Liquid chromatography with real-time video fluorometric monitoring of

using a laminar flow cell to minimize dead volume and scat- tered light. A total ..... for data storage on hard disk and real time analysis of the EEM...
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Anal. Chem. 1981, 53, 971-975

airtight syringe without any prior sample treatment. Results from the chromatographic method are compared to atomic absorption results obtained with an air-acetylene flame. The results from the two techniques are fairly coincident with each other within 3% of relative errors.

ACKNOWLEDGMENT The author is indebted to Professor Kyoji T6ei of Okayama University and to Professor Toyokichi Kitagawa of Osaka City University for valuable advice and discussion.

LITERATURE CITED (1) Fritz, J. S.; Story, J. N. Anal. Chem. 1974, 46, 825-829. (2) Argueilo, M. D.; Fritz, J. S. Anal. Chem. 1977, 4 9 , 1595-1598.

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(3) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801-1809. (4) Freed, D. J. Anal. Chem. 1975, 47, 186-187. (5) Zenki, M. Anal. Chlm. Acta 1978, 8 3 , 267-274. (6) Zenki, M. Anal. Chim. Acta 1977, 93, 323-326. (7) Samuelson, 0. “Ion Exchange Separation in Analytical Chemistry”; Wiley: New York, 1963; Chapter 15. (8) Flaschka, H. A.; Barnard, A. J., Jr. “Chelates In Analytical Chemistry”; Marcel Dekker: New York, 1969; Vol. 2, p 1. (9) Ferguson, J. W.; Richard, J. J.; O’iaughlin, J. W.; Banks, C. V. Anal. Chem. 1984, 36, 796-799. (IO) Michayiova, V.; Kouleva, N. Talanta 1974, 21, 523-532. (11) Sawin, S. 8.; Petrova, T. V. Zh. Anal. Khlm. 1989, 2 4 , 177-185.

RECEIVED for review November 21, 1980. Accepted March 2, 1981.

Liquid Chromatography with Real-Time Video Fluorometric Monitoring of Effluents L. W.

Hershberger, 9. B. Callis, and G. D. Christian*

Department of Chemistry BG- 10, University of Washington, Seattle, Washington 98 195

Real-time fluorescence monltorlng at multiple wavelengths of excitation and emission is performed by Interfaclng a highperformance liquid chromatograph to the video fluorometer using a laminar flow cell to mlnimlre dead volume and scattered Ilght. A total fluorescence chromatogram, as well as two selected excitation-emlssion wavelength chromatograms, are displayed in real time. For perylene, the linear dynamic range covers at least 2 orders of magnitude wlth a detectlon limit of 1 ng. Selected fluorescence monitoring Is capable of spectrally separating and quantifying benzo[a]pyrene and benro[elpyrene whose chromatographic retentlon profiles overlap slgniflcantly. Flnally, a complex mixture, shale oil, is analyzed for benzolalpyrene.

The combination of a separation technique with a molecular fingerprinting technique offers the most powerful means of analyzing the complex materials with which today’s analytical chemist must be concerned ( I ) . Obviously, the combination against which all others must be compared is gas chromatography/mass spectrometry (GC/MS). In cases where the components to be analyzed lack sufficient volatility for gas chromatography, liquid chromatography combined with a fingerprinting technique such as mass spectrometry or rapid-scanning UV-Vis absorption spectrometry offers a path to analysis. Very recently, Winefordner and co-workers (2)and Talmi and co-workers (3, 4 ) have evaluated the efficacy of rapid scanning fluorescence detection of high-performance liquid chromatography (HPLC) effluents. Winefordner (2) showed with simulated chromatography data that multichannel imaging detectors based upon the SIT vidicon provided sufficient sensitivity for trace analysis. Talmi and coworkers (3,4)showed that an HPLC-multichannel fluorometer could easily be assembled from commercial parts, and their preliminary work showed that a qualitative analysis of the aromatic hydrocarbon fraction was quite feasible. Independent of these and other efforts (5-10) to use multichannel imagers as fluorescence detectors, we developed the video fluorometer (11). This instrument is unique because i t uses a novel irradiation geometry to obtain excitation and emission

