Low-Temperature Fluorescence Spectrometric Determination of Polycyclic Aromatic Hydrocarbons by Matrix Isolation Robert C. Stroupe, P. Tokousbalides, Richard B. Dickinson, Jr., E. L. Wehry,” and Gleb Mamantov* Department of Chemistry, University of Tennessee, Knoxville, Tennessee 379 16
The performance of quantltatlve low-temperature fluorometry by the technique of matrlx isolation ts reported. Matrtxisdated samples of polycycllc aromatic hydrocarbons (PAH) are prepared by mixing the PAH vapors effuslng from a Knudsen cell with a large excess of nltrogen gas; the gaseous mlxture is then deposited onto an optical window at 15 K. Detectlon llmlts for PAH In nltrogen matrices are on the order of I O - ” g. Quantitative worklng curves for PAH are linear over five decades or more in concentratlon. Linear worklng curves can readlly be obtained for Individual PAH In mlxtures of lsomerlc compounds. The use of matrix-isolation fluorometry for ldentlflcation and quantltation of PAH In complex samples derived from coal liquids Is demonstrated.
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Analysis of polycyclic organic matter continues to receive widespread attention, in view of the possible production of polycyclic aromatic hydrocarbons (PAH) in coal conversion and combustion processes and the carcinogenicity of a number of the compounds ( 1 , 2). Because of their high sensitivity, fluorometric techniques have been widely applied to the analysis of PAH (3). However, fluorescence measured in liquid solution is not broadly applicable to quantitative analysis of mixtures of fluorophores, for two reasons. First, fluorescence spectra of aromatic molecules in liquid media are usually broad and relatively featureless; thus, severe difficulties with spectral overlap often arise in mixtures. Second, quenching and intermolecular energy transfer occur with high efficiency in fluid media; hence, the relation between observed fluorescence intensity and concentration for a particular analyte often depends upon the identities and concentrations of other species present in the sample. Consequently, determination of PAH in complex mixtures can usually be accomplished only in low-temperature matrices. There has been special interest in the fluorescence of PAH in frozen solutions of n-alkanes (commonly known as “Shpol’skii matrices” ( 4 , 5 ) ) due , to the exceptionally sharp “quasilinear” spectra often observed for PAH in such media. It is now apparent that Shpol’skii spectra can be very useful for “fingerprinting” specific compounds present at very low levels in mixtures of PAH (6-14). Quantitative analyses of PAH by Shpol’skii spectroscopy have also been reported (7, 10-12, 15-21). However, a number of difficulties can be encountered in performing precise quantitative analyses in Shpol’skii matrices (22, 23). In the production of a Shpol’skii matrix (or any frozen solution), formation of microcrystalline aggregates of solute may occur unless the freezing process is very rapid (24) and the solute concentration is low. Fluorescence quantum efficiencies in microcrystallites are usually different from those in solution, and other undesirable effects, such as excimer fluorescence (251, can be observed when microcrystallite production accompanies frozen solution formation. Moreover, intermolecular energy transfer proceeds with very high efficiency in crystals, thus magnifying the importance of quenching and sensitization phenomena (25). Though the
production of microcrystallites in frozen-solution luminescence experiments has received little experimental study, it is clear that the phenomenon can occur at surprisingly low solute concentrations (e.g., lW5 M for pyrene in cyclohexane (25)and M for pyrazine in CCll (26)). The irreproducibility of microcrystallite formation is a potential cause of quantitative imprecision in Shpol’skii matrices. Both the intensities and half-widths of individual “quasilines” in Shpol’skii spectra may depend upon the freezing rate (27-30). For some solutes, the observed Shpol’skii fluorescence intensity is strongly dependent upon the final temperature of the frozen solution (27). Bandwidths in Shpol’skii spectra are in certain cases dependent upon the fluorophore concentration (27,31). In most cases in which analytical working curves have been reported for Shpol’skii matrices, the linear region is restricted, even when careful standardization (usually a combination of standard addition and internal standard methods (10,11,15,16))is employed. There appears to be only one published report (10) in which a linear working region exceeding three decades in concentration has been claimed for Shpol’skii