of' trans- [IrCl(CO)(PPh3)2]with molecular oxygen (Equation 1) ( 9 ) .
trans- [IrCl(CO)(PPh&] + 0
2
-
[1rCl(Oz)(CO)(PPh3)~1(1) The chromatographic analysis a t successive time intervals of a T H F solution of trans- [IrCl(CO)(PPh3)2]under an 0 2 purge (Figure 2) clearly demonstrated the occurrence of the reaction by the diminution of the trans- [IrCl(CO)(PPh&] peak and the simultaneous appearance and growth of the [IrCl(02)(CO)(PPh&] peak. Since the oxygen adduct has limited solubility in THF, it began to precipitate a t a concentration around M and its corresponding peak no longer increased. A further demonstration comes from our investigation into the photochemical properties of [IrH3(PPh&]. The published synthesis of this complex results in a mixture of meridional and facial isomers and separation of these isomers can easily be achieved using a Zorbax column and 95% n-hexane/5% T H F as the mobile phase (Figure 3). When this mixture is irradiated with 366 nm, infrared evidence suggests that the meridional isomer loses H2 with a much higher quantum yield
than does the facial isomer to yield [IrH(PPh&] or a derivative thereof (10).This is confirmed by HPLC analysis of the irradiated solutions (Figure 3) which shows that the peak assignable to the meridional trihydride decreases much faster with increased irradiation time than does the peak corresponding to the facial isomer.
LITERATURE CITED (1) L. R. Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography", John Wiley and Sons, New York, 1974. (2) J. F. K. Huber, J. C. Kaak, and H. Veening, Anal. Chem., 44, 1555 (1972). (3) W. J. Evans and M.F. Hawthorne, J. Chromatogr. Sci., 88, 187 (1974). (4) J. M.Greenwood, H. Veening, and B. R. Willeford, J. Organometal. Chem., 38, 345 (1972). (5) J. S. Fritz and L. Goodkin, Anal. Chem., 46, 959 (1974). (6) L. C. Hansen and R. E. Sievers, J. Chromatogr. Sci., 99, 123 (1974). (7) N. Ahmad, S.D. Robinson, and M. T. Uttley, J. Chem. SOC.,Dalton Trans., 843 (1972). (8) D.Evans, J. A. Osborn, and G. W. Wilkinson, lnorg. Synth., 11, 99 (1970). (9) L. Vaska, Acc. Chem. Res., 1, 335, (1968), and references cited therein. (IO) G.L. Geoffroy and R. Pierantozzi, unpublished observations.
RECEIVEDfor review December 11, 1975. Accepted March 1,1976. We thank the National Science Foundation (Grant MPS 75-05909) for support of this research.
Determination of Polycyclic Aromatic Hydrocarbons in Atmospheric Particulate Matter by High Pressure Liquid Chromatography Coupled with Fluorescence Techniques Marye Anne Fox and Stuart W. Staley" Department of Chemistry, University of Maryland, College Park, Md. 20742
Polycyclic aromatic hydrocarbons (PAH) present in atmospheric particulate matter have been analyzed by high pressure liquid chromatography (hplc) coupled with on-line fluorescence detection. Efficient separations and a significant increase in sensitivity over other methods were achieved. Hplc fractions were identified by comparing retention times and fluorescence spectra of lndlvidual components with data for authentic samples and also by obtaining mass spectra of these fractions. Derivative fluorescence spectroscopy and selective modulation of the fluorescence spectra were investigated as complementary techniques in difficult identifications. .The use of these techniques In the quantitative analysis of eleven PAH components of the organic material present in atmospheric particulate matter collected in the Baltimore, Md., Harbor Tunnel and in College Park, Md., is discussed.