spectra simultaneously. Recent studies have demonstrated that the video fluorometer is capable of determining selected components in a 12-component mixture, even under circumstances where spectral overlap is severe and intensities vary over a couple of orders of magnitude (12-14). Thus, the combination of the video fluorometer and HPLC seems especially appealing because the resulting system would use three dimensions to separate mixtures (excitation wavelength, emission wavelength, and chromatographic retention time). In this exploratory paper, we report on the potential of the liquid chromatograph/video fluorometer (LC/VF) for analysis of complex mixtures. Our capability to display the data as EEMs and as selected excitation-emission wavelength chromatograms is shown to be extremely helpful to the operator of the instrument.

EXPERIMENTAL SECTION Flow Cell. A modified version (Figure 1) of the sheath flow cell previously described (15)was used as the flow cuvette in the video fluorometer. In the modified version, the quartz windows (G) and the laminar flow path were increased in length to 17 mm to permit placement in the cuvette position of the video fluorometer without modification to the optical arrangement. Standard HPLC tubing and fittings were used to simplify connections to the flow cell. The sheath alignment (A) and exit bores (I) were 1/8 in. in diameter. The two 1/32 in. diameter sheath inlets (B) were drilled into the top of the sample alignment bore. Connections to the sheath inlets were made using a ‘/I6 in. Swagelok male plug (C) soldered with the cap end to each sheath inlet and a hole drilled through the cap. The chromatographic effluent was introduced into the sample alignment bore from a piece of 0.02 in. i.d. and ’/I6 in. 0.d. stainless steel tubing (D). The sample inlet tube was aligned and held in the center of the inlet bore by a ‘Ile in. Altex male fitting (E) and a Teflon ferrule (F). The outlet from the in. Swagelok male plug (H) attached flow cell used a drilled by the same method as the sheath inlets. Each of the four l/s in. thick quartz windows (G)was held and sealed against a Teflon in. stainless steel plate (J)using screws. The gasket (K) by a four steel plates contained openings for the windows. Under the laminar flow conditions, the diameter of the sheath and sample stream was that of the entrance and exit (I) ports, Le., 1/8 in. The flowing stream was surrounded by stagnant sheath solvent that filled the void between the stream and the windows.

0003-2700/81/0353-C971$01.25/00 1981 American Chemical Society

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Figure 1. Diagram of laminar flow cell: (A) sheath alignment bore; (B) '/,* in. diameter sheath inlets; (C) drilled in. Swagelok male plug; (D) in. stainless steel tube for chromatographic effluent inlet: in. Teflon ferrule; (G) ' l ein. quartz (E) In. Altex male fitting; (F) windows (GI= left side, G2 = front side, G, = right side); (H) drilled In. stainless steel 'I, in. Swagelok male plug; (I) exit bore; (J) plate; (K) 1 mm thick Teflon gasket (K, = left side, K2 = front side, K, = right side).