fluorescence; other quantitative studies (7,11,15,18,20)have demonstrated a very restricted linear working range (2.5 decades or less). A further disadvantage of frozen-solution luminescence spectroscopyis the fact that, when very sensitive spectroscopic instrumentation is used, detection of small quantities of analyte may be hindered by background luminescence from the solvent, which can be very difficult to eliminate even when the best commercial solvents are subjected to additional purification (32). For these reasons, though the Shpol’skii effect will continue to encounter widespread use in the analysis of PAH, it appears desirable to examine other forms of low-temperature spectroscopy for their applicability to quantitative analysis. We have chosen to explore the utility of matrix isolation (MI) fluorescence spectroscopy in the qualitative and quantitative analysis of PAH. In MI, sample species, in the gas phase, are mixed with a large excess of “inert” diluent gas; the gaseous mixture is then deposited onto a cold surface. The deposited solid is subjected to spectroscopic analysis, usually a t temperatures of 20 K or less. Matrix gases commonly employed include nitrogen, argon, and xenon, all of which can readily be purged of luminescent contaminants. While MI has been widely employed in absorption spectrometry, particularly in the infrared (33, 34), few reports of MI fluorescence or phosphorescence spectra have appeared (35-38). While the potential advantages of MI fluorometry as an analytical technique were discussed as early as 1969 (39), no analytical applications of MI luminescence techniques have yet appeared. The purpose of the present study is to evaluate the utility of MI as a sample preparation technique for low-temperature fluorometry, with specific application to the qualitative and quantitative analysis of PAH in mixtures.
EXPERIMENTAL Matrix Isolation. PAH can be vacuum sublimed at tem-
peratures not greatly above ambient (40). Thus, the Knudsen ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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Figure 1. Room-temperature (RT) and matrix-isolation (16 K) fluorescence spectra of benzo[a]fluorene. The RT spectrum was obtained in nheptane (c= 3 X lo4 M); the MI spectrum was obtalned In nitrogen
cell method (41, 4 2 ) , wherein a “molecular beam” of solute is formed by effusion of vapor through am orifice, can be employed for MI of PAH. Design features of the Knudsen cell and cryostat were similar to those described previously for infrared measurements (43, 44). The deposit was collected on a sapphire window at ca. 15 K. In a typical continuous deposition MI experiment, the total deposition time ranged from 0.5 to 1.0 h. The matrix-to-sample mole ratio was ca. 107:l;the quantity of matrix gas was controlled by an external vacuum line, equipped with Teflon-packed ball valves (to eliminate fluorescent impurities which tend to distill from rubber O-rings) and a Fischer-Porter flowmeter. If the sample was a liquid solution, the appropriate volume (usually 10 pL) was introduced into the Knudsen cell by GC syringe; the solvent was stripped by passage of a stream of air through the cell, following which the cell was inserted into the vacuum system and heating to sublime the PAH was commenced. Fluorescence Spectrometer. A versatile high-resolution fluorescence spectrometer was constructed in this laboratory. Light from either of two sources, a 2500-W xenon or mercuryxenon lamp, was dispersed by a high-intensity monochromator (GM-250,Schoeffel Instruments, Westwood, N.J., reciprocal linear dispersion 33 A/mm), normally operated at a bandpass of 70 8, FWHM. The exciting light was directed onto the optical window in the cryostat (“Spectrim”, CTI Cryogenics, Waltham, Mass.). Fluorescence was viewed from the front surface of the window, and focused by a dual lens system onto the entrance slit of the emission monochromator (Model 1702,Spex Industries,Metuchen, N.J., reciprocal linear dispersion 11 A/mm), normally operated at a bandpass of 2 A FWHM. Analog detection was accomplished via a 1P28 photomultiplier tube and Aminco “photomultiplier microphotometer” (Model 510-280, American Instrument Co., Silver Spring, Md.). For photon counting, pulses from an RCA 8850 photomultiplier were processed by an Ortec “NIM” system (Ortec, Inc., Oak Ridge, Tenn., Models 9315 and 9320);the resulting signal was subjected to D/A conversion and displayed on a strip-chart recorder. No attempt was made to correct spectra for wavelength dependence of detector response or source output. Internal Standardization. It was necessary to employ internal standardization to compensate