The problem of the determination of polycyclic aromatic hydrocarbons (PAH) in atmospheric particulate matter and other environmentally significant mixtures is a 2-fold one. First, the compounds of interest must be adequately separated from related substances in the complex mixtures which are often encountered. Second, a high sensitivity of detection is required to analyze small samples collected over a relatively short period of time. Analyses of PAH mixtures have typically entailed various partitioning sequences followed by column, thin layer, or paper chromatography prior to detection by luminescence or absorption techniques (1-30). Such time-consum992
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
ing preliminary separations tend to reduce detectability and analytical reproducibility from the optimum inherent in fluorescence techniques and, furthermore, often permit the determination of only a few compounds in a sample. Microsublimation (31) and gas-liquid partition chromatography (32-35) have also been used as separation techniques prior to quantitative fluorescence analysis, but these methods are often limited by the low volatility of higher molecular weight PAH, by poor recovery ( 3 6 ) , and by the difficulty of completely separating all fractions of interest, e.g., benzo[e]pyrene (BeP) from benzo[a]pyrene (BaP) (37). High pressure liquid chromatography (hplc) is a particularly promising technique for the separation of high molecular weight PAH (16, 38-55). The advantages of hplc have been greatly enhanced by the recent development of bonded octadecylsilyl columns of micro particle size (56-58). For example, near baseline separation of the carcinogenic BaP and its noncarcinogenic isomer BeP is easily accomplished, as shown in Figure 1. With regard to the problem of detection, fluorescence spectroscopy has become well established as a sensitive and selective technique for PAH. Several groups have found it to be a t least an order of magnitude more sensitive than absorption spectroscopy (44, 59, 60). I t is also more sensitive (and less expensive) than mass spectral detectors, which normally have a nanogram detection limit (52, 53, 61, 62), although integrated ion current techniques which reduce this limit to the subpicogram range have been reported (63, 64). Since fluorescence spectroscopy is nondestructive, individual fractions can easily be collected and subjected to
21
8
+ .ln
-z
6
0
' 35
I
I
I
40
45
50
Time after injection ( m i d Figure 1. Hplc/fluorescence trace of a mixture of benzo[e]pyrene (1) and benzo[ a] pyrene (2) Conditions: 0.25 m X 8 mm i.d. Zorbax ODS column, 13:7(v/v) Me0H:HZO. 65 "C. 1400 psi (flow rate, 1.5 mllmin)
further analysis by other spectroscopic techniques. Furthermore, fluorescence detection gives the analyst the added benefits of selectivity. While many interfering molecules may absorb light, a much smaller fraction of these will fluoresce. By judicious choice of a filter system, selectivity or sensitivity can be emphasized. The use of the selectivity of fluorescence has been effectively employed by others ( 3 5 , 5 4 )in the analyses of PAH mixtures. Our interest in pinpointing the sources of atmospheric organic matter has forced us to develop techniques in which small samples could be collected over relatively short periods of time and be analyzed quantitatively. We describe here our techniques for the quantitative determination of PAH in atmospheric particulate samples which couple highly efficient separations by high pressure liquid chromatography on octadecylsilyl (ODS) stationary phases of micro particle ( 5 wm) size with on-line fluorescence detection. We are using this technique to determine the distribution of various PAH components on air-borne particulate matter and, hence, to approach the evaluation of the potential health hazard posed by carcinogenic PAH associated with respirable particulate matter (65).
EXPERIMENTAL Reagents. The methanol used in this study was Spectroquality (Matheson Coleman, and Bell) or certified ACS reagent grade (Fisher Scientific) purified by a double distillation from sulfanilic acid (66). The water was distilled, deionized water shown to be free of fluorescent impurities. The extraction solvent was certified ACS Reagent Spectranalyzed (Fisher Scientific) grade benzene. PAH standard samples were obtained from the following suppliers and were used without purification: fluoranthene, pyrene, and benz[a]anthracene from Eastman; chrysene and BeP from the National Cancer Institute Carcinogenesis Reference Compound Bank, Frederick Cancer Research Center, Frederick, Md.; BaP from Aldrich; and benzoperylene, 2,3,6,7-dibenzanthracene, 1,2,4,5- and 3,4,9,10-dibenzopyrene from K and K (Hydrocarbon Kit No. 2). (CAUTION: Since many of these compounds are recognized carcinogens, work with them should be done in a well-ventilated area with appropriate protective clothing. The work area should be scanned with uv light to be sure that contamination has not occurred.) Procedure. Sample Collection. Atmospheric particulate samples were obtained by pumping a calibrated volume of air through 102-mm diameter Gelman Type A glass fiber filters which had been previously cleaned as follows. First, the filters were immersed in a refluxing concd " 0 3 bath for 15 min. The filters were then immersed a t room temperature for an additional 3-4 days after which they were washed six times with deionized water and dried by heating overnight in a muffle furnace maintained a t ca. 290 "C. The dried filters were then extracted overnight in a Soxhlet extraction apparatus with spectral grade benzene and dried a t room temperature in a "clean" room.