The sample diameter could be varied by adjusting the relative flow rates of sheath and sample flow. The normal operating diameter was about 1mm, which corresponds to an optical volume of 13 pL. We found that maintenance of laminar flow was considerably facilitated if the ensheathing fluid had a slightly higher density than the chromatographic solvent. Materials. The benzo[a]pyrene (BaP),benzo[e]pyrene (BeP), and 9-methylanthracene (9MA) were obtained from Aldrich Chemical Co. and were used as purchased. The zone-refined perylene was obtained from James Hinton (Valparaiso,FL). Both the perylene and 9MA were made up as saturated solutions in ethanol with their concentrations being 58.2 and 173 pg/mL, respectively. The concentrations were calculated from measured absorbances on a Varian Superscan 3 and molar absorptivity values in the literature for perylene (16) and 9MA (17). BaP and BeP solutions were made by dissolving several milligrams of each in 100 mL of cyclohexane. Working stock solutions were made by diluting by a factor of 30 with absolute ethanol. The concentrations calculated as above for BaP (18) and BeP (19) were found to be 7.0 and 19.2 pg/mL, respectively. The BaP standard used for analysis of the shale oil sample was a 100 pg/mL solution obtained from Chem Service (West Chester, PA). Standard shale oil sample was obtained from the National Bureau of Standards. The eluting solvent was a mixture of acetonitrile and distilled water. The acetonitrilewas spectral grade obtained from Burdick and Jackson and the distilled water was filtered with a 0.5-pm fiiter. The sheath solvent was distilled water and was used without filtration. Sample Preparation. For testing the dynamic range of the LC/VF, we injected 20 pL of varying concentrations of perylene. 9MA at constant concentration (69.9 ng/20 pL) was added as an internal standard to all the perylene solutions. For analysis of chromatographically overlapping compounds, a varying amount of BaP was injected with a 100-pL loop. A constant amount of BeP (480 ng/100 pL) was added to each solution along with 9MA (54 ng/100 pL) as an internal standard. The shale oil sample was cleaned up by using a modification of a liquid chromatographic technique described by Wilkinson and co-workers (20). A 25 X 1cm column was prepared by dry packing with 10 g of Woelm neutral alumina (3% H20 w/w) and then wetting with cyclohexane. To the top of the column was added 0.75 in. of Na2S04. The column was washed with 20 mL

of methylene chloride and reconditioned with 20 mL of cyclohexane. The oil sample was added using enough cyclohexane to transfer 0.1 g of oil to the column. Elution was then accomplished with 25 mL of cyclohexane followed by 20 mL of a 75% cyclohexane/25% methylene chloride solution. The latter fraction contained the polycyclic aromatic hydrocarbon (PAH) and was taken to dryness on a rotary evaporator and transferred to a 2.0-mL volumetric flask with three rinses of tetrahydrofuran and made to volume. Twenty microliters of this was injected. Recoveries were calculated by following the same clean-up procedure for 2.5 pg of BaP. Chromatographic Equipment. The modular HPLC consisted of two Altex Model lOOA pumps, an Altex Model 400 solvent programmer, an Altex Model 905-42 loop sample injector, and an Altex Model 153 absorption detector. The column was a 250 mm X 3.2 mm bore column packed with 5-pm Spherisorb ODS packing. For all work except for the analysis of the shale oil sample, only one HPLC pump was used, and the acetonitrile and water were premixed at a ratio of 8020. The pump outlet went directly to the injector and then on to a 10-pm RP-18 precolumn before going to the column. The outlet of the column went to the UV detector for UV monitoring of effluents at 254 nm and then to the video fluorometer for fluorescence monitoring. For the dynamic range study, the flow rate was 1.0 mL/min, and for the study of chromatographically overlapping components the flow rate was 0.7 mL/min. Later work on the shale oil sample used the second pump and solvent programmer to deliver a gradient of acetonitrile and water at a flow rate of 0.8 mL/min. A 25-min exponential gradient (exp = 3) from 40% solvent B and 60% solvent A to 100% of solvent B was used. Solvent A was a mixture of 40% acetonitrile and 60% water and solvent B was 100% acetonitrile. The solvents were degassed before use by bubbling helium through them and then storing under an atmosphere of helium during the chromatographic runs. The water used as sheath solvent was delivered from a 500-mL glass tank which was pressurized by nitrogen to 7 psi. This pressure was found to be the lowest pressure which would deliver sufficient sheath flow to maintain laminar flow. Fluorescence Monitoring. For fluorescence detection of effluents, the video fluorometer described by Johnson and coworkers was used (11). The flow cell described above was accurately positioned by means of a five-dimensional translation stage from NRC, Model LP-1. One major improvement was made to the video fluorometer. Noise arising from 60-Hz ac line pickup was eliminated by phase locking the camera drive oscillator to the line. This eliminated base-line oscillation arising from the beat frequency between the ac line and the 59.9-Hz vertical retrace frequency and reduced the single pixel noise by a factor of 10. Data acquisition and chromatographic timing were under control of a PDP 11/04 minicomputer. The raw video signal from the SIT vidicon camera consisted of 256 horizontal lines and was amplified, filtered, and sampled by a high-speed eight-bit A/D converter at a rate of 64 times/line. Four horizontal lines were then summed together for each pixel. The resulting excitationemission matrix (EEM) of 64 X 64 elements was collected in 16.7 ms and stored in the buffer memory. Individual frames were summed together in the buffer memory into an EEM of 64 X 64 pixels, with each pixel contained in a 32-bit word. Each EEM collected during the chromatographic run consisted of 148frames of fluorescence summed together, with 148 frames of dark current subtracted. The dark current was obtained after blocking the fluorescence with an automatic shutter and allowing the camera to stabilize, The total elapsed time for measurement and dark current correction was 5.4 s, with 0.5 8 being used to stabilizethe camera between fluorescence and dark current frames. Another 1.1 s was allowed for data storage on hard disk and real time analysis of the EEM just acquired. For the perylene dynamic range study and the two-component system, only floppy disks were available for data storage. Therefore, the data storage time was increased to 3.5 s to allow for the slower write cycle of a floppy disk. For analysis and storage, the size of each EEM was reduced to 60 X 60 by discarding two columns on each side and the bottom four rows, This reduction in matrix size was done for three reasons: (1)the bottom four rows were blanked for the camera