for nonquantitative deposition of sample constituents and drift in the single-beam fluorometer. Each internal standard had to satisfy three criteria. First, its luminescence spectrum had to be located in a spectral region in which overlap with emission spectra of sample constituents was minimal. Second, each standard had to sublime at approximately the same temperature as the compound(s)for which it was to serve as standard. Third, whenever possible, it was desired to employ the same excitation wavelength for the standard and analyte(s). A known weight of the standard was either added directly to the Knudsen cell or else dissolved in a known volume of liquid solution which was then introduced into the Knudsen cell. Fluorescencedata for all analytes were compiled as ratios of fluorescence intensities for individual solutes to the 702
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X(L, Figure 2. MI fluorescence spectra for six-component PAH mixture in nitrogen (16 K) at three excitation wavelengths. Sample contained 0.25 pg benzo[b]fluorene (BbF), 0.38pg benzo[a]fluorene (BaF),0.40 pg chrysene (C), 0.50 pg pyrene (PX 0.50 Mg phenanthrene (Bh), and 1.37 pg triphenylene (T)
fluorescence intensity of the standard.
RESULTS AND DISCUSSION
MI Fluorescence Spectra. Fluorescence spectra of one PAH, benzo[a]fluorene (BaF), in liquid solution and in a nitrogen matrix are shown in Figure 1. In comparison with the solution spectrum, the MI spectrum exhibits the expected shift to lower wavelength (46)and the fine structure anticipated in a low-temperature matrix. In the MI fluorescence spectrum of BaF, one specific band (at 342 nm) is much more
4 - R I N G PAH MIXTURE INT. STD.; B E N 2 0 [8] FLUORENE
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Figure 3. Working curves for four-ring PAH in synthetic mixture. For each compound, the fluorescence intensity for the compound is ratloed
to that of the internal standard, benzo[b]fluorene. Each sample was equimolar in each of the four PAH. Shown for comparison is a working curve, obtained under identical conditions, for pure chrysene (0) intense than any other band in the spectrum. This appears to be a general characteristic of MI fluorescence spectra of PAH; the “principal” fluorescence band is usually, but not always, the lowest-wavelength band in the spectrum. The MI fluorescence spectra of a six-component synthetic mixture of PAH, measured at three different wavelengths of excitation, are shown in Figure 2. The appearance of the mixture spectrum is drastically altered by changing the excitation wavelength. For example, the mixture spectrum is virtually devoid of pyrene (P) emission for A,, = 280 nm, but is rich in pyrene fluorescencewhen 313-nm excitation is used. The positions and relative intensities of the principal MI fluorescence bands for specific PAH are unaffected by the presence of other PAH in the sample, provided that the matrix-to-sampleratio is sufficiently large to negate inner-filter absorption (46). MI fluorescence spectra may therefore be employed as diagnostic criteria for identification of PAH in complex samples, as described in a subsequent section. It has been our experience that nitrogen is the most generally suitable matrix for MI fluorometry of PAH;a similar conclusion has previously been noted for MI infrared spectrometry of PAH (44). Individual bands in fluorescence spectra obtained in argon tend to be much broader than those observed in nitrogen. Quantitative Analysis. A portion of the quantitative working curve for MI fluorometric determination of pure chrysene is shown in Figure 3 (black points). The internal standard was benzo[b]fluorene (BbF). The full working curve is linear from a minimum quantity of 20 pg chrysene (the detection limit) to an upper limit of 5 pg, i.e., over five decades in weight of chrysene. An estimate of the precision of the MI method was obtained by multiple determinations of one point on the working curve; the relative standard deviation for eight replicate samples, each containing 100 ng chrysene and 10 ng
