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, 54, 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 C C , 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, 62, 1812. Ferrell, T. L. Phys. Rev. B: Condens. Matter 1982, 25, 2930. Vo-Dinh, T.; Hiromoto. M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1984, 56, 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, 29, 129. Zahradnik, R.; Bocek, K. Collect. Czech. Chem. Commun. 1961, 26,
1733. Mecke, I?.;Freiburg, W. 2.Nektrochem. 1961, 65, 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, 27, 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, 89, 1843. Goudonnet, J. P.; Begun, G. M.; Arakawa, E. T. Chem. Phys. Lett. 1982, 92, 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
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ANALYTICAL CHEMISTRY. VOL. 58, NO. 6, MAY 1986
is capable of yielding fast, a m a t e estimates of luminescence lifetimes. The technique exploits the fact that a fluorescence decay from a pure compound is most often a well-defined system, so only two points on the decay are necessary to describe the lifetime completely. For example, a decay a r i s i i from an ideal (infinitely narrow) excitation function is described by a simple exponential Z ( t ) = k exp-'/'
where t is the time, Z ( t ) is the response at time t , T is the fluorescence lifetime, and k is a proportionality constant depending upon, among other things,concentration and source intensity (2). Equation 1implies that a plot of the natural logarithm of the intensity vs. time will yield a straight line with a slope of -1 f T . Since only two points on the decay are needed to obtain the slope, only two points are needed to obtain the lifetime, i.e., the negative reciprocal of the slope. For real excitation, where the pulse is broad with respect to the lifetime, the observed decay is a convolution of the ideal decay function with the excitation function (I,2). Hence, the measured signal can no longer be linearized by a simple log plot for short-lived decays. Although one cannot compute the lifetime from two points on the decay, two points still contain enough information 80 that the lifetime can be obtained from a suitable calibration curve as will be described below. The most useful way to combine these two decay values is by using their ratio. There are three reasons for this. First, if the two values on the decay are obtained for the same excitation puke, their ratio should be the same regardless of the intensity of the excitation. Second, the ratio should be independent of concentration. These two independencies can be justified by a further examination of eq 1 for two points in time
Z ( t z ) / I ( t l )= R = (k, exp-'S/3/(k, In
(R)= In (k,/k,)
exp-tl/r)
+ ( t ,- t , ) / ~
(2)
For an infinitely narrow excitation, k, equals k,, and the constant term, i.e., the term which is dependent upon the concentration and source intensity, drops out. With temporaUy broad excitation, k, and k, h m e complicated functions of the experimental variables, and a direct proportionality between the natural logarithm of the ratio and the reciprocal of the lifetime does not hold until the excitation pulse bas decayed away completely. However, a smooth relationship still exists between the two-point ratio and the fluorescence lifetime as long as the excitation pulse shape does not vary. As a result, a calibration curve can be constructed. The method, then, is somewhat similar to that of Rockley using a two-point nomogram with postrun data manipulation (3). T o capitalize on the concentration and source-intensity independencies mentioned above, it is necessary to acquire two values on a decay from the same excitation pulse. For this purpose, a two-channel sampling oscilloscope was used to monitor the photomultiplier anode current (Figure 1). The input to one channel was delayed with respect to the other so that they would monitor simultaneously two different points on the same decay. The output from each channel of the oscilloscope waa digitized and stored as an array of numbers in computer memory. When the measurement was completed, a ratio of the two arrays was computed, and a calibration curve was used to obtain the excited-state lifetime of the sample. It was determined that the ratio method yields an accurate estimate of the fluorescence lifetime. The ratio is indeed concentration independent, and the manner in which it is obtained eliminates most of the uncertainty due to sourceintensity fluctuations and amplifer drift. The method reduces the need for mathematical manipulation of the data, making
0
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Apparatus for twogoint decay measurements. The dashed lines are the path of the laser beam. Thin arrows indicate the flow of data. Heavy arrows represent computer control. The following symbols apply: M1 and M2, mirrors; QP, quartz plate; PD, fast photodiode: F, 3374111bandpass finer: L, focusing lens; F,, neuhal&nsity power filter; PMT. photomuiiiplier; D1. optical coaxial delay cable; i. divider: D2. 9-ns delay cable; scope. two-channel sampling oscillp scope; DAS and 8080, computer interfaces. F C m 1.
