Anal. Chem. 1986. 58. 1119-1123
1119
coagulation of the colloids, leading to a decreased signal and, ultimately, to formation of visible particles. For the highest enhancement silver colloid a detection limit ( S I N = 2) of 3 X lo-" M is obtained. A similar series of measurements was made for citrate colloid deposited on glass microscope slides. In this case detection limits are approximately 1pg of crystal violet, and working curves are linear (relative standard deviation 2%) to at least 1 ng.
Registry No. AgN03, 7761-88-8; NaBH,, 16940-66-2;Na3CSH507,68-04-2; Ag, 7440-22-4;crystal violet, 548-62-9.
CONCLUSIONS The citrate reduction of silver ion is superior to the borohydride method for preparation of silver colloids for surface-enhanced Raman spectroscopy. The fraction of the preparation that is coagulated to the required extent is greater, and the final colloid shows a more uniform particle size distribution. Sedimentation provides a simple and effective method for selecting the fraction of a colloid batch that is useful for SERS. Sedimentation appears to be about as effective as ultracentrifugation for isolation of the active fraction of the colloid. The colloids prepared by this technique can be used for measurement of samples in solutions or for samples spotted onto a glass slide. The latter procedure may prove appropriate for samples that are dissolved in or extracted into water-immiscible solvents. The detection limits obtained in this study, and similar results obtained for rhodamine 6G (6),place SERS among the most sensitive available analytical techniques for molecules in solution, not far behind laser-excited fluorescence. The 3-10 cm-' bandwidths of Raman lines and the qualitative information inherent in a Raman spectrum suggest that SERS may play an increasing role in trace analysis.
(5) Hildebrandt, P.; Stockburger, M. J . fhys. Chem. 1984, 8 8 , 5935-5944. (6) Creighton, J. A. In "Surface Enhanced Raman Scatterlng"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 315-337. (7) Kerker, M.; Wang, D.-S.; Chew, H.; Siiman, 0.; Bumm, L. A. In "Surface Enhanced Raman Scattering"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 109-128. (8) Ritchie, G.; Chen, C. Y. In "Surface Enhanced Raman Scattering"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 361-379. (9) Goudennet, J. P.; Begun, G. M.; Arakawa, E. T. Chem. fhys. Lett. 1982, 9 2 , 197-201. (10) Meier, M.; Wokaun, A.; Vo-Dinh, T. J . Chem. Phys. 1985, 89, 1843-1846. (11) Birke, R. L.; Lombardi, J. R. I n "Advances In Laser Spectroscopy"; Garetz, B. A., Lombardi, J. R., Eds.; Heyden and Son: London, 1982; VOI. 1, pp 143-152. (12) Creighton, J. A.; Blatchford, C. G.; Aibrecht, M. G. J . Chem. Soc., Faraday Trans. 2 1979, 75, 790-798. (13) Fabrikanos, A.; Athanassiou, S.; Lieser, K. H. 2. Naturforsch., B : Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 1963, lBB, 612-617. (14) Lee, P. C.; Meisel, D. J. J . fhys. Chem. 1982, 86, 3391-3395. (15) Tran, C. D. Anal. Chem. 1984, 5 6 , 824-026. (16) Tran, C. D. J . Chromafogr. 1984, 292, 432-438. (17) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 1 1 , 55-75.
LITERATURE CITED (1) Gantner, E.; Steinert, D.; Reinhardt, J. Anal. Chem. 1985, 5 7 , 1658-1662. Jennings, C.; Aroca, R.; Hor, A,-M.; Loutfy, R. 0.Anal. Chem. 1984, 56. 2033-2035 -___ Vo-Dinh, T.; Hiromoto, M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1984, 5 6 , 1667-1770. Siiman, 0.;Lepp, A.; Kerker, M. J . fhys. Chem. 1983, 8 7 ,
__.____
5319-5325 - - . - - -.
RECEIVED for review July 19, 1985. Accepted December 26, 1985. Financial support was provided by the National Science Foundation through Grant CHE-8317861.