A low volume Gast lVBF-lO-N-100X piston pump and a high volume Cadillac HP-3 Hurricane type pump which were used in the study were calibrated on a Singer American Meter Co. AT-210 gas flow meter. Cylindrical plugs (43-mm diameter X 50 mm and 65-mm diameter X 30 mm, respectively) fashioned from polyurethane packing material were used as gas phase filters behind the glass fiber (particulate) filter. Plugs for samples collected in the Baltimore Harbor Tunnel and in College Par,k, Md., had been cleaned by overnight extraction with spectral grade benzene. Both the particulate and gas phase filters were protected from sunlight and air currents during collection. Filters containing the crude particulate matter samples were extracted overnight (-16 h) with 250 ml of spectral grade benzene in a Soxhlet extractor. The cooled extract was reduced in volume by flash evaporation below 40 "C and was further concentrated to a known volume by careful evaporation under a slow stream of dry nitrogen. The volume of the concentrated extract was measured with a calibrated syringe. Alternatively, the absolute fraction of PAH subjected to hplc/ fluorescence analysis could be determined by use of an internal standard. Typically, 50 pg of triphenylmethane was added to an air filter extract and subjected to concentration and analysis. Equivalent results were obtained when the triphenylmethane was added after the concentration step. Triphenylmethane could be detected with the absorption detector, but its presence did not affect the hplc/fluorescence trace a t these concentrations since its fluorescence was filtered out. A parallel extraction of an identical blank filter which had been placed in contact with the filter mounting apparatus (contact blank) was conducted as a control to exclude the possibility of adventitious PAH contamination. An air filter sample collected from 255 m3 of air (high volume pump) in the Baltimore Harbor Tunnel (May 22, 1975, 26 "C, 70% rel. humidity) contained 573 mg of particulate matter of which ca. 75 mg (13%) was benzene-soluble. An air filter sample collected from 257 m3 of air (low volume pump) on the roof of wing one of the University of Maryland Department of Chemistry in College Park, Md. (April 20-23, 1975, average temp. ca. 16 "C, average rel. humidity ca. 60%) contained.46.3 mg of particulate matter, of which ca. 4 mg (9%) was benzene-soluble. High Pressure Liquid Chromatography. The particulate extract was injected (by syringe) into an injection port connected to a horizontally mounted 0.25 m X 6 mm i.d. stainless steel Du Pont Zorbax ODS column (-3200 plates, plate height -0.08 mm, for BaP elution with 7:3 (v/v) MeOH/H20 a t 65 "C in a Du Pont Model 630 liquid chromatograph equipped with a thermostated oven. In a typical analysis, the mobile phase was introduced by shaking together appropriate volumes of methanol and water, adding the mixture to the solvent reservoir, and recycling with the pump until all trapped bubbles were cleared from the solvent reservoir. "Deoxygenated" mobile phase was prepared by first bubbling nitrogen through the well-mixed solution of appropriate volumes of methanol and water for 20 min. This solution was then transferred under a nitrogen atmosphere to the solvent reservoir where a vacuum (-12 mm) was applied during solvent recycling. When the solvent was cleared of all gas bubbles, the vacuum was released and a nitrogen atmosphere, which was maintained through rhe chromatographic separation, was introduced. Separztion was typically effected upon elution with 7:3 (v/v) MeOH/H20 at 1400-2000 psi at 65 "C (flow rates of 1.5-2.0 ml/min). A Du Pont Model 836 absorbance/fluorescence detector (filter fluorometer) equipped with a Corning 7-60 (310 nm < X < 390 nm) excitation filter and a 3-72 ( X > 440 nm) emission cut-off filter was used to detect both uv absorption and fluorescence emission from eluting fractions. Signal intensities (absorption and fluorescence) were recorded on a dual pen recorder. The time required for a typical analysis under these conditions (where BaP is completely separated from BeP; Figure 1) is about 65 min. Relative retention times for standard PAH samples were obtained in an analogous fashion and are summarized in Table I.
