Environ. Sci. Technol. 2002, 36, 4424-4429
Laser-Induced Multidimensional Fluorescence Spectroscopy in Shpol’skii Matrixes for the Analysis of Polycyclic Aromatic Hydrocarbons in HPLC Fractions and Complex Environmental Extracts ADAM J. BYSTOL,† TIM THORSTENSON,‡ AND A N D R E S D . C A M P I G L I A * ,† Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105-5516, and Bismarck State College, Bismarck, North Dakota 58506-5587
Fluorescence measurements are easily made with the aid of a cryogenic fiber optic probe. Emission wavelength time matrixes, excitation emission matrixes, and timeresolved excitation emission matrixes are rapidly collected with a pulsed tunable dye laser, a spectrograph, and an intensified charged-coupled device. Compound identification is based on spectral and lifetime information. The potential of this approach for supporting high-performance liquid chromatography analysis and for the direct determination of benzo[a]pyrene without previous separation is demonstrated.
Introduction Polycylic aromatic hydrocarbons (PAH) represent a diverse class of environmentally important chemicals originated from a wide variety of natural and anthropogenic sources. Because of the carcinogenic nature of some PAH, their environmental monitoring is recommended. Among the hundreds of PAH present in the environment, the U.S. Environmental Protection Agency (EPA) lists only 16 as “consent decree” priority pollutants. Because one of the primary sources of human exposure to PAH is the contact with potable water, the U.S. EPA recommends routine monitoring of municipality wells to ensure concentration levels below the EPA drinking water criteria (1). If coal liquids contaminated a groundwater or surface water, the polluted sample would probably exhibit high compositional complexity. The environmental chemist then faces the challenge to determine the EPA PAH concentrations in highly complex samples. Current EPA methodology for PAH analysis in water samples is based on solid-liquid extraction (SLE) and highperformance liquid chromatography (HPLC) (1). SLE preconcentrates PAH, simplifies matrix composition, and facilitates analytical resolution in the chromatographic column. HPLC is mostly used in concert with single-wavelength absorption and fluorescence detectors. The major drawback of this approach is the use of nonspecific detection schemes. Because PAH identification is solely based on retention times, * Corresponding author phone: (701)231-8702; fax: (701)231-8831; e-mail:
[email protected]. † North Dakota State University. ‡ Bismarck State College. 4424
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unambiguous compound identification requires complete chromatographic resolution of sample components. For the analysis of complex samples, the U.S. EPA recommends using a supporting analytical technique. The usual recommendation is gas chromatography-mass spectrometry (GC-MS), which often provides accurate compound identification and peak purity check of HPLC fractions. However, GC-MS requires additional chromatographic separation. Elution times of 30-60 min are typical, and standards must be run periodically to verify retention times. If the concentrations of target species are found to lie outside the detector’s response range, the sample must be diluted and the process repeated. These are important considerations when routine analysis is under the scope. Several strategies exist to improve the specificity of HPLC analysis. Because most PAH exhibit vibronic structure in their room temperature absorption, fluorescence, and excitation spectra, some strategies include collecting on-the-fly multidimensional spectra (2-7) and fluorescence lifetime analysis (8-11). To a certain extent these strategies improve specificity, but their application to highly complex samples is limited by the broad nature of room temperature spectra. On the other end, high-resolution luminescence techniques have shown the specificity for determining PAH in HPLC fractions (12-14) and complex environmental extracts without chromatographic separation (12, 15). Reducing the sample temperature often improves vibronic resolution of PAH fluorescence and excitation spectra. However, the widespread use high-resolution techniques for routine analysis of environmental samples has been hampered by several reasons. These include inconvenient sample freezing procedures, questions about signal reproducibility for calibration purposes, and relatively long freezing times for sample analysis below 77 K. In recent papers. we presented improved methodology for laser-excited time-resolved Shpol’skii spectroscopy (LETRSS) of PAH at liquid nitrogen (77 K) and helium (4.2 K) temperatures (16, 17). The well-known disadvantages of conventional low-temperature measurements were eliminated using cryogenic fiber optic probes with the distal end frozen directly into the sample matrix. Upon sample excitation with a frequency-doubled tunable dye laser, fluorescence spectra, wavelength time matrixes (WTM), and fluorescence