it fast. The result is the ability to obtain lifetimes in studies of transient species or of species whose concentrations are in constant flux. EXPERIMENTAL SECTION Instrumentation. The 337.1-nm line of a pulsed nitrogen laser (Princeton Applied Research Model 2100) was used as the excitation sou~ce.The pulse widths were approximately 1.5 ns fwhm. The overall instrument response was typically 3.5-4 ns fwhm. The laser was operated at a repetition rate of 10 Hz. A portion of the beam was split off with a quartz plate and used to trigger the sampling oscilloscope via a photodiode (Texas Instruments TIED 56). The main part of the beam was focused into the sample from the underside, thus eliminating much of the scatter, and providing a vertical image that matched the emission monochromator entrance slit (4). Depending on the experiment being performed, the sample cell was either a conventional 1-em quartz cuvette or a custom-built quartz flow cell with a vertical flow channel (Hellma Cells, Inc.). For flowing measurements, an autosampler (Technicon AutoAnalyzer Sampler IV)and a peristaltic pump (Gilson Minipulse 2) were used. The flow rate was 0.8 mL/min, with a 45-s sample plug and a 15-8 wash plug. Each bolus, sample or wash, was separated from the next by au air bubble (segmented flow). Emission was collected and focused onto the entrance slit of a J-Y H-20monochromator, which passed the desired wavelength range to an RCA 931A photomultiplier tube (PMT). For these experiments, a 2-nm bandwidth was used. Before reaching the oscilloscope, the signal was split evenly by a power divider. To one branch of the power divider output a 9.2-ns length of coaxial delay cable was added, while the other branch was connected directly to the oscilloscope. This enabled the monitoring of two points, separated in time by 9.2 ns, on the fluorescence decay. The oscilloscope was a Tektronix 5103N mainframe with a 5S14N dual-channel sampling head that permits simultaneous determination of two points on one trigger. Less expensive sampling systems using a single sampling head to display alternate channels on each trigger would not work for this application. The 5S14N sampling circuitry was characterized by a sampling aperture approximately 350 ps wide. Outputs from the two channels were obtained by bringing leads from test points on the Vertical Board to BNC connectors added to the front panel of the sampling head. This was done to bypass the channelswitching display circuitry, which added an unacceptable amount of noise to the normal outputs. The signals from the channel 1 and channel 2 outputs were digitized by means of a Keithley DAS series 500 interface, and the digitized data were then passed to an IBM PC-XT microcomputer. The entire data collection portion of the experiment was under computer control. Programs written m M i m f t Pascal were used to store,manipulate, and display data. The actual data collection was controlled by interrupt-drive0 assembly language routines linked to the main Pascal program. An internal timer
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
was used to generate the interrupts at the desired digitization frequency. The laser was used to trigger the sampling oscilloscope only. No attempt was made to synchronize the data collection frequency with the excitation frequency, since the sample-and-hold circuitry of the oscilloscope was capable of holding a signal with no droop until the next trigger 0.1 s later. The digitization frequency used was 16 conversions per channel per trigger, in order to minimize any possible noise in the voltage level generated by the sample-and-hold circuitry. The 16 conversions were averaged to give a single value for each trigger. This data acquisition protocol was used for static and unsegmented flowing measurmenta. For the segmented-flow measurements, which required up to 10 min, it was necessary to use an average of five triggers for each datum (still at an acquisition rate of 16 conversions/ trigger) to keep file size manageable and to avoid exceeding the computer’s memory limit. At a 10-Hz excitation frequency, and with two channels of input at 16 conversions each per pulse, only 320 conversions/s were necessary. Since the data collection was interrupt-driven at this relatively slow rate, old data could be processed and stored while simultaneously collecting new data in a foreground/background mode that was transparent to the user. A second interface, actually an 8080 microprocessor-based computer built by the Purdue University Chemistry Department Instrument Shop, was used to control the emission monochromator and could also be used to control the aperture delay on the sampling oscilloscope via its external scan input. Reagents. Water was distilled in glass and checked periodically to ensure that it did not have any significant fluorescence. Gentisic acid (2,5-dihydroxybenzoicacid), obtained from the Aldrich Chemical Co., was used without further purification. It was selected because of its absorbance maximum at 337 nm, because it fluoresces strongly, and because its lifetime of approximately 6 ns makes it a good candidate for testing the ratio method with an instrument having an impulse response of 3.5-4.0 ns. Solutions of gentisic acid were made in distilled water. Solutions of quinine bisulfate (Eastman) were prepared in 0.05 M sulfuric