ANALYTICAL CHEMISTRY. VOL.

beam vertical retrace so that no meaningful data were contained in these four rows, (2) the two columns on each side contained data geometrically distorted and severely reduced in intensity, and (3) reduction of the matrix size reduced the storage area needed. Data Storage. Data were saved for postrun analysis in two forms. First, the fluorescence intensity for each of two selected combinations of excitation and emission wavelengths (two pixels) was saved for each EEM acquired. Also saved for each EEM was the total fluorescence intensity obtained hy summing all pixels together. Second, ensembled EEMs consisting of one or more EEMs summed together were stored on either floppy or hard disk. These EEMs were saved starting at a predetermined chromatographic time input hy the operator. Also saved was an ensemhled EEM of scattered light acquiredjust before injection of the sample, to he used for scattered light subtraction before further data analysis. Graphics. A real-time display of the two selected pixel fluorescence intensities (SPFI) and the total fluorescence intensity (TFI) was provided on a graphics terminal and was updated after acquisition of each EEM and subtraction of the scattered light. The display consisted of three different araDhs of fluorescence intensity as a function of time. Several methods of display were available for postrun graphics. The most useful for checking on the general elution of analytes was based on the selected pixel and total fluorescence intensity data and was similar to the real-time display with several modifications. First, the three plots displayed were normalized before display. Second, the operator could change the normalization factor for any or all of the plots. Third, any combination of the two SPFIs and the TFI could he displayed, including the option of displaying the same chromatogram more than once with the scaling increased by a factor of 5 for each additional display. The ensemhled EEMs could he presented in several forms. Each EEM could he displayed on the television monitor. Each EEM could also be displayed on the graphics terminal as a three-dimensional plot or contour map. Quantitative Analysis. Postrun data analysis could be based on either of the two data types, selected pixel or ensemhled EEMs. To obtain quantitative information from selected pixel data, we calculated the area or height of the elution peak and corrected for the background. The boundaries for background regions and peaks for the SPFIs or the TFI were selected by the operator. Boundaries were specified in an interactive mode and were stored after all three chromatograms had been previewed. Another program calculated the peak areas and heights and corrected for the background drift, as well as calculating the standard deviation of the residuals for the background. The peak heights were compared to the residuals to see if a peak had a signal-to-noise ratio greater than 2. Other schemes of quantitation were based on analysis of the ensemhled EEMB. Least-squares fitting of standards to individual ensembled EEMs based on the method described hy Warner et al. (21) was used. The method was modified to select only a fraction of the pixels on which to base the least-squares fitting. Another least-squares method involved fitting of all ensembled EEMs in a chromatographic run to the standarh simultaneously, as described by Johnson et al. (12). In this case, each point was now a function of three independent variables: excitation wavelength, emission wavelength, and chromatographic retention time. As described above, only selected pixels in each EEM were used. The final scheme of data analysis using ensembled EEMs was rank annihilation, described by Ho et al. (13). Each ensemhled EEM in a chromatographic run was analyzed for the component of interest and results for all EEMs in an elution peak were summed together for quantitative calculations. Comparison to a standard analyzed in the same manner was used to quantify the amount of unknown in a sample. The method of rank annihilation had the advantage over least-squares fitting of the data in that it was not necessary to know all components present to quantitate the one of interest.