lifetimes were collected with a multichannel detection system consisting of a spectrograph and an intensified chargedcoupled device (ICCD). The simplicity of the experimental procedure, the short analysis, and the excellent analytical figures of merit were demonstrated for several PAH. In a later paper, SLE was coupled to 77 K LETRSS, and 15 EPA PAH were determined in a spiked Red River sample without previous separation (18). The analysis of more complex samples was not attempted. For the analysis of heavily contaminated samples, we are proposing LETRSS at liquid helium temperature. In comparison to 77 K, lowering the temperature to 4.2 K promotes significant spectral narrowing and enhances the specificity of WTM, excitation emission matrixes (EEM), and time-resolved EEM (TREEM) analysis. Because 4.2 K measurements are still made with a fiber optic probe, the selectivity enhancement has no cost in analysis time. The feasibility of this approach is demonstrated for the analysis of heavily contaminated water. Its analytical potential is evaluated for PAH determination in HPLC fractions and as a stand-alone technique for the direct determination of target compounds without previous separation. 10.1021/es020691u CCC: $22.00
2002 American Chemical Society Published on Web 09/19/2002
Experimental Section Sample Collection. The output water stream of the American Petroleum Institute (API) separator at a North Dakota refinery was selected as the sample source for these experiments. Water at this point has been largely stripped of visible petroleum but has received no further treatment to remove or destroy dissolved components, which ensures the collection of samples with highly complex PAH composition. The samples were collected from a sampling valve on the output pipe of the API separator. Prior to sample collection, the valve was allowed to thoroughly flush to ensure a sample consistent with the throughput water. Samples were then collected in 1-L glass bottles with PTFE-lined caps and stored at 4 °C. PAH extraction and HPLC analysis were completed within 72 h of sample collection. SLE. Samples from the API separator contain a significant quantity of suspended solids. For the sample under consideration, the total residue analysis per EPA Method 160.3 revealed a total residue of 2520 mg/L. Because separate prefiltration and solid removal treatment indicated PAH loss by adsorption on suspended particles, subsequent extraction for HPLC analysis was performed in the presence of suspended solids. SLE and concentration of the extract solvent were both performed per EPA Method 550.1. SPE was carried out processing 0.5 L of water sample through 47-mm C-18 SPE disks (Ansys Diagnostics) with a Supelco ENVI-Disk filtration apparatus. The compounds were eluted from the disk with a small quantity of methylene chloride, dried, and concentrated further to 1 mL. A 3.0-mL portion of acetronitrile was added to the extract and concentrated to a final volume of 0.5 mL. HPLC Analysis. It was performed using a Beckman HPLC system with model 110B gradient pumps, a model 166 UV detector, and a model 406 control interface. HPLC operation was controlled with Beckman System Gold software. A Supelco Supelcosil LC-PAH column (25 cm length, 4.6 mm diameter, and 5 µm particle size, catalog no. 58229) was selected for optimum PAH resolution. Flow conditions were as stipulated by Supelco; i.e., 1.5 mL/min flow rate, isocratic elution with water/acetonitrile (60:40 v/v) for 5 min, and then linear gradient to 100% acetonitrile over 20 min. Detector response was monitored for 20 min following the latest eluting component to ensure the absence of any additional components. The column was allowed to re-equilibrate for a minimum of 15 min at initial conditions between all runs. All sample and standard injections were at a volume of 20 µL using a fixed-volume injection loop. HPLC fractions were collected using 25-mL test tubes. All solvents were Aldrich HPLC grade. An AccuStandard PAH mixture (Product M-610A) was used as a reference standard for the 16 EPA PAH. For compound identification, a working standard was prepared by diluting 1.00 mL of the AccuStandard mix to a final volume of 10.00 mL in acetonitrile. Laboratory reagent blanks were run in conjunction with each series of samples using identical conditions of glassware, equipment, solvents, and analysis to ensure absence of interfering contamination. 4.2 K LETRSS Analysis. The fiber optic probe and the instrumental setup for LETRSS analysis has been described in detail elsewhere (16, 17). Standard PAH solutions were prepared in the appropriate Shpol’skii solvent. Acenaphthylene, acenaphthene, anthracene, benzo[ghi]perylene, dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene, naphthalene, phenanthrene, fluorene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, pyrene, fluoranthene, and benzo[a]pyrene were purchased from Aldrich at their highest purity available. HPLC grade n-pentane, n-hexane, n-heptane, and n-octane were acquired from EM Science.
