (435) 415 (405) 440 (445) 477 (425) 494 (485) 372 (375) 390 (390) 389 (390) 370 (375) 430 (435)
relative intensity 1.00 (1.0) 0.52 (1.0) 6.15 (25) 0.05 (0.4) 9.87 (121) 33.67 (13) 0.01 (0.08) 2.52 (0.96) 0.006 (0.12) 672 (200)
a The values in parentheses were determined “on the fly” in a preliminary experiment; see text for details.
add even more selectivity, as pointed out in ref 18), only instruments with presettable fixed wavelengths were available for this work. It was necessary, therefore, to determine the optimum wavelengths for monitoring individual NPAH compounds. A preliminary study was carried out by pumping a He-degassed 100 µM solution of the NPAH compound in the mobile phase, holding the EC detector at -2 V and manually locating the excitation and emission maxima on the flow-through fluorometer. Based on these results, NPAH solutions, ranging from 0.1 to 100 µM in concentration, were prepared in the mobile phase, He-degassed, and put through the EC detector in the same manner as above, except at a flow rate of 0.3 mL/min to achieve more complete reduction. The effluent was collected in the dark under a helium blanket and then transferred to a cuvette, and complete fluorescence spectra were recorded. The results are shown in Table 1,
Figure 3. Chromatogram of an NPAH test mixture. The peaks are due to (amounts injected in nanomoles are given in parentheses): 1, 1-amino-4-nitronaphthalene (10); 2, 9-hydroxy-3-nitrofluorene (10); 3, 1-nitronaphthalene (1); 4, 2-nitronaphthalene (2); 5, 2-nitronaphthol (0.05); 6, 9-nitroanthracene (0.05); 7, 2-nitrofluorene (0.05); 8, 2-nitro-9-fluorenone (2); 9, 1-nitropyrene (0.01); and 10, 1-amino-7-nitrofluorene (0.2). Peaks 7′ and 8′ are due to unknown impurities that are present in compounds 7 and 8. The traces show the electrochemical detector signal and the fluorescence detector signals under three different excitation/emission conditions. In each case, the chromatofluorograms are accompanied by a trace immediately below it which shows the fluorescence detector signal with the electrochemical detector deliberately turned off. See text for details.
where the preliminary values are shown in parentheses. While the relative order of the product fluorescence intensities is the same in both the experiments, the exact values differ, especially for poorly fluorescent products, e.g., that resulting from 2-nitro9-fluorenone, because high concentrations, as used in the first set of experiments, allow impurities to play a more important role. In a few cases, the optimum excitation and emission wavelengths between the two sets of experiments differ markedly. This occurs because of impurities, optical filtering by excess unreacted product, and inner filter effects from the relatively large concentrations of the fluorophore generated during the reduction step. In most others, the maxima are well within the bandwidths of the monochromators in the two instruments. The data show that the relative fluorescence intensities vary by more than 4 orders of magnitudesthe method is likely to be particularly attractive for NPAHs like nitroanthracene, nitrofluorene, or nitropyrene and not for an NPAH that already contains an amino group. The occurrence of the latter class of compounds in ambient aerosol or diesel exhaust has actually never been reported, while simple nitro-substituted PAHs have been repeatedly described.10,18 Regarding the choice of specific wavelengths, all of the compounds tested in this study will be adequately detected with broad band excitation at 260-300 nm and detection of all the emitted light at wavelengths above 350 nm. Subsequent to the completion of the bulk of this study, a filter fluorometer was tested for the purpose and found to be satisfactory. However, all of our other experiments were conducted with the monochromator-equipped detector, and these are the data reported. Selection of Chromatographic Conditions. It is well known that the NO2 group substituted on an aromatic ring is reduced to the NH2 group through the intermediate formation of the NHOH group; unless the pH is sufficiently low, complete reduction to