RESULTS Identification of Components. Typical partial hplc/ fluorescence traces (Figures 2 and 3) show the large number of compounds present in urban air filter samples collected in the Baltimore Harbor Tunnel and on the roof of the University of Maryland chemistry building, respectively. At least 35 well-resolved fluorescent fractions are ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
993
'i i I
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ot-
d
I
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5
10
I
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I I 1 I I 15 2C 25 50 35 Time after injection (mid
I
I
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I
40
45
'1 1 1I 50
Flgure 2. Partial hplc/fluorescence trace of a benzene extract of an atmospheric particulate matter sample collected in the Baltimore Harbor Tunnel Peak identification as listed In Table Ii. Conditions: 0.25 m X 8 rnrn 1.d. Zorbax ODS column, 7:3 (v/v) MeOH:H20, 65 O C , 1600 psi (flow rate, 1.7 rnl/ min)
Time after Injection ( m i d
Flgure 3. Partial hplc/fluorescence trace of a benzene extract of an atmospheric particulate matter sample collected on the roof of wing 1 of the Department of Chemistry, University of Maryland, College Park, Md. Conditions: 0.25 rn X 8 mrn 1.d. Zorbax OOS column, 7:3 (v/v MeOH:H20), 65 OC, 1600 psi (flow rate, 1.7 rni/rnin)
present in the Baltimore Harbor Tunnel sample. Of these, 11 (those listed in Table I) have been identified. Fractions corresponding to the retention times of phenanthrene and anthracene have also been detected, but extra peaks present in their fluorescence emission and excitation spectra suggest that these compounds are not unambiguously identified. For peak identification, the eluting sample fractions were collected and subjected to further anlysis. The hlpc retention times and fluorescence spectra (excitation and emission) were compared with those of authentic samples and molecular weights were obtained by mass spectrometry. Fluorescence spectra (uncorrected for photomultiplier response) were recorded on a Perkin-Elmer model 204 fluorescence spectrophotometer equipped with a Perkin-Elmer model 150 xenon lamp and power supply. Fluorescence spectra are obtained by manually scanning the excitation monochromator range to determine the maximum fluorescence signal with, typically, the emission monochromator set a t 400 nm. Then, with the excitation monochromator 994
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Table I. Relative Retention Times for Standard PAH Compound Rel. retention time0 Fluoranthene Pyrene Benz [alanthracene Chrysene Benzo[e]pyrene Benzo[k]fluorantheneb Benzo[a]pyrene Benzoperylene 2,3,6,7-Dibenzanthracene 1,2,4,5-Dibenzopyrene 3,4,9,10-Dibenzopyrene
(1.0) 1.5 2.0 2.3 3.8 3.8 4.2 5.7 6.2 7.5 7.9
7:3 (v/v) MeOH/H20, 1400 psi, 6 5 O , 0.25 m X 8 mm i.d. Zorbax-ODScolumn. Tentatively identified in air particulate matter from its fluorescence excitation, emission, and mass spectra. However, a standard sample was not available.