BbF, was 4.0%. Detection limits for more intensely fluorescent PAH, such as benzo[a]pyrene, are in the 5-10 pg range. Figure 3 also shows working curves for each compound present in synthetic mixtures of four-ring PAH. Each sample was equimolar in the four compounds. The excellent linear working curves for all four compounds demonstrate that MI fluorometry is substantially free from interference by intermolecular energy transfer or inner-filter effects (46),even for samples containing microgram quantities of several closely related PAH. Particularly striking is the fact that the working curve for chrysene in the four-component mixture is virtually superimposable upon that for pure chrysene (Figure 3). Thus, in samples of moderate complexity, working curves obtained for samples of pure PAH may be directly applicable to quantitation of that PAH in an unknown sample. It is noteworthy that three of the four compounds (benz[a]anthracene, triphenylene, and chrysene) are isomeric (CI8Hl2), and that all quantitative data in Figure 3 were obtained by use of a single internal standard (BbF). The respective fluorescence intensities for each PAH in the mixture were measured from a single deposit of the sample, with the only variable parameter being the excitation wavelength. These results demonstrate the applicability of the MI fluorescence procedure to determination of individual PAH present in mixtures at nanogram or subnanogram levels. “Real” Samples. Goldstein (47,423) has described a liquid chromatographic (LC) procedure for fractionation of complex samples of PAH, such as coal-derived liquids and shale oils. This LC procedure (using cross-linked poly(vinylpyrro1idone) as the stationary phase and isopropanol as eluant) separates PAH in complex samples into groups of several compounds (generally having the same number of aromatic rings). Chromatographic fractions of this type are readily amenable to examination by MI fluorometry. Figure 4 shows MI fluorescence spectra of a 50-ILLaliquot portion of one such fraction (of “Synthoil”, a catalytic hydrodesulfurizationprocess coal liquid) at two excitation wavelengths. The spectra unequivocally demonstrate the presence of pyrene (Py), benz[a]anthracene (BaA),and chrysene (C) in the LC fraction. Quantitation, using BbF as internal standard, shows the concentrations of the three compounds in the original LC fraction to have been 16 ppb Py, 110 ppb BaA, and 120 ppb C. A number of other peaks in the spectrum do not match MI fluorescence spectra in our current library and must be regarded as unknown (U).The use of MI fluorescence and Fourier transform infrared spectrometry in the characterization of synthetic fuels will be described in detail elsewhere (49, 50). Comparison of MI w i t h Shpol’skii Fluorometry. An instructive comparison of the analytical capabilities of MI and frozen-solution Shpol’skii fluorometry may be obtained by comparison of Figures 5 and 6, which show Shpol’skii and MI fluorescence spectra, respectively, of BaA a t three different concentrations. The concentrations of the frozen solutions were chosen such that the absolute amounts of BaA closely matched those used to obtain the MI spectra in Figure 6. Two conclusions emerge from comparison of Figures 5 and 6. First, much sharper fine structure is obtained in Shpol’skii matrices than by MI in nitrogen. (However, if the MI experiment is performed using an organic solvent, such as n-heptane, as the matrix, then MI spectra with bandwidths comparable to those obtained in Shpol’skii frozen solutions can be observed @I).) Thus, Shpol’skii spectra should be more generally useful thmMI spectra in nitrogen for identification of PAH in complex mixtures. Second, the BaA spectrum is observed to be strongly concentration-dependent in a Shpol’skii matrix. Figure 5 shows that the relative intensities of the long- and shortwavelength branches of the Shpol’skii spectrum are drastically ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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X,nm Flgure 4. MI fluorescence spectra of liquid chromatographic fraction from “Synthoil”. Identified components: BbF, benzo[&]fluorene(added as internal standard):BaA, benz[a]anthracene:C, chrysene; Py, pyrene. “U” denotes a fluorescence band which cannot at present be assigned to a specific compound: “Hg” denotes stray light (a mercury line emitted by the xenon-mercury arc source) altered by a hundredfold change in BaA concentration. In contrast, the appearance of the BaA fluorescence spectrum is essentially independent of concentration in a nitrogen matrix; the intensity ratio, FdO3nm/F381 nm, varies from 0.28 to 0.30 as the amount of BaA is increased by a factor of 150. The difficulties of performing quantitative analyses by the Shpol’skii effect have been discussed by other workers (22); 704
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
it seems evident that MI is the more promising low-temperature fluorometric technique for quantitative determination of PAH in mixtures. Future improvements in the MI fluorescence method will include use of organic solvents as matrices (51),as well as coupling of MI with time-resolution techniques (52),which should enhance both the selectivity and sensitivity of the technique (53).