Detection of Nitro Polynuclear Aromatic Compounds by Surface-Enhanced Raman Spectrometry Paul D. Enlow, Milan Buncick, Robert J. Warmack, and Tuan Vo-Dinh*
Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
The various nitro polynuclear aromatic compounds, including 1-nitropyrene, 9-nitroanthracene, 2-nitronaphthalene, and 2nitrofiuorene have been investigated by use of the surfaceenhanced Raman scattering (SERS) spectrometry technlque. Silver-coated substrates consistlng of latex spheres on glass and filter paper and prolate SIO, posts on quartz were used. The llmit of detectlon of 1-nitropyrene was found to be 0.3 ng. The SERS signals were enhanced over conventional Raman signals by a factor of 5 X 10' to 9 X 10' for 1-nitropyrene adsorbed on various SERS-active substrates. The production and practlcality of SERS-active substrates with glass, paper, and quartz supports are discussed.
Nitro polynuclear aromatic (nitro-PNA) compounds have recently received intensive interest. These compounds are often produced in atmospheric reactions in PNAs with NO, or during incomplete combustion in automotive engines. 0003-2700/86/0358-1119$01.50/0
Nitro-PNA species have been detected in a variety of products including ambient particulates (1-3), diesel exhaust particulate ( 4 , 5 ) ,and carbon black and xerographic toners (6, 7). Correlations between the presence of nitro-PNAs in environmental extracts and mutagenic activity have been reported in a number of studies (8,9). A recent study has also reported the carcinogenicity of some of the nitro-PNAsin rats (10). Due to the biological and environmental importance of these nitro-PNA compounds, a variety of analytical techniques have been developed for the identification and quantification of these species. Most of these techniques involve chromatographic methods such as gas chromatography-mass spectrometry (4), glass capillary gas chromatography (11),highperformance liquid chromatography (12), and thin-layer chromatography (13). In this paper we evaluate the detection technique based on the surface-enhanced Raman scattering (SERS) technique using silver-coated solid substrates. A number of observationshave recently indicated enhancement in the Raman scattering efficiency by factors of 103-106where 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY. VOL. 58. NO. 6. MAY 1986
a compound is adsorbed on rough metallic surfaces having submicrometer protrusions. The technique associated with this phenomenon is known as SERS spectrometry. Total enhancement is believed to he a result of a combination of several electromagnetic and chemical effects between the molecule and the surface (14). Surface plasmons, quanta associated with collective electron resonances induced by incident light on rough metallic surfaces, is one of the electromagnetic effects believed to contribute to the SERS phenomenon (15,16). Practical procedures for preparing SERS-active surfaces with well-defined roughness and structure have been previously reported (28, 29). In a previous communication, we described a practical SERS method using filter paper covered In this work with silver-coated submicrometer spheres we describe further evaluation of the SERS method and investigation of the use of other types of solid supports for the microspheres such as glass and quartz. We also investigated SERS signals obtained by using a solid SERS-active media based on etched quartz substrates having prolate SiOz posts coated with silver developed by Buncick e t al. (19). Special attention was devoted to the detection of nitro polynuclear aromatic compounds and especially 1-nitropyrene (1-NP). a potent direct-acting bacterial mutagen often found in lightduty diesel exhaust particulate extracts. The results indicate that this SERS technique provides an efficient tool to detect 1-NP. SERS enhancement factors, calculated relative to conventional Raman signal intensities, are about 106-107.
(In.