the NH2 group does not occur.15,22,23 Dilute aqueous acetic, monochloroacetic, phosphoric and sulfuric acids, in conjunction with acetonitrile or methanol as the organic modifier, were studied with respect to maximizing fluorescence and electrochemical signals while keeping the background current of the EC detector at a manageable level. The EC signal increased with increasing acidity. Sulfuric acid appeared to be the best choice for acidifying the eluent; 1.0 mM was the highest practical concentration that could be used (beyond this, the background current was too high: 50 µA at a cathodic potential of 2 V). An overall concentration of the order of 0.1-0.2 mM in a mixed aqueous-organic eluent appeared to be optimum in terms of S/N. Greater EC and fluorescence signals were observed with methanol as the organic modifier relative to acetonitrile. Under isocratic elution conditions, at least 50% of the organic modifier was needed to accomplish chromatographic elution. At any given applied potential on the coulometric detector, and with the aqueous phase component of the eluent being 1.0 mM H2SO4, the highest fluorescence was observed at 85:15 methanol-sulfuric acid. Since decent chromatographic separations for most of the analytes of interest could be attained, this was selected as the eluent of choice. The apparent pH of this eluent was 2.8. Initially, an acid-resistant poly(styrenedivinylbenzene)-based reversed-phase column (PRP-1, 4.1 mm × 250 mm, Hamilton Co.) was chosen for separations. However, even with 99% methanol, 1-NN, which elutes in an intermediate place among the analytes we investigated, took more than 40 min to elute. A silica-based reversed-phase column was thence used for separations; this column allowed the elution of 1-NN in ∼11 min. (22) Pearson, J. Trans. Faraday Soc. 1948, 683-692. (23) Southwick, L. M.; Willis, G. H.; Dasgupta, P. K.; Keszthelyi, C. P. Anal. Chim. Acta 1976, 82, 29-35.
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Figure 4. Calibration behavior of selected NPAH compounds under indicated chromatographic and detection conditions. Left panel, ECD response; central Panel, fluorometric response (see text for individual ex/em settings); right panel, signal ratio.
Choice of Coulometric Detection Conditions: Extent of Conversion. Hydrodynamic voltammetric data and fluorescence signals were collected to determine the effect of the applied potential on system performance with 100 µM analyte dissolved in the methanol-H2SO4 eluent continuously flowing through the system. The data are shown for four compounds of interest in Figure 2. It will be observed that for none of the compounds is the onset of significant reduction seen until a cathodic potential of 0.5-0.7 V is applied. The current then increases monotonically (until the experimental limit of ∼2.5 V). Voltages above 2 V are impractical due to the large contribution of the background current and the consequent deterioration of S/N. A plateau for the fluorescence signal is not observed for 2-NF, even at the maximum voltage applied, while the fluorescence signal does appear to reach a plateau level by 1.6-1.9 V for the others. While the NO2 group may be reduced in a two-step fashion as noted earlier, the reduction potentials of the individual steps appear to be too close under our operating conditions to be resolved in the voltammetric data. However, the fluorescence properties of the NHOH- and NH2-substituted PAHs might be significantly different, and careful observation of the fluorescence signals in Figure 2 will indicate that a two-step development of the fluorescence signal is observed for all but 2-NF and is most easily visible for the 1-NN case. It may be observed that the EC signals are all of the same order of magnitude for the different analytes. The extent of reductive conversion achieved appears to be small. In chromatographic experiments (vide infra), 1 nmol of injected 1-NN produces an EC peak area of ∼25 µC, only ∼5% of the charge that would be expected if six electrons are consumed per mole. This is also consistent with the magnitude of the fluorescence signal (EC detector on or off) observed when 1 mol of authentic 1-aminonaphthalene (1-AN) is injected in the system; this is ∼20 times higher than the signal produced by the same amount of injected 1-NN. Based on the fluorescence spectra, we have confirmed that the fluorescing species formed in the reduction of 1-NN is 1-AN, nevertheless. The reduction appears to be limited due to kinetic reasons; if the flow rate is reduced, the EC and fluorescence 1230
Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
Figure 5. Chromatofluorogram (Ex 260 nm, Em 485 nm) of a diesel engine exhaust under no load condition (a) with the ECD off, (b) with ECD on, and (c) the difference signal (b - a).