Note: Use extreme cautions when handling PAH that are known to be extremely toxic. 4.2 K LETRSS Analysis of HPLC Fractions. It was performed as follows: for each fraction tested, 300 µL of the mobile phase was pipetted into the sample vial followed by 200 µL of n-alkane solvent. The sample vial was vigorously shaken for ∼2 min and then allowed to stand for ∼1 min to separate the two liquid layers. The sample vial was attached to the fiber optic probe, and the fiber assembly was adjusted vertically so the tip was ∼1 cm below the surface of the n-alkane layer. Sample freezing was accomplished by lowering the copper tubing into the liquid helium held in a Dewar flask with a 60-L storage capacity. The 60 L of liquid helium would typically last for 3 weeks of daily use, averaging 15-20 samples per day. Complete sample freezing took less than 90 s. The approximately 1-min probe cleanup procedure involved removing the sample vial from the cryogen container, melting the frozen matrix, warming the resulting solution to approximately room temperature with a heat gun, rinsing the probe with n-alkane, and drying it with warm air from the heat gun. The entire freeze, thaw, and cleanup cycle took no longer than 5 min. Direct Analysis of SLE Extracts. It was carried out as follows: 10 µL of the extract was pipetted into the sample vial followed by 200 µL of n-alkane solvent. The sample vial was manually shaken for ∼3 min and then allowed to stand for 1 min. The freezing procedure was then performed as previously described. Lifetime Analysis. The 4.2 K fluorescence lifetimes were determined via a three-step procedure: (i) full sample and background WTM were collected; (ii) the background decay curve was subtracted from the fluorescence decay curve at the wavelength of maximum fluorescence for each PAH; (iii) the background-corrected data were fit to single exponentials. In cases where the exact sample composition was unknown and the formulation of a correct blank for lifetime background subtraction was impossible, the fluorescence decay at the base of the target peak was used for background subtraction at the target wavelength. The accuracy of this procedure was investigated previously (18). Origin software (version 5, Microcal Software, Inc.) was used for curve fitting of fluorescence lifetimes. The decay curve data were collected with a minimum 10-ns interval between the opening of the ICCD gate and the rising edge of the laser pulse, which is sufficient to avoid the need to consider convolution of the laser pulse with the analyte signal. Fitted decay curves (y ) y0 + A1 exp-(x-x0)t1) were obtained by fixing y0 and x0 at a value of zero.
Results and Discussion HPLC Analysis. Figure 1 shows a chromatogram of a typical API sample. Table 1 relates the retention times of the 16 EPA PAH under the same chromatographic conditions as Figure 1. The comparison reveals at least 40 definitive detector responses in the chromatogram within the retention time region bracketed by the 16 EPA PAH. In an effort to confirm the identity of at least some EPA PAH, a spiked sample was prepared and analyzed by HPLC. A quantity of the concentrated SLE extract was mixed with an equal volume of the working standard. The expectation here was that those sample peaks corresponding to an EPA PAH would show an increase in relative height (absorbance). On the other end, new peaks would be observed in cases where an EPA PAH was not present in the initial sample to a significant extent. The attempt provided the following information: (i) the height of the peak at 16.62 min increased in the spiked sample (in the naphthalene “window”); (ii) the earliest eluting of the three components that appear in the 21-min region (21.19 min) increased significantly in relative detector response in the spiked sample (in the fluorene response region); (iii) the VOL. 36, NO. 20, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Chromatogram of API water extract using Supelcosil LC-PAH column. Identified components are (I) naphthalene, (II) fluorene, and (III) phenanthrene. Dashed vertical lines indicate HPLC fractions. Fractions further analyzed by LETRSS are 4-8 and 10.
TABLE 1. Average HPLC Retention Timesa for the 16 EPA PAH Using Supelco Column Conditionsb PAH
retention time (min)
naphthalene acenaphthylene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
16.38 ( 0.19 18.18 ( 0.15 20.30 ( 0.09 20.98 ( 0.18 22.62 ( 0.10 24.32 ( 0.03 25.87 ( 0.12 27.10 ( 0.17 31.00 ( 0.29 32.18 ( 0.47 34.84 ( 0.36 36.61 ( 0.44 38.29 ( 0.59 41.10 ( 0.85 43.06 ( 0.86 45.26 ( 1.00
a Standard deviation from three standard runs bracketing the corresponding sample analysis. b Column conditions include a 1.5 mL/ min flow rate, isocratic elution with 60:40 water/acetonitrile for 5 min and then linear gradient to 100% acetonitrile over 20 min.