acid. Sufficient reagent grade NaCl was added to give lifetimes ranging from 4.2 to 18.5 ns (5). The final concentration of each of those solutions was 1.4 X lo4 M with respect to quinine. M e t h o d . For static measurements, a cuvette of sample was placed in the sample holder, and signals from two fixed points (in time) on the emission, either overlapped or 9.2 ns apart, were monitored by the sampling oscilloscope. Emission from gentisic acid and quinine bisulfate was monitored at 449 and 447 nm, respectively. These analog signals were digitized and stored in memory. Upon completion of data collection, the raw data could be stored on magnetic disk, and a ratio of the two channels could be obtained. The mean and standard deviation of the ratio file, or the raw data, could be obtained over the desired time interval through software control. For two files obtained simultaneously, the covariance and correlation coefficient could also be computed. For studies on the effecb of varying concentration or lifetime, many solutions needed to be examined. In order to increase the sample throughput, an autosampler was used. The data collected resembled bar graphs, where the height of the “bars” was proportional to the signal intensity generated by the sample plug. For reasons to be discussed below, a dc offset was substracted from these plots until their base lines centered about zero. In order to avoid the large fluctuationsin the ratio fde due to division by values close to zero, only those values above a user-selected threshold were utilized in the ratio file. For those regions of the data that were below threshold, the ratia was arbitrarily given a value of zero. The instrument response was measured by scanning the emission (in time) with one channel of the oscilloscope. A water Raman line at 382 nm was used as a fluorescence-free source of scatter. Convolutions of the instrument response with exponential decay functions were performed with a BASIC program. Lifetimes of the standards were then determined by measuring their fluorescence decays and comparing these to the model decays generated by convolution (6). RESULTS AND DISCUSSION Source N o i s e and A m p l i f i e r Drift. Figure 2a shows the results obtained from the simultaneous collection R e d u c t i o n of
1125
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2
Figure 2. Instrument response: (a) upper traces, signals from twochannel monitoring of a water Raman line with both sampling oscilloscope apertures superimposed in time; lower trace, a point-by-point ratio of the two data files. (b) An expanded portion of a.
of two channels of data overlapped in time on the peak of the water Raman signal. The noisy trace at the top of Figure 2a is actually an overlay of the two simultaneously obtained signals, one from each channel. This is more clearly seen in the expanded view of Figure 2b. The large variations in signal amplitude are due mostly to fluctuations in source intensity. Because both channels are monitoring output generated from the same laser pulse at the same point in time, they correlate extremely well, and both channels will fluctuate with source intensity to the same degree. In fact, one may measure pulse-height distributions for the laser by means of this ability to monitor source intensity fluctuations. Correlation coefficients for simultaneously collected data such as those in Figure 2 are typically 0.97-0.98, indicating a high degree of correlation. The lower trace in parts a and b of Figure 2 is a ratio of the two data files. Due to the large amount of correlation between the channels, the ratio has a much narrower distribution. Using a zero covariance (as expected for completely uncorrelated data), and the measured variances for the two data sets in Figure 2, one can determine, through a propagation-of-error calculation, that the expected standard deviation of the ratio would be 0.228 for uncorrelated data seta (7). By use of the observed covariance of 1.21 instead of zero, the expected standard deviation of the ratio is 0.0448. The actual value is 0.0498, which corresponds to an 80% reduction in noise over the uncorrelated case. This was confirmed experimentally by taking a ratio of two water Raman data files obtained sequentially instead of simultaneously. The standard deviation for the ratio of these uncorrelated files was 0.238, close to the predicted value of 0.228, and 5 times the measured value of 0.0498 for the simultaneously acquired (correlated) case. It has been determined that for fluorescence measurements that are not shot-noise limited, source intensity
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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0.972
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0.971
0.970 0.969 0.260
"Correlation coefficient computed from 2401 replicates. Gentisic acid. Ox)
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6.0
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Figure 3. Gentisic acid reproducibility studies: (a) emission data for eight repetitive autosamples of 1.4 X 10-'7M solutions-upper trace, channel 1; middle trace, channel 2; lower trace, channel 2/channei 1. (b) Expanded display of the ratio showing good stability despite