EXPERIMENTAL SECTION Instrumentation. Surface-enhanced Raman scattering measurements were conducted with a Spex (Model 1403) double-grating spectrometer equipped with a gallium arsenide photomultiplier (RCA, Model C31034) used in the single-photon counting mode, The 514.5-nm excitation source was a Spectra Physics 164 argon ion laser. Data storage and processing were performed on a SPEX Datamate DM1 processor. The light scattering from the substrate was focused by appropriate lenses onto the spectrometer entrance slit. The total light collected by the system was optimized by adjusting the sample and lens on an optical rail until the image of the entrance slit entirely filled the spectrometer grating. All spectra were recorded with slit widths of 400 pm providing a 0.2-nm spectral resolution. Photographs of the substrate surfaces were taken with a Novascan 30 scanning electron microscope. All etching of substrate surfaces was done with a Vacuum Industries, Inc., plasma etcher using a CHF, plasma. The thickness of the silver layers on the substrates was measured during depositionwith a quartz crystal thickness monitor (Kronos, Inc.. Model QM-311). Methods for SERS-Active Substrate Preparation. We have investigated experimental procedures for producing SERS-activesubstrates that a n he easily prepared and yet yield results with good sensitivity and reproducibility. Two practical approaches involve (1) coating various solid surfaces first with submicrometer spheres and then depositing a layer of silver to produce a uniformly rough metal surface and (2) etching a crystalline SiOI surface to produce submicrometer prolate posts that are also coated with silver. Preparation of Substrates of Silver-CoatedSpheres. A 100-pL volume of a suspension of 0.364-,tm latex microspheres was applied to the surface of the substrate of interest. The substrate was then placed on a spinning device (Headway Research, Inc.) and spun at 8W2ooO rpm for 20 s. The solid substrates investigated in this work were filter paper and quartz microscope slides. The silver was deposited on the sphere-coated substrate in a vacuum evaporator at a rate of 1.5-2 nm/s. The depth of silver deposited was 158-200 nm. Figure 1 is a scanning electron microscope (SEM) photograph of a microscope slide that has been coated with 0.364-pm spheres, spun at 2000 rpm, and coated with a 150-nm silver layer thickness. The boundaryof the spheres seen a t the right of Figure 1 demonstrates that the cohesion of the spheres as well as their adhesion to the glass is good.
Figure 1. Scanning electron microscope (SEM) photograph of 0.364pm latex spheres on glass coaled with a 150-nrn silver layer.
Preparation of Substrates of Siluer-CoatedProlate Posts. The preparation of siopprolate posts involves plasma etching of sioz with a silver island film as an etch mask (19). This process is similar to that used by Liao (18),except that we use a stochastic silver island film as an etch mask that produces smaller posts with higher density than Liao's method. Since fused quartz etches much more slowly than thermally deposited quartz, a 500-nm layer of SiOzwas thermally evaporated onto fused quartz at a rate of 0 . 1 4 2 nm/s. The resulting crystalline quartz was annealed to the fused quartz for 45 min at ca. 950 OC. A 5-nm silver layer was then evaporatedonto the thermal SiOzlayer, and the substrate was flash-heated for 20 s at ca. 500 O C . This heating causes the thin silver layer to head up into small globules that act as etch masks. The substrate was then etched for 3 M O min in a CHF, plasma to produce submicrometerprolate SiO, posts, which were then coated with an 80-nm silver layer at an evaporation angle normal to the substrate (30). Figure 2 shows a schematic diagram of the substrate preparation procedures (top) and a SEM photograph of a prolate post substrate made by the process described above (bottom). It is significant that these posts are much smaller than the silver-coated spheres shown in Figure 1, since the SERS signal intensity should increase with decreasing particle size. Materials and Reogents. All chemicals employed were purchased at their purest grade commercially available and were used without further purification. The 1-nitropyrene and 9-nitroanthracene were purchased from Pfaltz and Bauer. The 2nitronaphthaleneand 2-nitrofluorenewere obtained from Aldrich. The solvent used was ethanol purchased from Aager Chemical. The 0.364-rrm latex spheres used were purchased from Dow Chemical. Materials used to support the spheres were Whatman 50 hardened filter paper and polished microscope slides. The prolate pasts were supported on quartz microscope slides from ESCO Products, Inc
RESULTS AND DISCUSSIONS SERS Spectra of I-Nitropyrene on Substrates Having Silver-Coated Spheres. A single-scan SERS spectrum of 1-nitropyrene is depicted in Figure 3a. The sample consisted M solution of 1-nitropyrene in of a 3-pL spot of a 5 X ethanol applied to 0.364-pm latex spheres on glass coated with 200 nm of silver. The power of the 514.5-nm laser line used for excitation was 67 mW. For comparative purposes, a conventional 10-scan Raman spectrum of solid 1-NP in a of capillary tube is also shown in l?igure 3b. The agreement the peak positions between the SERS and solid spectrain parts a and b of ~i~~~~ 3 is fairly good as shown in Table 1. Note that the peak at 1128 cm-l in Figure 3b is due to a mercury line from fluorescent lights in the laboratory. The Peaks a t 1335 cm-l for the adsorbed compound and a t 1343 em-' for the hulk 1-NP sample appear to correspond to the symmetric NOz stretch. The peaks a t 1509 cm-' could cor-
ANALYTICAL CHEMISTRY. VOL. 58. NO. 6, MAY 1986 lbl
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DEPOSITEO S i 0 2 LAYER,
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Table I. Comparative Studies of Raman Peak Positions for 1-Nitropyrene on Various Surfaces
SILVER ISLAND,
I conventional raman peak positions," cm-'
I1 SERS peak on silver-coated spheres on glass: em-'
111 SERS peak position on prolate post surface,' em-'
1158 1191 1227 1243 1313 1343 1394 1411 1426 1445 1509 1600 1630
1162 1198 1226 1243 1301 1335 1383
1157 1196 1224 1239 1295 1332 1377
1423 1509 1598 1629
1506 1595 1622
"Column I lists the wavenumbers of peaks obtained by a conventional spectrum. *Column I1 lists the wavenumbers of peaks obtained by SERS on 0.364-pm latex spheres coated with 200 nm of silver. 'Column I11 lists the wavenumbers of peaks obtained by SERS on prolate posts coated with 80 nm of silver.