peak at 22.74 min, which is part of an incompletely resolved pair of components, increased in relative response in the spiked sample (in the elution time frame for phenanthrene). Because no additional peaks appeared in the spiked sample within the time windows of naphthalene, fluorene, and phenanthrene and assuming that no other PAH coeluted, the results from the spiked sample identify the presence of these three compounds. Continuing on to the later portion of the chromatogram, it was not possible to make assignments and positive identifications with a certain degree of confidence due to the overwhelming number of components found in the chromatogram. The inability to monitor EPA PAH in complex samples demonstrates the need for supporting techniques for HPLC. Our studies will demonstrate the simplicity of interfacing our approach with liquid chromatography. 4426
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LETRSS Parameters for Identifying EPA PAH in HPLC Fractions. Six HPLC fractions per water sample were collected for subsequent LETRSS analysis. Table 2 groups the EPA PAH expected to appear in each chromatographic fraction according to their retention times. Following the criteria that matches the length of the solvent molecule to the effective length of the PAH (12), the best spectral resolution for each EPA PAH is obtained with one of five organic solvents. These include n-pentane (naphthalene, acenaphthene, and acenaphthylene), n-hexane (phenanthrene and pyrene), n-heptane (fluorene, fluoranthene, benzo[ghi]perylene, benzo[b]fluoranthene, and anthracene), n-octane (benzo[a]pyrene, dibenz[a,h]anthracene, chrysene, benz[a]anthracene), and n-nonane (indeno[1,2,3-cd]pyrene and benzo[k]fluoranthene). Cross examination with Table 2 shows that, with the exception of fraction 4 (chrysene and benz[a]anthracene), the remaining fractions grouped PAH with sufficient disparity in their molecular dimensions to dictate the use of at least two solvents for best spectral resolution. Seeking for convenience and simplicity of analysis, we arbitrarily chose one of the optimum solvents per fraction. The selected solvents are related in Table 2 along with the excitation and emission wavelengths that should be used for identifying each compound in the HPLC fraction. The target wavelengths for each compound were selected from a synthetic mixture containing all the PAH expected to appear in that particular fraction and provide wavelengths with no spectral interference. The only PAH that showed no fluorescence was acenaphthylene. Its behavior was not surprising because lack of fluorescence had also been observed at 77 K. At present, we have no explanation for the observed phenomenon, which persisted in several solvents. Purging its standard solutions with nitrogen gas for 15 min showed no effect on fluorescence enhancement. In addition to highly resolved spectra, our approach provides easy access to fluorescence lifetimes of target PAH in HPLC fractions. Fluorescence decays are obtained from the WTM of the mixture, which consist of a series of emission spectra assembled in a three-dimensional plot. The WTM is easily built up because our instrument automatically increments the time interval between successive fluorescence
TABLE 2. 4.2 K Fluorescence Wavelengths and Lifetimes for Identifying EPA PAH in HPLC Fractions
a Wavelength of excitation. b Wavelength of emission used for identification. c Fluorescence lifetime based upon three replicate measurements of standard solutions extracted into appropriate Shpol’skii solvent. d No fluorescence was observed for this PAH-solvent combination.
TABLE 3. 4.2 K LETRSS Limits of Detection for EPA PAH PAH
solvent
λexc/λema
LOD (ng/mL)b
naphthalene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
n-pentane n-pentane n-hexane n-hexane n-hexane n-hexane n-hexane n-octane n-octane n-octane n-octane n-octane n-octane n-octane n-octane
291.5/319.8 289.0/320.8 289.0/302.2 287.1/347.2 362.2/377.4 360.4/408.9 328.0/370.2 362.2/385.2 326.2/359.1 370.4/396.8 380.4/402.3 290.0/402.9 302.5/393.0 290.0/419.5 302.5/461.2
5.8 4.1 7.3 7.2 17.2 10.1 0.5 7.4 0.5 2.9 2.3 0.1 7.4 1.1 4.0
a Wavelengths of excitation (λ exc) and emission (λem). detection is based on a signal-to-noise ratio of 3.
b
Limit of
scans. As shown in Table 2, single exponential decays were observed for all the studied PAH. The residuals between the calculated and the observed points were less than 1% within the first two lifetimes of the decays and showed no systematic errors. The lifetime values from the mixtures matched those from individual standard solutions, demonstrating that fluorescence lifetimes can be used to monitor spectral purity. 4.2 K Limits of Detection for EPA PAH. Table 3 lists the limits of detection (LOD) for the 15 EPA PAH under the experimental conditions for HPLC fraction analysis. The LOD calculations were based on signal-to-noise ratios of 3. The noise was measured at the base of the target peak. The spectra were recorded from mixtures containing all the PAH expected to appear in the HPLC fraction. Previous to fluorescence measurements the standard mixtures were shaken for 3 min with acetonitrile to account for potential mobile phase effects on the fluorescence intensity of PAH. The volumes of standard mixtures and acetonitrile were 300 and 200 µL, respectively.