RESULTS AND DISCUSSION Sensitivity a n d Dynamic Range. In view of the rather disadvantageous photon collection geometry in our system

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Flgure 2. (A, top) Fluorescence chromatograms obtained from injection of perylene (1.16 ng) together with 69.2 ng 01 9MA added as an internal standard. Pixel 28.24 (172 countsldivision) shows the elution 01 perylene. and pixel 21.36 shows the elution of 9MA (1320 countstdivision). (El. Bottom) EEM 01 perylene at top of peak. with scattered light subshacted,

compared with previously described systems (22,23), were interested in comparing the sensitivity of our system with that reported hy others. Figure 2A shows a set of chromatograms recorded hy our system for an injection of 1.16 ng of perylene together with 9MA (69.2 ng) added as an internal standard. This figure shows three chromatograms: one derived from the total fluorescence, one from the pixel at which the fluorescence of 9MA is maximal, and one from the pixel a t which perylene fluorescence is maximal. At this concentration, the perylene peak is only marginally apparent in the total fluorescence intensity (TFI) chromatcgram but quite discernible in the selected pixel fluorescence intensity (SPFI) chromatogram which is optimal for perylene detection. The oscillating base line in these chromatograms was found to arise from 60-Hz line pick up and in later work was reduced considerably as described in the Experimental Section. Figure 2B shows the EEM for 1.16 ng of perylene as it appears at the mean retention time, photographed on the television monitor. A dynamic range study showed that one could easily quantitate perylene injections in amounts from 116 to 1.16 ng. The results were obtained by finding the peak areas of the selected pixel chromatograms. A linear least-squares fit of the logarithms of concentration vs. signal was linear with a slope of 0.96 f 0.01 and a correlation coefficient of 0.938. If the internal standard was used to correct the results, the slope was 0.99 f 0.02 and the correlation coefficient was 0.998. Thus only a slight improvement in results was obtained with the internal standard.

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Figure 3. Fluorescence chromatograms of a mixture of anthracene (900 ng) and 9-methylanthracene (173 ng). Pixel 14,32 was chosen to be maximal for the fluorescence of BaP while pixel 24,23 was maximal for 9-methylanthracene.