mure 2. (a)Schematic diagram of me experimental steps to produce SiO, posts on a quartz plate. (b) SEM photograph of a prolate post
substrate. id SERS
I
SPECTRUM
WAVENUMBER
I
I
I
I
I
l b ) R4MAN SPECTRUM
(ern-')
ngue 3. (a)S E W spectrum of37 ng of I-nitropyrene on 0.364-wm spheres on glass coated with a 200-nm silver layer. (b) Conventional 10-scan Raman spectrum of solid 1-nitropyrene.
respond to an NO2stretch mode in analogy to other reported Raman spectra of nitro polynuclear aromatic compounds (20). It is of interest to compare Raman spectra of 1-NP and pyrene and note that the SERS spectrum of pyrene is much simpler than that of 1-NP. Pyrene displays only two peaks (1171 and 1295 cm-') in comparison to the 11peaks exhibited by 1-NP (see Figure 3a and Table I). The pyrene peaks a t 1171 and 1295 cm-' are apparently due to vibrations that are of Raman and infrared active symmetries only in the solid
rather than in the gas or liquid state due to solid-state distortion. It is evident from Figure 3 that the SERS signal for the 3-pL spot of 5 X M 1-NP is much greater than the Raman signal for the hulk analyte. Calculations based upon signal intensities of the peaks at 1243 cm-"corresponding to the 4 vibration reported for pyrene (21) have indicated that the SERS signal for 1-NPis enhanced relative to the conventional Raman signal by a factor of 5 X lofi. The enhancement is based on the comparison between the SERS and the Raman signals, normalized to the number of 1-NP molecules estimated to he in the laser illumination molecules in bulk 1-NP. This result is in agreement with SERS enhancement factors observed in previous studies (22). A SERS spectrum for 1-NP has also been observed on a substrate consisting of 0.364-pm spheres on filter paper with a ZOO-nm silver layer. However, the SERS enhancement factor obtained by using the spheres on filter paper is an order of magnitude lower than the enhancement factor exhibited by the spheres on glass. This is noteworthy in that this high degree of signal enhancement for the silver-coated microspheres is not expected from electromagnetic calculations for silver microstructures as large as 0.364 pm because the theoretical models differ from the actual configurations. Most electromagnetic calculations treat models of isolated and solid particles on a surface, whereas the silver-coated microspheres involve a spherical metal layer covering dielectric cores in a densely packed configurations. Also it appears that the SERS effect is very specific to 1-NP and involves chemical as well as electromagnetic interaction with the substrate. This interpretation is supported by the fact that the SERS spectra of 1-NP differ considerably with other nitro polynuclear aromatic compounds in both signal intensity (Le., 1-NP signal is much more intense) and bandwidths (more narrow bandwidths for 1-NP). SERS w i t h Substrates Using Silver-Coated Prolate Posts. Figure 4 is a single-scan SERS spectrum of 9 x M I-nitropyrene on a substrate consisting of prolate posts coated with an 80-nm layer of silver. The peak positions are essentially the same as those obtained from the silver-coated sphere substrates as shown in Table I. The peak widths and shapes obtained on the prolate posts are also quite similar to those obtained with surfaces using spheres. The peak at 1239 cm-l on the prolate posts coated with 80 nm of silver exhibits a signal enhancement of a factor of 1.8
1122
ANALYTICAL CHEMISTRY, VOL.