instrumental drift in the raw data. fluctuations are the primary source of noise (8). Using fluorescence decay ratios is thus a potential way of greatly reducing this major component of the noise in lifetime measurement. Decay ratios for a series of identical solutions were obtained under flowing conditions, using segmented-flow analysis, with gentisic acid as a model fluorophore. The output from each channel is now a crude bar graph where each "bar" represents the fluorescence intensity observed as the sample plug passes through the detect& (Figure 3). In this case, the apertures were not superimposed, but instead were separated in time by 9.2 ns. The 9.2-11s delay was chosen because it would give a 60% intensity difference between the two channels for this fluorophore, thus generating ratios far from unity. If the apertures are too close together in time, the signal seen at both channels will be approximately the same, and the ratio will fluctuate about unity, If they are too far apart, the aperture a t t 2 will see no signal and will be effectively monitoring only base line. The 9.2-11s delay is thus a compromise value between two extremes. The degree of correlation between the two channels, now separated in time and under flowing conditions, deteriorated. However, the ratio still showed a reduction in noise over that expected for uncorrelated data. A small amount of low-frequency drift between sample plugs was often observed, as evidenced by the changing height of the "bars" even though identical solutions were being examined. However, the ratios, averaged over each sample plug, remained essentially unchanged, and what little deviation is present is random with respect to changes in channels 1and 2. This indicates that two-point decay measurements are rugged toward drift. The unexpected deterioration in correlation for the two nonoverlapping channels of input indicated that some new source of uncorrelated noise was being introduced in the
flowing experiments with gentisic acid compared with static monitoring of a Raman signal with the apertures superimposed. In order to isolate this new source of noise, the degree of correlation of the raw data between two channels was examined under a variety of experimental conditions. The correlation coefficient between channels was used as a measure of the correlation (Table I). As shown, no significant decrease in between-channel correlation was encountered when changing from overlapping observation of the water Raman signal to overlapping observation of a fluorescent sample. The change from Raman to fluorescence was thus eliminated as the source of uncorrelated noise. Similarly, no significant decrease in correlation was observed UPQP switching from static to flowing solutions as long as the apertures were superimposed in time. This was true for both fluorescence and Raman emission. Hence, the change from a static to a flowing system was also ruled out. However, when the oscilloscope apertures were separated in time, a significant deterioration in the correlation coefficient was observed, indicating that the source of uncorrelated noise was somehow related to this separation. A detailed investigation into the source of this error is being conducted. Although not yet fully understood, it should be noted that many sources of noise that are correlated when the apertures are overlaid are uncorrelated when the apertures are separated, including signal shot noise and high-frequency Johnson noise. These could be expected to be major contributors to the deterioration in correlation observed when the apertures are separated. The correlation coefficient is not necessarily a good measure of the signal-to-noise ratio (SIN) observed in the ratio file. The S I N of the ratio depends not only on the degree of correlation but also on the magnitude of the noise in the raw data, i.e., their variances. Subjecting the oscilloscope output to a low-pass filter decreases the variances without affecting the correlation coefficient. The 5S14N sampling head has a "low-noise" option, which is essentially a low-pass filter. Although use of this option during sampling tended to superimpose an RC time constant on the data (Figure 4), it still yielded the same ratios as the unfiltered case, with even narrower distributions about the mean. For this reason, unless otherwise specified, the low-noise option was used for all subsequent experiments. Additional software filtering of the raw data was shown to yield no further improvement in the Sf N of the ratio file. Reduction of Sample Concentration Effects. Ratios were obtained for a series of eight solutions of different concentrations of gentisic acid. Although there was an increasingly wider distribution in the data as the concentration decreased, the changes in the mean of the ratio were not statistically significant (Table 11). This was true regardless of the order in which the various concentrations were sampled. Therefore, it is safe to conclude that, even under flowing conditions, the ratio of two values on the same decay is independent of concentration. The ratio can, in turn, yield a concentration-independent lifetime as shown below. It is
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986 1.0 I
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X
lo8,M
4.4 7.0 8.8 11 14 18 21 28 a
Pa
6
0.382 0.314 0.334 0.297 0.311 0.308 0.314 0.322
0.104 0.039 0.032 0.025 0.024 0.023 0.019 0.019
Values are comDuted bv using 80 redicates.