gk
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Figure 5. Frozen-solution (Shpol’skii) spectra for benz[a]anthracene in +heptane at three different concentrations at 16 K. The apparent attenuation of the bands at ca.390 nm is not produced by self-absorption
(11) G. F. Kirkbright and C. G. de Lima, Analyst (London), 09, 338 (1974). (12) L. M. Shabad and 0. A. Smirnov, Atmos. Environ., 6 , 153 (1972). (13) C. A. Parker and C. G. Hatchard, Photochem. Phofobiol., 5, 699 (1966). (14) P. P. Dikun, Russ. J. Appl. Spectrosc., 6, 130 (1967). (15) G. E. Fedoseeva and A. Y. Khesina, Russ. J. Appl. Spectrosc.,0, 838 (1968). (16) P. P. Dlkun, N. D. Krasnitskaya, N. D. Gorelova, and I. A. Kalinina, Russ. J . Appl. Spectrosc., 8, 254 (1968). (17) J. Jager, Chem. Listy, BO, 1184 (1966). (18) 0. E. Danil’tseva and A. Y. Khesina. Russ. J. Appl. Spectrosc., 5 , 196 (1966). (19) R. I. Personov and T. A. Teplitskaya, Russ. J. Anal. Chem.. 20, 1176 (1965). (20) R. I. Personov, Russ. J. Anal. Chem., 17,503 (1962). (21) 6. Muel and G. Lacroix, Bull. SOC. Chlm. Fr., 2139 (1960). (22) R. J. Lukasiewicz and J. D. Winefordner, Talanta, 10, 381 (1972). (23) E. L. Wehry, fluorescence News, 8 , 21 (1974). (24) R. A. Kelier and D. E. Breen, J. Chem. Phys., 53,2562 (1965). (25) R. J. McDonald and B. K. Selinger. Aust. J. Chem., 24, 249 (1971). (26) R. J. McDonald, L. M. Logan, I. G. Ross, and B. K. Selinger, J. Mol. Spectrosc., 40, 137 (1971). (27) N. S.Dokunikhln, V. A. Kkel, M. N. Sapozhnikov, and S. L. Solodar, Opt. Spectrosc., 25, 42 (1968). (28) . . D. M. Grebenshchikov, N. A. Kovizhnykh, and S. A. Kozlov, Opt. Spectrosc., 37, 155 (1974). (29) G. L. LeBei and J. D. Laposa, J. Mol. Spectrosc., 41, 249 (1972). (30) A. Colmsjo and U. Stenberg, Chem. Scr., 9, 227 (1976). (31) E. V. Shpol’skii, L. A. Klimova, G. N. Nersesova, and V. I. Giyadkovskil, ODt. SDeCtfOSC.. 24. 25 (1968). (32) . , C: A. Parker. “Phbtoluminescende of Solutbns”. American Elsevier. New York, 1968,’ p 287. (33) H. E. Halhm, ‘Vibrational Spectroscopy of Trapped Species”, Jchn Wiley, London, 1975. (34) S. Cradock and A. J. Hinchcliffe, “Matrix Isolation; A Technique for the Study of Reactive Inorganic Species”, Cambridge University Press, New
Figure 6. M I fluorescence spectra (in N1, 16 K) for benz[a]anthracene for three different sample sizes. The quantities of benz[ alanthracene correspond closely to the total amount of the compound used to obtain the frozen-solution spectra in Figure 5
ACKNOWLEDGMENT We thank G. Goldstein (Oak Ridge National Laboratory) for the LC fractions from “Synthoil”. LITERATURE CITED (1) National Academy of Sciences, “Particulate Polycyclic Organic Matter”, Washington, D.C., 1972. (2) R. Freudenthal and P. W. Jones, “Polynuclear Aromatic Hydrocarbons:
York, 1975. (35) 6. Meyer, “Low Temperature Spectroscopy”, American Elsevier, New York, 1971. (36) B. Meyer, Sclence, 168, 783 (1970). (37) 6. Meyer and J. L. Metzger, Specfrochlm. Acta, Pari A, 28, 1563 (1972). (38) J. L. Metzger. 6. E. Smith, and B. Meyer, Spectrochim. Acta, Part A , 25, 1177 (1969). (39) J. S.Shirk and A. M. Bass, Anal. Chem., 41 (Il),103A (1969). (40) J. L. Monkman, L. Dubois, and C. J. Baker, Pure Appl. Chem., 24, 731 (1971). (41) M. J. Linevsky, J. Chem. fhys., 34, 587 (lg61). (42) L. Brewer, G. D. Brabm, and 8. Meyer, J. Chem. Phys., 42, 1385 (1965). (43) E. L. Wehry, G. Mamantov, R. R. Kemmerer, H. 0. Brotherton, and R. C. Stroupe, in Ref. 2, p 299. (44) G. Mamantov, E. L. Wetny, R. R. Kemmer, and E. R. Hinton, Anal. W m . , 49. 86 (1977). (45) E. L. Wehry, in “Practical Fluorescence”, G. G. Guilbauit, Ed., Marcel Dekker, New York, 1973,p 125. (46) C. A. Palker, “Photoluminescence of Solutions”, American Elsevier, New York, 1963,p 220-229. (47) G. Gddstein, Abstracts, 271h Pittsbuw Conference on A ~ l y t i c aChemistr] l and Applied Spectroscopy, Cleveland, Ohio, March 1976,Paper 279. (48) 0. Goidstein, J. Chromatogr., 120, 61 (1976). (49) G. Mamantov, E. L. Wehry. R. R. Kemmerer, E. R. Hinton, R. C. Stroupe, and G. Goldstein, paper presented at 173rd National Meeting, American Chemical Society, New Orleans, La., March 1977. (50) E. L. Wehry, G. Mamantov, R. R. Kemmerer, E. R. Hinton, R. C. Stroupe, and G. Goldstein, paper presented at 28th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March
1977. (51) P. Tokousbalides, E. L. Wehry, and 0. Mamantov, paper presented at 28th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1977. (52) J. M. Harris, R. W. Chrisman, F. E. Lytle, and R. S. Tobias, Anal. Chem., 48, 1937 (1976). (53)R. B. Dickinson, Jr. and E. L. Wehry, unpublished work, University of Tennessee, 1976-1977.
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1976. (3) E. Sawicki, Talanta, 16, 1231 (1969). (4) C. A. Parker, “Photoiuminescence of Solutions”, American Elsevier, New York, 1968,p 379-386. (5) R. A. Passwater, Fluorescence News, 5 (5),4 (1971). (6) G. F. Kirkbright and C. G. de Lima, Chem. Phys. Leff., 37,165 (1976). (7) 6. S.Causey, G. F. Kirkbright, and C. G. de Lima, Analyst (London), 101, 367 (1976). (8) R. Farooq and G. F. Kirkbright, Analyst (London), 101, 566 (1976). (9) A. P. D’Silva, G. J. Oestrelch, and V. A. Fassei, Anal. Chem., 48, 915 (1976). (10) T. Y. Gaeyava and A. Y. Khesina, Russ. J. Anal. Chem., 29, 1913 (1974).
RECEIVED for review December 27,1976. Accepted February 14, 1977. This work was supported by t h e Electric Power Research I n s t i t u t e (Contract 741011-RP-332-1) and t h e N a t i o n a l Science Foundation (Grant MPS75-05364). Portions
of the work were presented at the 27th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, M a r c h 4, 1976 and t h e 173rd N a t i o n a l M e e t i n g , American Chemical Society, New Orleans, La., March 21,1977.
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