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58, NO. 6,
MAY
1986
r7-7
E
h c
I
3
1 - NITROPYRENE
20,000
a
8 C
+ .e a v
t
L
II
z w [r
W
m
-
0
i
402[e/e
I400
4 700
Figure 4. SERS spectrum of 74 ng of 1-nitropyrene on SiO, prolate posts on quartz coated with an 80-nm silver layer. 4000
600
l ' il.
3500
I
4200
900
40'
1 (o-~ CONCENTRATION ( M )
40,000
1100
WAVENUMBER
1100
i
I-
WAVENUMBER (crn-')
Figure 6. Diagram of the centration.
1700
(cm-')
Figure 5. (a) SERS spectrum of 56 ng of 9-nitroanthracene on 0.364-pm spheres on glass coated with a 200-nm sllver layer. (b) SERS spectrum of 47 ng of 2-nltronaphthalene on 0.364-pm spheres on glass coated wlth a 200-nm silver layer. (c) SERS spectrum of 59 ng of 2-nitrofluorene on 0.364-pm spheres on glass coated with a 200-nm sliver layer. over the signal obtained from the spheres coated with 200 nm of silver using the 514.5-nm laser line. It appears that the conditions for obtaining strong SERS signals (e.g., silver thickness and excitation wavelength) are different for the prolate posts and spheres, and it is likely that the enhancement conditions for the prolate posts have not yet been optimized. Previous theoretical calculations for the electromagnetic effect indicated that the size of particles is an important parameter affecting SERS intensities. The optimal particle sizes for observing maximum SERS signals are determined by several factors, such as the magnitude of the induced electric field at the particle surface, the amount of Raman-shifted light scattered into the spectrometer, and two energy loss mechanisms,viz., surface scattering of conduction electrons, which becomes important for small particles, and radiation damping, which occurs for large particles (23-25). As previously discussed the chemical effect probably also contributes to the enhanced mechanism. SERS Studies of Other Nitropolynuclear Aromatic Compounds. 9-Nitroanthracene. Figure 5a is a the SERS M 9-nitroanthracene on spectrum of a 3-pL spot of 9 X 0.364-pm latex spheres coated with 200 nm of silver. Peaks appear at 690,979,1054, and 1167 cm-'. No peaks were seen in the 1200-1700-~m~~ range where nitropyrene displays very strong absorptions. The SERS peaks for 9-nitroanthracene are fairly ill-defined and broad compared to typical SERS M signals for 1-nitropyrene. A t a concentration of 9 X
SERS
intensity of 1-nltropyrene vs. con-
the peak at 690 cm-' in Figure 3 is no longer seen and the shoulder of the 979 cm-' signal is resolved into a peak at 1008 cm-l. 2-Nitronaphthalene. Figure 5b is a SERS spectrum of 1 X M 2-nitronaphthalene under the same conditions as the 9-nitroanthracenediscussed above. The only visible peaks appear at 974 and 997 cm-l. The signal to base line noise ratios for 2-nitronaphthalene and 9-nitroanthracene are approximately equal at the M level ( S I N = 3.1). 2-Nitrofluorene. The SERS spectrum of 1 X M 2nitrofluorene (shown in Figure 5c) exhibited peaks only at 1174,1335,1603, and 1717 cm-'. Although the signal to base line noise ratio is very good for these four peaks, they are extremely broad relative to 3-nitropyrene. Analytical Figures of Merit. The SERS response for 1-nitropyrene is linear with concentration within the limits of 10-6-10-4 M on 0.364-pm latex spheres coated with 200 nm of silver and using 514.5-nm excitation at a power of 70 mW. Since the nitropyrene appeared to be thermally degraded slightly by the laser, all measurementswere taken immediately after the substrates had been exposed to the laser beam for the same amount of time. In spite of the degradation of nitropyrene under laser excitation, it is noteworthy that the SERS technique is sensitive enough to detect this compound at the subnanogram level. The SERS intensity of the 1243 cm-l 1-nitropyrene signal as a function of concentration for a 3-pL volume of solution spotted on silver-coated spheres is shown in Figure 6. If solutions more concentrated than M are used the plot is no longer linear, since so many layers of analyte molecules are present on the surface that conventional Raman signals are also observed from analyte molecules not adjacent to the substrate. This observation is consistent with results obtained by using other surface emission techniques where adsorption of the analyte molecules to the surface is required (26). It is significant that the SERS reM, since that is the region of sponse is linear below analytical interest. We have easily detected a 3-pL spot of lo4 M 1-nitropyrene. This represents only 0.7 ng of material. The limit of detection for 1-nitropyrene in a sample spot is
Anal. Chem. 1986, 58,1123-1128
0.3 ng if we use a five-scan spectrum to obtain a signal-to-noise ratio of 2. Since the sample area illuminated by the laser beam is only 1/100 of the total sample spot, the actual limit of detections for 1-NP is only 3 pg.