important to note at this point that concentration effects will be seen unless the base-line offset is subtracted from each channel of input before a ratio is obtained. Once the raw data have been corrected for this artifact, concentration independence is assured. The concentration independence of these ratios indicates that this technique would be an ideal detection method for liquid chromatography, since a fluorescence lifetime could be determined a t several points across the elution profile of a peak regardless of the continually changing concentration. Determination of Fluorescence Lifetimes. The final system examined with the two-point decay method consisted of eight solutions of quinine bisulfate quenched with chloride ion (5). The ratios obtained from the raw data decreased with decreasing lifetime, as expected (Figure 4). The usefulness of these ratios as an indication of fluorescence lifetime was determined as follows. First, the instrument response was obtained and convolved with synthetic exponential functions to yield a family of curves that represent normalized fluorescence decays (6). By comparison of synthetic decays
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Flgure 5. Relationship between ratios and lifetimes: (a) open circles represent ratios of two points on the Synthetic convolutes vs. the "lifetime" of the convolute. The open circles thus represent a callbratbn curve for expected results. Filled clrcles represent ratios for autosamples of chioridaquenched quinine bisulfate vs. lifetimes determined by the convolute9nbcompare method. The ratios ykid the true lifetimes to within 0.5 ns In all cases. The error bars are the standard deviatkn for 60 replicates, each an average of five excitation pulses. (b) Linearization of the data shown in part a.
to observed decays (the so-called "convolute and compare" method), lifetimes of the eight quinine solutions were determined. A ratio determined from two points 9.2 ns apart on a synthetic convolute should equal the ratio produced by a real decay from a fluorophore of the same lifetime. Therefore, a calibration curve of ratio vs. lifetime (Figure 5a, open circles) was constructed from the same convolutes used to determine the lifetimes of the quinine solutions as mentioned above. The two points chosen for the ratios were 5.5 and 14.7 ns after the peak of the instrument impulse response. The quinine ratios generated from the flowing measurements depicted in Figure 4 were plotted on the same graph (filled circles) using the previously determined lifetimes as the abscissas. Note the excellent correlation between the measured ratios and the ratios calculated separately from convolutions of the instrument response. This is true despite the fact that the convolutes were obtained from an instrument response determined with a standard 1-cm cuvette instead of the flow cell. It can be concluded that once an instrument response has been measured and a calibration curve has been constructed from the computer-generated convolutes, ratios can be measured for unknown solutions, and lifetimes can then be determined from the calibration curve with accuracy. In fact, because of the large number of replicates, the mean values of the lifetimes are well-defined and are accurate to within 0.5 ns despite the deceptively wide error bars shown in the figure. If standards of known lifetime are available, one need not even measure the instrument response and perform the convolutions in order to generate a calibration curve. Rather, ratios can be obtained for these standards, and a calibration
1128
Anal. Chem. 1986, 58, 1128-1133
curve of ratio vs. known lifetime can be constructed. Equation 2 implies that this calibration curve can be linearized. A plot of In R vs. 1 / for ~ the synthetic decays is linear with a slope of -8.09 f 0.0033 for lifetimes 1 8 ns, but significant deviation from linearity occurs at short lifetimes (Figure 5b). The nonlinearity at short lifetimes is due to the large contribution made by the trailing edge of the instrument response to the observed decay. Equation 2 implies that the slope of the line should be -At. The actual At chosen for the convolutes was 9.2 ns, compared to the observed slope of -8.09 ns. The difference can be attributed to the fact that these ratios were obtained with tl positioned over the trailing edge of the instrument response. If tl is placed beyond the last vestiges of the excitation pulse, the calibration curve will be linear and of the correct slope. For comparison purposes, the ratios obtained from the quinine bisulfate experiments are also plotted in Figure 5b. The major advantages of the ratio method over more conventional methods of obtaining fluorescence lifetimes are as follows. The ratio method does not require an extensive amount of postrun data processing onCe an appropriate calibration curve has been obtained. The technique is fast, with applications to automated analyses or other areas where a high sample throughput is desired. The ability to measure lifetimes on the fly and the concentration independence of the method lend themselves well to liquid chromatography. The ratio
technique is less sensitive to drift, and it removes most of the contribution of source intensity fluctuations to the total noise. The major disadvantages are that the presence of multiple decays cannot be detected unless the component concentrations change and that complex instrumentation is required.