set at this absorption maximum, the emission monochromator was mechanically scanned and the signal from the photomultiplier was recorded (emission spectrum). Excitation spectra were obtained by recording the photomultiplier signal at the observed fluorescence emission maximum while scanning the excitation monochromator (typically 200 nm < X 5 400 nm). First- and second-derivative fluorescence spectra (electronic differentiation) and selective modulation fluorescence spectra were obtained by the methods of Green and O'Haver (67) and O'Haver and Parks ( 6 8 ) ,respectively. Mass spectra were obtained on a Du Pont 21-492 mass spectrometer after concentration of the eluted fraction by extraction into pentane, removal of most of the solvent by flash evaporation under 40 "C, and final concentration by passing a slow stream of dry nitrogen over the sample. The collected fractions were identified on the basis of the fluorescence and mass spectral data in Table 11. Quantitative Analysis. Quantitative analyses were obtained by comparing the hplc peak area with that of a standard calibration curve constructed for each hydrocarbon. The calibration was periodically checked by injecting a 1-ng sample of BaP. In all cases examined, the hplc/fluorescence response was linear with PAH concentration in the range from 1 to 100 ng of each hydrocarbon injected onto the column. (The response continued to be linear a t lower concentrations of benzo[a]pyrene, the only PAH examined at levels less than 1 ng.) Different, but linear, Calibration curves were obtained for BaP in the presence and absence of dissolved oxygen in the mobile phase. Hplc/fluorescence peak areas of standard samples were reproducible to f2%. Precautions required for collection and workup during the quantitative analysis of atmospheric particulate samples are described elsewhere (69). Polyurethane back-up filters prevented loss by vaporization from deposited particulate matter during collection and samples were shielded from room light during workup and analysis. Extracts were stored a t 0 O C in the dark before analysis. The efficiency of overnight extraction with benzene was established by two methods. First, reextraction as above (-16 h, spectral grade benzene) of two atmospheric particulate samples which had previously been extracted as above and dried at room temperature failed in each case to give measurable hplc/fluorescence signals (Le., >99.5% removal of benzene-soluble fluorescent species occurred in the first 16 h of spectral grade benzene extraction). Second, an air filter sample was cut in half and 100 ng of BaP was injected onto one half. The doped sample was allowed to
Table 11. Fluorescence and Mass Spectral Data of Analytical PAH Fractions“ Fractionb Compound Excitation maximaC(nm) 1 2
3 4
5 6 7
8 9 10 11
Fluoranthene Pyrene Benz[a]anthracene Chrysene Benzo[e]pyrene Benzo[k]fluoranthened Benzo[a]pyrene 1,12-Benzoperylene 2,3,6,7-Dibenzanthracene 1,2,4,5-Dibenzopyrene 3,4,9,10-Dibenzopyrene
291,330,346,360 295,322,337 274,296,330,346,360 299,307,321,360 307,323,336,365 307,361,383 298,364,381 350,371,387 337 302,335, 357,373 297, 312, 329, 358, 371,390
Emission maximac (nm)
Ma+ ( m l e )
409,440,465 367,385,396 385,405,435 362,380,404,427 392 404,425,451 405,428,455 420,455 390,408,550 394,414,442,471 431,457,493,532
202 202 228 228 252 252 276 278 302 302
Obtained by hplc/fluorescence separation of an atmospheric particulate matter sample collected in the Baltimore Harbor Tunnel. See Figure 2. c In 7:3 (v/v) MeOH/H20; calibration: f 2 nm. Fluorescence spectra were determined by selective modulation. a
dry (-2 h) a t room temperature. Each half was then subjected to the extraction procedure described above, triphenylmethane (internal standard) was added, and the quantity of BaP present was determined by hplc/fluorescence. After subtracting the quantity of BaP present on the undoped sample from that found on the sample containing added BaP, 94-97 ng of BaP could be accounted for (i.e., recovery of 94-95%). This small apparent loss probably occurred during the concentration of the extracted solution. Reduction in the volume of standard solutions of BaP and pyrene in benzene, accomplished by flash evaporation to about 10 ml, followed by either careful distillation or concentration under a stream of dry nitrogen to a known solution volume, led to recoveries of 94-100% and 92-loo%, respectively. When flash evaporation of solvent from extracts was allowed to proceed to dryness before dissolving the residue in a known volume of solvent, only 8 1 4 3 % of the BaP was recovered (70). The concentrations of BaP determined by hplc/fluorescence and by collection of an eluent fraction and subsequent standard fluorometry agreed to f 6 % . Finally, the reproducibility of the technique was examined by subjecting identically sized portions of an air filter sample to the entire analysis (extraction, concentration, injection, hplc/fluorescence). The quantity of BaP obtained agreed to within fll%.By use of an internal standard (triphenylmethane) which was detected by uv absorption, the precision could be increased to f 6 % . Sensitivity. With our filter system and with air saturated mobile phase, 90 pg of BaP was required to obtain a signal-to-noise ratio of 2 with the hplc/fluorescence detector. When the mobile phase was “deoxygenated”, only 25 pg of BaP was required for this response.