CONCLUSIONS This study shows that a number of nitro-PNA compounds can be detected by the SERS technique using solid substrates covered with silver-coated spheres and surfaces having SiOz prolate posts coated with silver. The results of this work also show that the SERS technique is particularly efficient and specific for 1-nitropyrene. The feature is of great environmental interest, since this compound is commonly found in indoor air and an occupational environment and in diesel engine emission; 1-NP has also been found to exhibit strong mutagenic activity. Techniques to further develop the new SERS technique for the direct characteristion of complex environmental samples are under study. In this research phase, we have investigated the use of two practical procedures for preparing surfaces having known and well-defined roughness and structure. The first method, involving deposition of submicron spheres on solid surfaces, has been found to be very convenient and simple. This technique is also quite practical, since measurements conducted with the scanning electron microscope have shown that these commercial spheres are very uniform in size and in shape. The surface roughness can also be easily controlled by selection of the sphere size. And last but not least, the materials involved in the substrate preparation of silver-coated spheres are inexpensive due to the small amount of spheres required and the low cost of the substrates such as filter paper, glass, and quartz plates. Other types of substrates that were found to be SERS active include the silver-coated prolate posts on etched quartz plates. We have recently evaluated the utility of the etched quartz and substrates by producing silver particles on top of the quartz posts by non-normal-angle evaporation (27). Registry No. SOz,7631-86-9;Ag, 7440-22-4;1-nitropyrene, 5522-43-0; 9-nitroanthracene, 602-60-8; 2-nitronaphthalene, 581-89-5;2-nitrofluorene, 607-57-8.
LITERATURE CITED (1) Pitts, J. N., Jr.; Canwenberghe, K. A. K.; Grosjern, D.; Schmidt, J. P.; Fitz. D. R.; Belser, W. L., Jr.; Knudson, J. B.; Hynds, P. M. Sclence (Washingon, D . C . ) 1978, 202, 515.
1123
Wang, C. Y.; Lee. M. S.; King, C. M.; Warner, P. 0. Chemosphere 1980, 9 , 83. Jager, J. J. Chromatogr. 1978, 152, 575. Newton, D. L.; Erickson, M. D.; Tomer, K. B.; Pellizzari, E. D.: Gentry, P.; Zweidinger, R. B. Environ. Sci. Technol. 1982, 76. 206. Riley, T.; Prater, T.; Schuetzle, D.; Harvey, T. M.; Hunt, D. Anal. Chem. 1982, 5 4 , 265. Fitch, W. L.; Smlth, D. H. Envlron. Sci. Technol. 1979, 13, 341. Rosenkraz, H. S.; McCoy, G. C.; Sanders, D. R.; Butler, M.; Kiriazides, D. K.; Mermeistein, R. Science (Washington, D . C . ) 1980, 209, 1039. Henderson, T. R.; Royer, R. E.; Clark, C. R.; Harvey, T. M.;Hunt, D. F. J. Appl. Toxlcol. 1982, 2 , 231. Henderson, T. R.; Li, A. P.; Royer, R. E.; Clark, C. R. Envlron. Mutat. 1981, 3 , 211. Ohgaki, H.; Matsukura, N.; Morino, K.; Kawachi, T.; Sugimura, T.; Morita, K.; Tokiwa, H.; Hirota, T. Cancer Lett. 1982, 15, 1. Randalill, T.; Kueseth, K.; Becher, 0. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1982, 5 , 19. Rappaport, S. M.; Jiu, 2. L.; Yu, X. B. J. Chromatogr. 1982, 240, 145. Jager, J. J. Chromatogr. 