In all cases, the LOD were at the low parts per billion level. Although it was not attempted, better LOD could have been obtained by optimizing the volume of Shpol’kii solvent used to extract the PAH from the HPLC fraction. The fiber optic probe allows one to use as low as 50 µL of organic solvent. In addition, it is important to keep in mind that the LOD in Table 3 do not represent the minimum detectable concentrations in the water sample. The reported values only correspond to the LOD dictated by the blank and the instrumental noise. If one were to consider the preconcentration step achieved with SLE, the LOD in the water sample would be at the parts per trillion level. 4.2 K LETRSS Analysis of HPLC Fractions. Table 4 summaries the LETRSS results obtained from the API sample that provided the chromatogram in Figure 1. Nine priority pollutants were found, including the three PAH identified by HPLC. Spectral purity at the target wavelengths was checked with lifetime analysis. In all cases, single exponential decays were observed, indicating no spectral interference at the target wavelengths. For a confidence interval of 95% (R ) 0.05) and six determinations (N ) 6) (19), the standard lifetimes in Table 2 were obtained for all the PAH but two. The exceptions were acenaphthene and pyrene. Considering the possibility of synergistic effects among the fraction components, the volume of extracting solvent was increased from 200 µL to 300 µL as a tentative means of reducing PAH concentration in the analytical sample and, therefore, decreasing energy transfer processes. For R ) 0.05 and N ) 6, the lifetimes after sample dilution matched the lifetimes of acenaphthene and pyrene in Table 2. Quantitative analysis was performed with the standard addition method. The standard addition (5 µL of standard in acetonitrile) was made to the HPLC fraction (300 µL) prior to PAH extraction with 200 µL of Shpol’skii solvent. The standard composition included all the PAH expected to appear in the fraction. The PAH concentrations were calculated based on the linear regressions of the calibration curves generated with six standard additions. The signals were measured using optimum delay and gate times. The optimum delay (10 ns) accounted for the time-resolution of VOL. 36, NO. 20, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 4. PAH Concentrations in HPLC Fractions PAH
λexca (nm)
λemb (nm)
tgc (ns)
τHPLCd (ns)
Cse (µg/L)
naphthalene acenaphthene fluorene pyrene chrysene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene
291.5 289.0 289.0 328.0 326.2 380.4 290.0 302.5 290.0
319.8 320.8 302.2 370.2 359.1 402.3 402.9 393.0 419.5
2000 200 50 2000 200 50 200 200 2000
197.8 ( 2.1 74.3 ( 1.8 (49.1 ( 0.5) 6.4 ( 0.1 420.0 ( 5.6 (498.8 ( 9.3) 54.7 ( 1.4 9.2 ( 0.5 36.8 ( 1.7 44.7 ( 0.9 118.5 ( 1.1
2.835 ( 0.227 3.811 ( 0.373 40.217 ( 3.56 0.032 ( 0.003 0.880 ( 0.075 0.040 ( 0.003 7.249 ( 0.717 8.495 ( 0.787 60.275 ( 4.967
a Wavelength of excitation. b Wavelength of emission. c Gate time. d Observed lifetime upon extraction with 200 µL of n-alkane. Values in parentheses indicate lifetime observed upon extraction with 300 µL of n-alkane. e Concentration in the water sample.