Table 111. Concentrations of Some Polycyclic Aromatic Hydrocarbons in Atmospheric Particulate Mattera Baltimore Harbor Tunnel Compound Fluoranthene Pyrene Benz[a]anthracene Chrysene Benzo[e]pyrene Benzo[a]pyrene Benzoperylene 2,3,6,7-Dibenzanthracene 1,2,4,5-Dibenzopyrene 3,4,9,10Dibenzopyrene
College Park, Md.
ndng BaP
ng/m3 c,d
wing BaP
93 f 10
1.4
4.1 f 0.5
1.3
120 i 1 2 102 I 10
1.8
5.2 f 0.5 4.6 f 0.5
1.6 1.4
106 f 9 69 f 7
1.6
4.8 f 0.5 4.6 f 0.5
1.5
1.1
66 f 7
(1.0)
3.2 f 0.4
(1.0)
85 f 9
1.3
3.9 f 0.5
1.2
ng/m3 b~
1.5
3 f 0.5
0.05
7 f 0.9
0.11
3 10.5
0.05
1.4
a PAH concentrations were determined by comparison of peak areas obtained upon elution from the column with a calibration curve for air saturated samples. b Average of four samples. Experimentally determined values are corrected for losses found in collection (4 & 2%) and in extraction, concentration, and analysis (4 f 2%). The error limits reflect observed variation (after analysis) among samples. Average of two samples.
DISCUSSION The concentrations of several polycyclic aromatic hydrocarbons present in atmospheric particulate matter in a heavily-traveled traffic tunnel (Baltimore Harbor Tunnel) and in “relatively clean” suburban air (in College Park, Md., a suburb of Washington, DC.) have been determined. Although the ratios of the concentrations of several PAH to the concentration of benzo[a]pyrene are similar (as shown in Table 111), the significance of this observation remains unclear until a variety of PAH sources are examined. The concentration of benzo[a]pyrene found in the Baltimore Harbor Tunnel (66 ng/m3) is high compared to typical concentrations in urban areas in the United States (0.1-60 ng/m3) (2, 71, 72).
The analytical technique used in this study allows for the direct detection and quantitative determination of a wide variety of polycyclic aromatic hydrocarbons. Its dramatic improvement in sensitivity over chromatographic techniques using uv detection is apparent from Figures 4A and 4B which show simultaneous uv and fluorescence traces of a typical atmospheric particulate extract subjected to liquid chromatographic fractionation. Hlpc/fluorescence fractions were unambiguously identified by comparing the fluorescence spectra of the analytical samples with standard fluorescence spectra. A typical spectrum of an analytical sample corresponding in retention volume to that of benzo[a]pyrene is shown in Figure 5A. ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
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.-+
u)
c W
5 IC W > .e
-0 nW
5
Time ofter Injection (mid I
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I Wavelength (nm)
Wavelength (nm)
Figure 5. Fluorescence emission spectra of benzo [ a]pyrene.
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15 20 25 30 Time ofter injection (mid IO
(A) Analytical fraction collected upon elution from an hplc column after injection of a benzene extract of an atmospheric particulate matter sample collected in the Baltimore, Md., Harbor Tunnel. Chromatographic conditions as listed for Figure 2: ,A, = 380 nm. (B) Standard BaP solution (in methanol): ,A, = 297 nm
Figure 4. Partial h p l c h v absorption (A) and fluorescence (B) traces of a benzene extract of atmospheric particulate matter collected in the Baltimore Harbor Tunnel These traces were determined simultaneously. Conditions: 0.25 m X 8 mm i.d. Zorbax ODS column, 13:7 (v/v) MeOH:H20, 65 OC, 1400 psi (flow rate, 1.5 ml/min). Sensitivity: 0.01 A.U. full scale (A) and 8 @Afull scale (6)
The fluorescence spectrum of a standard solution of BaP is given for comparison in Figure 5B. Identification was also achieved by comparing fluorescence excitation spectra. Figure 6 compares both fluorescence emission and excitation spectra for the analytical fraction corresponding to the elution volume for pyrene with those of an authentic pyrene sample. In the absence of intermolecular effects, the excitation spectrum should exactly parallel the uv absorption spectrum (73).For compounds where fluorescence is very efficient, excitation spectra thus allow an easy extension of the detection limits imposed by the uv extinction coefficient. The technique could easily be extended to other fluorescent systems. In solving our problem, we chose to enhance selectivity by slightly reducing detector sensitivity by our choice of excitation and emission filters, thereby discriminating against one- and two-ring