ACKNOWLEDGMENT We acknowledge Harry Pardue for the use of the autosampler and the peristaltic pump.
LITERATURE CITED Demas, J. N. Excited State Lifetime Measurements ; Academic Press: New York, 1983; Chapters 1 and 2. Hieftje, G. M.; Vogelsteln, E. E. "A Linear Response Theory Approach to Time-Resolved Fluorometry". I n Modern Nuorescence Spectroscopy; Wehry, E. H., Ed.; Plenum: New York, 1981; Vol. 4, Chapter 2. Rockley, M. G. Biophys. J . 1980, 193-198. Matthews, T. G.; Lytle, F. E . Anal. Chem. 1979, 51, 583-585. Chen, R. F. Anal. Blochem. 1974, 57(2), 593-604. Lytle, F . E. Photochem. Photobloi. 1973, 17, 75-78. Bevingtbn, P. R. Data Reductlon and Error Analysis for the Physical Sclences; McGraw-Hill: New York, 1969; Vol. 1, p 62. Almelda, M. C.; Seitz, R. Appl. Spectrosc. 1985, 39(1),84-90.
Received for review July 29, 1985. Resubmitted January 14, 1986. Accepted January 14,1986. This work was supported in part by the American Cancer Society, the Indiana Elks, and the National Science Foundation, Grant CHE-8320158.
Sensitized Fluorescence Spectrometry Using Solid Organic Substrate T. Vo-Dinh* and D. A. White'
Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Thls study Investigateda slmple technlque based on sensltlred lumlnescence for detecting trace amounts of polynuclear aromatlc (PNA) compounds. Anthracene was used as the sensltizer deslgned to absorb excitation energy and funnel It to the guest analyte GOmpOundS spotted on anthracenetreated fllter paper. The data Indicate that anthracene can Improve the fluorescence slgnal of varlous PNA compounds such as perylene and benzo[a]pyrene by 2 orders of magnltude. For other compounds such as fluoranthene, no fluorescence sensltlratlon by anthracene was observed. The usefulness of the senslflred fluorescence detected by a fiberoptlcs lumlnoscope devlce for screening complex samples Is Illustrated In analyses of a coal llquld and an alr partlcular sample extract.
Sensitized luminescence by energy transfer in organic crystals has been a topic of extensive fundamental research (1-11). Although some earlier works have discussed the analytical features of sensitized luminescence (12-14) there has been, however, no current emphasis on practical applications of this photochemical process. A qualitative spot test procedure based on simple visual observation of sensitized Research Associate a t Oak Ridge N a t i o n a l Laboratory a n d T h e U n i v e r s i t y of Tennessee, Knoxville, TN.
fluorescence has been described for characterizing the content of polynuclear aromatic (PNA) compounds using naphthalene as the sensitizer (15, 16). Preliminary studies conducted in this laboratory further investigated the sensitized luminescence technique and led to the use of another efficient sensitizer, anthracene (17-19). Sensitized fluorescence based on naphthalene has also been recently reported (20). In this work we further evaluated the technique of sensitized fluorescence as a practical screening tool for detecting trace amounts of PNA compounds. Sensitized luminescence refers to the photophysical process by which the excitation energy absorbed by a donor molecule is transferred to an acceptor molecule, the luminescence of which is detected. This luminescence is not due to direct excitation of the acceptor molecules (usually present at low concentrations). Instead, acceptor molecules are intereacted with a large number of donor molecules (or sensitizers) acting as antennas collecting a higher amount of excitation energy and funneling this energy to the acceptor analytes. Under appropriate energy transfer conditions, this process could result in drastically increased luminescence of the acceptor analyte compounds. Emphasis is on anthracene used as the sensitizer. Since this technique has a' great potential as a simple tool for field use, we also evaluated the use of a portable fiber-optics luminoscope. This instrument was developed in this laboratory for monitoring skin contamination of workers and for remote sensing of pollutants in process streams at energyrelated technologies (21, 22). The results indicate that an-
0003-2700/86/0358-1128$01.50/00 1986 American Chemical Society