1978, 752, 575. Chang, R. K., Furtak, T. E., Eds. “Surface Enhanced Raman Scattering”; Plenum: New York, 1982. Philpot. M. R. J. Chem. Phys. 1975, 6 2 , 1812. Ferrell, T. L. Phys. Rev. B: Condens. Matter 1982, 2 5 , 2930. Vo-Dinh, T.; Hiromoto. M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1984, 5 6 , 1667. Liao, P. F. “Surface Enhanced Raman Scattering”; Chang, K. K., Furtak, T. E., Eds.; Plenum: New York, 1982; p 379. Buncick, M. C.; Warmack, R. J.; Little, J. W.; Farrell, T. L. Bull. Am. Phys. SOC. 1984, 2 9 , 129. Zahradnik, R.; Bocek, K. Collect. Czech. Chem. Commun. 1961, 2 6 , 1733. Mecke, I?.;Freiburg, W. 2.Nektrochem. 1961, 6 5 , 327. Van Duyne, R. P. “Chemical and Biochemical Applications of Lasers”; Moore, C. B. Ed.; Academic Press: New York, Vol. 4, Chapter 4. Wokaun, A.; Gordon, J. P.; Liao, P. F. Phys. Rev. Lett. 1982, 48, 957. Barber, P. W.; Chang, R. K.; Massoudi, H. Phys. Rev. B: Condens lclatter 1983, 2 7 , 7251. Meier, M.; Wokaun, A. Opt. Lett. 1983, 8 , 581. Vo-Dinh, T. “Room Temperature Phosphorimetry for Chemical Analysis”; Wiley: New York, 1984. Meier, M.; Wokaun, A.; Vo-Dinh, T. J. Chem. Phys. 1985, 8 9 , 1843. Goudonnet, J. P.; Begun, G. M.; Arakawa, E. T. Chem. Phys. Lett. 1982, 9 2 , 197. Goudonnet, J. P.; Inagaki. T.; Warmack, R. J.; Buncick, M. C.; Arakawa, E. T. Chem. Phys., in press. Buncick, M. C. Ph.D. Thesis, to be submitted to the Physics Department, University of Tennessee, Knoxville, TN.
RECEIVED for review February 28, 1985. Resubmitted November 22,1985. Accepted November 22,1985. This research is sponsored jointly by the Department of the Army under Interagency Agreement DOE 40-1294-82 and ARMY 33111450 and the Office of Health and Environmental Research, US. Department of Energy, under Contract DE-AC05840R21400 with Martin Marietta Energy Systems, Inc.
On-the-Fly Determination of Fluorescence Lifetimes from Two-Point Decay Measurements David J. Desilets, Joel T. Coburn, Douglas A. Lantrip, Peter T. Kissinger, and Fred E. Lytle*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
A slmpllfled, rapid technlque for measuring fluorescence lifetimes Is presented. The method uses a two-channel sampling oscllloscope to acquire slmuitaneously two values on a fluorescence decay generated by pulsed laser excitation. I t is shown that a ratlo of these values Is reiatlveiy insensltlve to fluctuations In source Intensity compared to conventlonal measurements, Is concentration independent, and is mathematically related to the fluorescence lifetlme. These propertles permit rapid and accurate estimation of lifetimes wlthoul extensive signal averaglng. Applications to fiowlng systems are also discussed.
Fluorescence lifetime measurements play an important role in experimental physical chemistry, biochemistry, and analytical chemistry. Yet, lifetimes can be difficult to measure, frequently requiring extensive signal averaging and/or sophisticated mathematical treatment of the data ( I ) . Often, the analyst may feel that the effort spent in acquiring lifetimes is not justified by the amount of information obtained. In addition, many experiments, such as liquid chromatography or the study of transient molecular species, would benefit from an ability to measure lifetimes in real time. These arguments led to the development of a method that 0 1988 American Chemical Society