Let us now place our instrument in context. As regards sensitivity, it clearly does not approach that of fluorescence detectors based upon single wavelengths of excitation and emission. Obviously, these latter have much more favorable irradiation and collection geometries. However, the selection of the wavelength pair inevitable must involve a compromise if multiple components are to be detected (see below) and the only confirmation one otherwise has of a particular component's identity is the retention time. In contrast, the EEM obtained by the video fluorometer will invariably contain the optimal wavelengths of excitation and emission for detecting the component of interest, and one has a three-parameter confirmation of a component's identity as welk retention time, excitation spectrum, and emission spectrum. Finally, one can also use the EEM to determine if more than one component is contributing to a single peak. When compared with UV-Vis absorption monitoring, our fluorescence system can obtain detection limits equal to or better than fixed-wavelength absorption for strongly fluorescent compounds. Moreover, the fact that two types of spectra are measured and the fact that not all materials are fluorescent confer certain selectivity advantage. It should be noted that with our unique irradiation geometry the absorption spectrum of each component is easily implemented by placing a diode array detector behind the flow cell to monitor the transmission of the polychromatic beam. Spectral Resolution. We next tested the capability of our system to simultaneously quantitate two components (anthracene and 9MA) in the case where they were not resolved chromatographically. Figure 3 shows a chromatogram of the two components. The total fluorescence chromatogram shows only a single peak with a shoulder, but the two selected pixel chromatograms chosen to optimize detection of each component, respectively, reveal that small differences do exist in retention times for the two compounds. In similar studies with mixtures of BaP and BeP under conditions where no chromatographic resolution was possible, we found that spectral overlaps were so low that satisfactory quantitation of 1.4-17 ng of BaP could be made in the presence of 480 ng of BeP. Least-squares analysis of the logarithms of signal vs. concentration for peak areas was linear with a slope of 1.00 f 0.03 and had a correlation coefficient of 0.991 after correction using the internal standard. The relative standard deviation for the BeP was calculated to be 9.2%. The data were also analyzed by three-dimensional least squares and compared to the selected pixel analysis. The precision as measured by the standard deviation of the residuals showed only a slight difference, but the detection limit was lowered by a factor of 2 to 0.7 ng. Use of an internal standard 9MA did not sig-

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nificantly improve the results in this case, indicating good stability. Complex Sample. Finally, we tested the capability of our system to quantitate BaP in a shale oil sample. Figure 4 shows the total fluorescence chromatogram and two selected pixel chromatograms, one optimized for BaP and the other pixel selected to fall into the range of fluorescence for anthracenes. Figure 5 shows data taken under identical conditions for 25 ng of authentic BaP. By comparison of Figures 4 and 5 one can see that neither the TFI chromatogram for shale oil nor the SPI chromatogram optimized for anthracene show evidence of a peak a t 22.5 min, where BAP is expected to elute. However, the SPI shale oil chromatogram optimized for BaF' does show a distinct peak at 22.5 min, albeit superimposed on a large background.. Figure 6 shows the EEMs taken at the top of the BaP chromatographic peaks for both chromatograms. Clearly the two components are similar and confirm that the spectrum of BaP in the shale oil sample is added to a large nonstructured background. Quantitation was based on the method of rank annihilation. The most intense EEM from the standard injection was chosen as the standard ensembled EEM for rank annihilation calculations. The recovery

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ACKNOWLEDGMENT We are indebted to Ed McArthur for fabricating the flow cell and to Sheldon Danielson for developing the line-locked oscillator. The standard shale oil sample was kindly supplied by Steven Wise of the National Bureau of Standards. We thank C.-N. Ho and M. L. Gianelli for useful discussion. Finally, we thank I. M. Warner of Texas A&M University for making available a preprint of his own work on this subject.