signals increase in a proportionate fashion. The fact that the fluorescence signal for 1-NN reaches a plateau in the manner observed in Figure 2 leads us to believe that the limitation is in the first reduction step to NHOH. Attempts were made to improve the extent of conversion by incorporating a metal ion redox mediator, most notably Fe2+, in the eluent; these were not successful. Undoubtedly, the investigation of a different electrode material is warranted; however, flow noise was the limiting factor in the electrochemical detector in the present study, and this aspect was therefore not pursued. pH Dependence of Fluorescent Product. An aspect of the fluorescence signal from the reduced product needs to be pointed out. The fluorescence of compounds such as 1-AN is pHdependent; the fully protonated form is less fluorescent. The fluorescence of 1-AN is about twice as large at pH levels >4
Figure 6. Difference chromatofluorogram (Ex 260 nm, Em 485 nm) of a sample similar to that in Figure 5, except obtained under 50% load conditions presented in logarithmic (solid trace, left ordinate) and linear (dashed trace, right ordinate) ordinate scaling.
relative to that at the operating eluent pH of 2.8.24 Obviously, the fluorescence signal will be improved if the pH is increased by incorporating a membrane reactor25 to raise the pH after the electrochemical detector to introduce, e.g., NH3. This approach was not attempted because the gain would be more limited for amino-PAHs with a larger ring system, where the lone-pair electrons on the N atom can be more effectively delocalized, making them weaker bases than 1-AN (for comparative purposes, the pKa values of anilinium and the 1-naphthylammonium ion are 4.63 and 3.92, respectively26 ). Chromatographic Performance: Calibration, Limits of Detection, and Reproducibility. Figure 3 shows the chromatogram of a mixture of 10 test NPAH species. The various NPAH compounds are identified in the legend. The first response observed in the electrochemical detector is a combination of an anodic and cathodic signal (not numbered) and occurs due to the difference in oxygen content and the matrix composition of the sample and the deoxygenated eluent. Peaks 7′ and 8′ are due to impurities present in compounds 7 and 8, which are apparently highly electroactive compounds. Note that neither produces a fluorescence signal uniquely associated with the ECD being turned on. At the injected concentrations, peaks due to compounds 5-9 are not easily discernible in the ECD signal due to the flow noise; they are, however, readily visible in the indicated regions when injected at higher concentrations. As the data in Table 1 may indicate, the three chromatofluorograms show that the individual compounds are best detected under individually tailored excitation and emission conditions. It is also interesting to note that peaks 7′ and 8′ are not due to any NPAH compound, because 7′ does not produce a fluorescence signal whether the ECD is turned on or off and peak 8′ shows a small but discernible fluorescence response (Ex 260 nm/Em 485 nm) that is due to (24) Dasgupta, P. K. Anal. Chem. 1981, 53, 2084-2087. (25) Dasgupta, P. K. In Ion Chromatography; Tarter, J. G., Ed.; Marcel Dekker: New York, 1987; pp 191-367. (26) Weast, R. C., Astle, M. J., Eds. Handbook of Chemistry and Physics, 62nd ed.; CRC Press: Boca Raton, FL, 1981; pp D139-140.