systems. In this way, for example, benzene (present in relatively high concentrations as the extraction solvent) was completely invisible to our method of detection since its emission signal was completely filtered out. The broad band-pass filters described above were thus effective in solving our problem, but the commercial availability of a wide variety of narrow band-pass and cutoff excitation and emission filters makes hplc/fluorescence potentially even more widely adaptable (54). The fact that hplc/fluorescence is nondestructive allows for easy application of newly developed techniques for luminescence analysis. When supplemented by hplc retention volumes, ordinary fluorescence spectra are usually sufficient for identification of a given analytical fraction. However, for difficult identifications, first and/or second derivative fluorescence techniques (67) can be used to emphasize minor features of the observed fluorescence spectra. Figure 7 illustrates an application of this technique to an unknown fraction from a Baltimore Harbor Tunnel sample. This compound (possibly an anthracene derivative by virtue of its elution volume and the general similarity of its fluorescence spectra to those of anthracene), has a 996
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Wovelenr"
'--I
350
450
550
Flguire 6. Fluorescence spectra of pyrene (A) Excitation spectra. (B) Emission spectra. (Al) Standard pyrene solution (in methanol);, ,A = 385 nm. (A2) Analytical fraction collected upon elution from an hpic column after injection of a benzene extract of an atmospheric particulate matter sample collected in the Baltimore, Md., Harbor Tunnel. = 387 nm. (61) StanChromatographic conditions as listed in Figure 2; ,A. dard solution: ,A. = 295 nm. (B2) Tunnel sample: ,A, = 343 nm
minor feature indicated by an arrow in the left trace (A) (a zeroth derivative fluorescence spectrum). This minor feature can be detected as a well-resolved peak in the first derivative fluorescence spectrum of this same compound, shown in the right trace (B). Selective modulation (68) fluorescence techniques are also readily adaptable to this study. Modulation of the excitation monochromator a t a maximum absorption peak of the interfering fluorescent species allows for the nulling of the interfering spectrum while the desired spectrum is observed. An application of this technique to the analysis of atmospheric particulate matter is illustrated in Figures 8-1 1. Figure 8 shows the fluorescence emission spectrum obtained for the hplc fraction from a Baltimore Harbor Tunnel sample corresponding in retention time to that of an authentic sample of chrysene. Standard fluorescence emission spectra of chrysene and benz[a]anthracene are shown for comparison in Figures 9 and 10, respectively. Although no impurities can be seen in the analytical sample (Figure 8 ) , application of the selective modulation technique (modulation of the excitation monochromator in the region of the absorption maximum of chrysene) resulted in the emission spectrum shown in Figure 11 (curve l),conditions under which a standard chrysene sample is completely nulled (curve 2). On comparing this residual spectrum
Wovelengt h (nml
Flgure 9. Fluorescence emission spectrum of a standard sample of chrysene (in methanol); Lx= 296 nm
Wavelength ( n m l
Wavelength(nrn1
Figure 7. Fluorescence emission spectra of an unidentified fraction collected upon elution from an hplc column after injection of a benzene extract of an atmospheric particulate matter sample collected in the Baltimore, Md., Harbor Tunnel Chromatographic conditions as listed for Figure 2; A,, = 350 nm (A) Normal (zeroth derivative) spectrum. (B) First derivative spectrum (same sensitivity) of the minor spectral feature indicated by an arrow in A. Bottom curve: sensitivity X 4
(L
350 400 450 W a v e l e n g t h (nrn)
Figure 10. Fluorescence emission spectrum of a standard sample of benz[a]anthracene (in methanol): A,, = 296 nm
20
;10
350
450 430
Wavelength lnrn)
Figure 11. Curve 1: Selectively modulated fluorescence emission spectrum of the same sample whose normal spectrum is shown in Figure 8; excitation modulation at the absorption maxima of chrysene. we, = 2.0 mm, we, = 0.5 mm, 1.5 nm/s, sens = 3 % full scale, J = 1 s. Curve 2: Selectively modulated (i.e., nulled) fluorescence emission spectrum of a standard chrysene sample (Figure 9) under conditions as in 1
W a v e l e n g t h (nmi