LITERATURE CITED (1) Hirchleld. T. Anal. Chem. 1978, 48. 16A. (2) Cwney, R. P.; VPDinh. T.; Walden. G.; Wlnelordner. J. D. Anal. Chem 1977, 49. 939. (3) Jadamec. J. R.; Saner. W. A,; Talml, Y. A M . Chsm. 1977, 49, 1316. (4) Jademec,J. R.; Saner. W. A,: Talml, Y. ACS Symp. Ser. 1070, No. 102, 115-133. ( 5 ) Vo-Dinh. T.; Johnson. D. J.; Winefwdner. J. D. Spechochim. Acta. Part A 1977, 33A. 541. (6) Cwney. R. P.; VoDlnh. T.; Winefwdner. J. D. Anal. CMm. Acta 1977. 89. 9. (7) Ryan. M. A,; Miller. R. J.; Ingle. J. D. Anal. C h m . 1978. 50, 1772. (8) Warner. I. M.; F W r t y . M. P.; Shelly. D. J. Anal. Chlm. Acta 1979. 109. 361. (9) Shelly, D. C.: Ilger. W. A,; Fogerty. M. P.; Warner. I. M. "Anex Chromatogram"; Anex Sclentllic Cwp.: W e l e y . CA. 1 9 7 9 VoI. 3, NO. 1. p 4. (10) Talmi. Y.; Baker, D. C.; JadBmc. J. R.; Saner, W. A. Anal. Chem. 1978. 50. 936A. (11) Johnson. D. W.; Gladden, J. A.; Callis. J. B.; Christian, G. D. Rev. Scl. Instrum. 1979. 50. 118. (12) Johnson. D. W.; Callis. J. 8.; Christian. 0.D. ACS Symp. Ser. 1979. No 102, 97-114. (13) Ho. C.-N.: Chrinian. G. D..; DavMson. E. R. Anal. Chem. 1078. 50.

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5 u m 6. Thrdiensional projection of EEMs from identical retention lines of chromatograms of Figures 4 and 5. corresponding to the peak Of Bap at pixel 29,16: (A, top) EEM from standard BaP chromatogram; (B,bonom) EEM from the shale 011 chromatogram. w&4calculated to he 85' and the amount Of BaP in the shale oil sample was calculated to be 38 + 2 ppm on a weight-weight basis. For comparison, the same sample was analyzed by GC-Ms using monitoring and found to have a concentration of 29 nom for BaP. The results are in satisfactoryagreement the National Bureau of Standards workers for the standard shale oil (24). ~~~

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CONCLUSION The results of this investigation and one other by Warner (9) show that the video fluorometer is a viable detector for HPLC effluents. First, it allows one to obtain detailed fluorescence fingerprints (EEMs) of materials "on-the-fly". Second, it provides sufficient sensitivity for many analytical applications. Third, i t has sufficient resolution for quantitation of specific species in very complex samples.

(14) Ho. C.-N.: Christian. 0. D.; Dsvldson. E. R. Anal. Chem. 1980, 52. 1071. (15) Hershberger, L. W.; Callis. J. B.: Christian. G. D. A d . Chem. 1979, 51. 1444. (16) Phillips, J. P.. Lyk, R. E., Jones. P. R.. Eds. "Organic E k c h o n l ~ SpecIra1 D a w ; Intersclence: New York. 1969 Vol. 5. p 656. (17) . . Berlman. 1. 8. "Handboak of Fluorescence S~ectra of Aromatlc MOleC~les";Academic FTesS: New York, 1971; 356. (18) Phillips, J. P., Nachcd, F. S..Eds. "Organic Elechonic Spectral D a w Interscience: New York, 1963: Val. 4, p 794. (19) Friedel, R. A,; Orchln, M. 'YJltravioiet Spectra of Aromatlc Compounds"; Wiley: New Yolk, 1951. Specha No. 553. (20) Wilkinson. J. E.; Strup. P. C.; Jones. P. W. In "PolynuclearAromaBc Hvdrocarbons": Jones. P. W.. Leber. P... Ed%:. Ann Arbw Sclence. I&: ~ n A& n MI,i 9 7 9 p i 217-529. (21) Warner. I. M.; Davason, E. R.; Christian. G. D. Anal. Chem. 1977; 49. 2155. (22) Steichen. J. C. J. Chromstogr. 1979. 104, 39. (23) Slavin. .. . .. W.; . Rhya Williams. A. T.; Adams. R. F. J. Chmmalogr. 1977,

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s.; &own. J. M.; chesiar. s. N.; ~uenmw,F. R.: ~iipert.L. R.; May, W. E.: Pards. R. M.; Wise. S. A. Anal. Chem. 1980. 52. 1650.

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R F C for~review Oct~ber6,1980. Accepted March 9,1981. This research was supported by the National Institutes of Health Grant GM-22311.