the parent compound (it persists even when the ECD is turned off). Figure 4 shows calibration data under chromatographic conditions for both the ECD and the fluorescence detector over several orders of magnitude of injected sample concentration and the corresponding signal ratio, which is relatively invariant and provides a further means of confirming of the identity of a peak beyond its retention characteristics. The fluorescence data presented were collected under the following excitation/emission conditions: 285/440 nm (1-NN, 2-NN, 2-NNOH, and 1-NP), 270/ 380 nm (1-A-7-NF, 2-NF, and 2-N-9-F), and 260/485 nm (9-NA). The noise in the difference signal (fluorescence signal with ECD on minus the fluorescence signal with the ECD off, vide infra) provides a realistic basis for determining the fluorometric detection limits, which under the chosen excitation/emission conditions listed above were determined to be (S/N)3) 0.75 (1-NP), 1.1 (2NF), 1.4 (2-NNOH), 1.3 (9-NA), and 4.5 (1-A-7-NF) pmol, respectively, on the basis of actual injections made near the LOD. The reproducibilities of chromatographic peak heights were also acceptable, e.g., 1.27% for 1-NN at 10 nmol injected, 3.5% for 9-NA and 5.3% for 2-NF at 300 pmol injected, and 1.6% for 1-NP at 30 pmol injected (n ) 8 in all cases). The flow noise in the present setup does not allow a fair assessment on the electrochemical detection limits and was not pursued. Application to Diesel Engine Exhaust Samples. Because of the presence of compounds that are already fluorescent prior to reduction (e.g., PAH compounds that are not necessarily nitrosubstituted), sometimes the chromatofluorograms can be quite complex. A relatively facile means to survey the presence of NPAH compounds is to generate the difference chromatofluorogram between two successive runs with the ECD turned on and off. A significant difference signal indicates the presence of an NPAH compound (as this implies, a corresponding ECD signal of varying magnitude is always observed and need not generally be considered, except for the purposes of ratio computation). The generation of the difference signal from two separate runs is Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
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Figure 7. Difference chromatograms of the sample in Figure 5, subsequently exposed to NO2 in sunlight, under three different excitation/ emission conditions. Possible NPAH responses are marked a-g. See text for details.
illustrated in Figure 5 for a diesel engine exhaust sample obtained under no load conditions (despite the flow noise, the experimental system exhibited sufficient flow stability and thence run-to-run retention reproducibility to permit such subtraction). It should be possible to directly generate such a difference signal by diverting half the flow through a dummy resistance, simulating the resistance and residence time of the active ECD cell, and thence through a second fluorometer acting as a reference channel. This was not experimentally investigated. Figure 5 clearly shows the presence of a significant amount of at least two (and possibly more) NPAH compounds. While unambiguous identification is not possible without mass spectrometry, the major peak (occurring at ∼14 min) elutes at the retention time corresponding to that of 9-NA, and spiking experiments as well as the ECD/fluorescence signal ratio indicate that this peak is due to 9-NA. Figure 6 shows the likely presence of several NPAH species in a diesel exhaust sample obtained under 50% engine load conditions and then exposed to sunlight in ambient air prior to extraction. The difference signal (Ex 260 nm, Em 485 nm) is presented in both logarithmic (left) and linear (right) ordinate scaling; the logarithmic scaling increases the ability to visually
1232 Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
identify small peaks. Figure 7 shows difference chromatograms of a diesel exhaust sample obtained under no load conditions and then exposed to sunlight in air spiked with NO2. 9-NA (peak b) remains the dominant peak, but a significant number of other peaks are discernible in more than one chromatofluorogram. Obviously, a multichannel fluorescence detector (or a spectrally imaging fluorometer) would provide even a greater wealth of information. Peak a occurs at the retention time of 2-NN, but the S/N is too poor to permit ratio calculation or other approaches to identification. The other peaks occur at retention times significantly different from the limited number of NPAH test species we investigated. In summary, we have presented here a powerful screening method for NPAH compounds that are of considerable environmental concern. This is a sensitive method with a unique selectivity that can be practiced with a modest capital investment. Received for review September 18, 1995. November 21, 1995.X AC950933R X
Abstract published in Advance ACS Abstracts, March 1, 1996.
Accepted