Figure 8. Fluorescence emission spectrum of a fraction (the retention time of which corresponds to that of chrysene) from a Harbor Tunnel sample. The chromatographic conditions were as listed in Figure 2;Lx= 298 nm
with that of a standard sample of benz[a]anthracene (Figure lo), one clearly detects a close correspondence with the emission expected from a mixture of the latter compound and an unidentified species emitting at ca. 345 nm, which was subsequently shown to be present in very low concentrations in the solvent. This may therefore represent a minor tailing of the benz[a]anthracene peak into the chrysene fraction which would have been undetectable by ordinary fluorescence techniques. In the same way, the fluorescence spectrum of a coelutant of benzo[e]pyrene could be identified as benzo[k]fluoranthene by comparison of the null mode selectively modulated fluorescence spectrum of the mixture with a published fluorescence spectrum of benzo[k]fluoranthene ( 5 3 ) .The contribution of BeP to the fluorescence of the coeluted mixture could similarly be determined by comparing the intensity of the enhancement mode selectively modulated fluorescence spectrum of the mixture with a calibration curve for BeP under modulation conditions. However, the absolute quantity of benzo[klfluoranthene present could not be established since we did not have an authentic sample of this compound. A sometimes overlooked method for the enhancement of fluorescence of very dilute PAH samples entails the removal of dissolved oxygen from solution, thereby reducing or eliminating oxygen quenching of the relatively long-lived PAH excited singlets ( 5 5 ) .Since this effect depends on the
fluorescence lifetime ( 7 4 ) , its magnitude will differ within a series of compounds. We have found that compared to pyrene, for which substantial oxygen quenching has been reported ( 7 5 ) , oxygen quenching of PAH fluorescence is less important in chrysene but more important in benzo- and dibenzopyrenes. The importance of oxygen quenching is obvious from the observation that a nearly fourfold enhancement of the fluorescence intensity of BaP is obtained by “deoxygenating” the aerated mobile phase in hplc analyses by the technique described in the Experimental section. Separate linear calibration plots of BaP were obtained with aerated and with “deoxygenated” mobile phases. Thus, the detectability of trace quantities can be enhanced by the extra effort required for “deoxygenation” of the mobile phase. However, we have not yet fully determined how effective or reproducible our “deoxygenation” process is. It should be noted that quantitative (or unambiguous qualitative) work is possible by the hplchluorescence technique only for samples for which an authentic sample is available. Quantitative determination of any species requires that a calibration curve be constructed for each compound identified, since fluorescence response in this detector depends on the filter system, elution solvent, fluorescence efficiency, fluorescence lifetime, and absorption characteristics (extinction coefficient) of the compound being analyzed. Furthermore, as in all luminescence studies, inner filter effects and self-quenching always impose an effective upper limit for concentrations of fluorescent species which can be directly determined quantitatively ( 7 3 ) . Nonetheless, the dramatic sensitivity and selectivity ANALYTICAL CHEMISTRY, VOL. 48, NO.
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of hplc/fluorescence techniques make them attractive for the rapid Of mixtures Of trace Organic components.
ACKNOWLEDGMENT Special thanks go to T. C. O'Haver and W. C. Parks for with fluorometry instrumentation and for conducting the selective modulation experiments. We gratefully acknowledge the generous gift of fourteen standard samples from the National Cancer Institute. Helpful technical assistance in the collection of samples was given by W. H. i l p.~~~l~~~~ ~ , and by D. R. ~~k~~ in the zoller, E. ~ ~ and initial hplc analyses. Finally, H. Hughes, Secretary of Transportation, State of Maryland, and D, ill^, superinh l d e n t of Bridges and Tunnels, were most cooperative in allowing us access to the Baltimore Harbor Tunnel.
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RECEIVEDfor review December 4, 1975. Accepted February 9, 1976. We are grateful to the RANN Program of the National Science Foundation (ESR-75-02667) for financial support.