FIGURE 2. Spectra of 10 µL of HPLC fraction 6 extracted into 300 µL of n-octane at 4.2 K. Delay and gate times were 10 and 2000 ns, respectively. Excitation wavelength was 302.5 nm. (Panel A) HPLC fraction. (Panel B) HPLC fraction spiked with 5 µL each of 1.3 µg/mL DBA, 4.0 µg/mL I[123cd]P, 3.6 µg/mL B[ghi]P, and 0.35 µg/mL B[a]P. Peaks are labeled as follows: DBA, dibenz[a,h]anthracene; B[a]P, benzo[a]pyrene; B[ghi]P, benzo[ghi]perylene; and I[123cd]P, indeno[123-cd]pyrene. background emission. At 10 ns, the fluorescence emission of the blank was equivalent to the instrumental noise. This delay was also sufficient to avoid the need to consider convolution of the laser pulse with the analyte signal. The optimum gate, which varied with the fluorescence lifetime of the PAH, collected most of the PAH fluorescence and still avoided instrumental noise. Figure 2 shows the spectrum of fraction 6 before (A) and after (B) the first standard addition. It demonstrates the low PAH concentrations that our approach can determine with excellent signal-to-noise ratios. Direct Analysis of Benzo[a]pyrene. The determination of benzo[a]pyrene in environmental samples is of outmost importance because it is the most toxic pollutant in the EPA priority list. The selectivity of our approach for the direct analysis of a specific PAH was then put to test using this 4428
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FIGURE 3. (Panel A) 4.2 K EEM of API water extract in n-octane. Delay and gate times were 20 and 200 ns, respectively. Contour lines represent an intensity range of 20 000-250 000 counts over 16 increments. Spectra were accumulated over 100 laser shots at each excitation wavelength using increments of 0.1 nm. (Panel B) 4.2 K EEM of API water extract spiked with 5 µL of 0.35 µg/mL benzo[a]pyrene using the same parameters as panel A. Intensity range is 20 000-100 0000 counts over 16 increments. pollutant as the target compound. A total of 10 µL of the SLE water extract was spiked into 300 µL of octane, and the mixture was vigorously shaken for 3 min prior to LETRSS analysis. The API extract was the same that generated the chromatogram in Figure 1.
Figure 3A shows the 4.2 K EEM from the octane layer. The EEM consists of 100 fluorescence spectra collected over a 10-nm excitation range in 0.1-nm excitation steps. The reason the analysis was done at an excitation range above the excitation wavelength (290 nm) recommended in Table 2 is because the EEM from the extract within the 285-295-nm range did not show the spectral resolution needed for benzo[a]pyrene’s direct determination. The presence of benzo[a]pyrene is clearly noticed around 403 nm emission wavelength. The maximum intensity is observed upon sample excitation at 387.4 nm. Fluorescence decay analysis at the target fluorescence peak (402.9 nm) yielded a singleexponential decay with fluorescence lifetime equivalent to that in Table 2. Figure 2B shows the EEM recorded from the water extract spiked with a standard solution of the target compound in methylene chloride: acetonitrile (25:75 v/v), i.e., the solvent composition of the extract. A clear enhancement of benzo[a]pyrene’s fluorescence intensity is observed upon standard addition. On the basis of three standard additions, the calculated concentration (7.0 ( 0.6 µg.mL-1) of benzo[a]pyrene in the water sample was statistically equivalent (R ) 0.05, N1 ) 6, N2 ) 3) to that in Table 4. Advantages of 4.2 K LETRSS. We have presented a unique tool to investigate the composition of HPLC fractions of heavily contaminated water samples. The fiber optic probe provides the analyst with a rapid and simple procedure to obtain accurate and reproducible data at 4.2 K. The probe and the combination of the pulsed tunable dye laser with the spectrograph and the ICCD allows one to collect highly resolved WTM, EEM, and TREEM for the analysis of complex PAH mixtures. Because the spectral resolution is not deteriorated by the volume of mobile phase partitioning into the Shpol’skii solvent, many coeluted PAH can be identified in an HPLC fraction. For the problem at hand, PAH identification was made using only three parameters, namely, one excitation and emission wavelength and fluorescence lifetime. However, the highly resolved vibronic spectra of PAH at 4.2 K certainly provide numerous excitation and fluorescence wavelengths for selective PAH identification. This could be an advantage in fractions with many coeluted PAH and strong spectral overlapping. In comparison to GC-MS, LETRSS presents the advantage of being easily interfaced with HPLC. Including the LETRSS measurement, the procedure takes less than 8 min per sample. For the direct analysis of PAH, LETRSS is still an open question. At present, our results show tremendous potential for quantitative PAH screening in complex environmental samples.
Acknowledgments This research was partially supported by EPA-EPSCoR. A.J.B. is grateful to the ND-EPSCoR Doctoral Dissertation Fellowship Program for his award.
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Received for review April 17, 2002. Revised manuscript received July 5, 2002. Accepted July 9, 2002. ES020691U
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