Rapid and Accurate Determination of Stern-Volmer Quenching Constants

1000 Volume 53, Number 8, 1999 APPLIED SPECTROSCOPY0003-7028 / 99 / 5308-1000$2.00 / 0

q 1999 Society for Applied Spectroscopy

Rapid and Accurate Determination of Stern± VolmerQuenching Constants

JOHN V. GOODPASTER and VICTORIA L. M CGUFFIN*Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322

In this work, a novel system has been designed, characterized , andvalidated for the determination of ¯ uorescence quenching constants.

Capillary ¯ ow injection methods are used to automate the prepa-

ration and mixing of the ¯ uorophore and quencher solutions. Be-cause of the small diameter of the capillary (75± 200 m m), ¯ uores-

cence measurements can be made without corrections for primary

and secondary absorbance effects. The ¯ uorescence spectrometer isequipped with a charge-coupled device (CCD) that has a detection

limit of 3.0 3 10 2 9 M (2.3 ppb) and a linear dynamic range of 105

for integration times of 0.01± 10 s. This spectrometer has a 300 nmspectral range with 1 nm resolution, allowing the ¯ uorescence

quenching constants to be calculated at single wavelengths or over

integrated wavelength ranges. This system was validated by com-parison to traditional methods for the determination of Stern± Vol-

mer constants for alternant and nonalternant polycyclic aromatic

hydrocarbons with nitromethane and triethylamine.

Index Headings: Fluorescence; Fluorescence quenching; Instrumen-tation; Polycyclic aromatic hydrocarbons.

INTRODUCTION

Quenching is usually considered to be a detrimentalphenomenon in ¯ uorescence spectroscopy. However, thedeliberate quenching of ¯ uorescence can be used to an-alytical advantage if applied in a judicious and carefullycontrolled manner. This method can provide valuablephotophysical and photochemical information about anindividual ¯ uorophore that can be used for its classi® -cation or identi® cation. In addition, when combined witha separation method, it can be used to simplify the qual-itative and quantitative analysis of complex samples byselective discrimination against interfering components.

Fluorescence quenching can be de® ned as any processthat decreases the observed ¯ uorescence power from asample.1 These processes include trivial phenomena suchas primary and secondary absorption as well as light scat-tering due to changes in the refractive index or turbidityof a solution. Of more use in analytical applications isdynamic quenching, which involves the collision andsubsequent formation of a transient complex between anexcited-state ¯ uorophore and a ground-state quencher.2,3

Because this complex must be formed during the excited-state lifetime of the ¯ uorophore, dynamic or collisionalquenching is diffusion controlled. The excited-state com-plex dissociates upon radiative or nonradiative deactiva-tion, leaving both the ¯ uorophore and quencher in theground state. This form of quenching is described by theStern±Volmer equation.2,3

F f 8 / F f ø P f 8 /Pf 5 1 1 kd t f 8 Cq 5 1 1 KdC q (1)

where F f 8 and F f are the quantum ef® ciencies of the ¯ uo-

Received 11 January 1999; accepted 10 March 1999.* Author to whom correspondence should be sent.

rophore in the absence and presence of the quencher, re-spectively. These quantum ef® ciencies are directly pro-portional to the measured ¯ uorescence powers (P f 8 andPf, respectively) provided that the source power, ef® cien-cy of optical irradiation and collection, and ¯ uorophoreabsorbance remain constant. Finally, t f 8 is the ¯ uores-cence lifetime of the unquenched ¯ uorophore, C q is themolar concentration of the quencher, kd is the bimolecularrate constant for dynamic quenching, and K d is the Stern±Volmer constant.

In static quenching, a complex is formed between aground-state ¯ uorophore and a ground-state quencher.2,3

This ground-state complex is stable with spectral prop-erties that will necessarily differ from those of the un-complexed ¯ uorophore. For static quenching, a linear re-lationship is observed between the quantum ef® ciency or¯ uorescence power ratio and the quencher concentration:2,3

F f 8 / F f ø Pf 8 /P f 5 1 1 K sC q (2)

where Ks is the equilibrium formation constant of the¯ uorophore±quencher complex, and all other variablesare as de® ned previously.

When only a single quenching mechanism is present,a Stern±Volmer plot of (P f 8 /P f) vs. Cq should yield astraight line with a slope equal to the quenching constantand an intercept of unity. However, if the quencher ab-sorbs at the excitation or emission wavelength, there willbe a positive deviation from linearity in the Stern±Volmerplot. This absorbance effect can be corrected through amodi® ed form of the Stern±Volmer equation:4,5

o(P /P ){[1 2 exp( 2 2.3Î bC )]/2.3Î bC }f f q,ex q q,ex q

3 {[1 2 exp( 2 2.3Î bC )]/2.3Î C }q,em q q,em q

5 1 1 K C (3)d q

where Îq,ex and Îq,em are the molar absorptivity of thequencher at the excitation and emission wavelengths, re-spectively; b is the pathlength of the cell; and C q is theconcentration of the quencher.

An alternative method that is not affected by staticquenching or by absorbance effects relies on the fact thatdynamic quenching is a nonradiative process that de-creases the observed ¯ uorescence lifetime. Hence, the at-tenuation of lifetime as a function of quencher concen-tration can also be used to determine quenching con-stants. In this approach, the ratio of unquenched toquenched ¯ uorescence lifetime ( t f 8 / t f) is calculated as afunction of the quencher concentration (Cq). This ap-proach can quantify the amount of dynamic quenchingdirectly, even in the presence of absorbance effects orground-state complexation.6,7

To date, the development of quenching as a tool for

APPLIED SPECTROSCOPY 1001

FIG. 1. Schematic diagram of the instrumentation for ¯ uorescencequenching studies. I, injection valve; T, mixing tee; F, ® lter; L, lens;CCD , charge-coupled device; PMT, photomultiplier tube.

chemical analysis has been relatively limited. This situ-ation is largely due to the methods for determining Stern±Volmer constants, which remain both time and labor in-tensive. In a typical study, a series of solutions with con-stant ¯ uorophore concentration and varying quencherconcentration is prepared, and the attenuation of the ¯ uo-rescence power (or lifetime) at a single wavelength ismeasured. While this technique is effective, it is alsoslow, tedious, and prone to experimental error. Hence,such an approach is not well suited for the evaluation oflarge numbers of ¯ uorophores and quenchers.

Recently, some novel approaches to quenching exper-iments have been proposed. For example, Desilets et al.used a gradient pump from a liquid chromatograph tolinearly increase the concentration of quencher in a ¯ ow-ing stream of a ¯ uorophore, measuring the decrease in¯ uorescence power at a single wavelength. 8 This methodrequired little solution preparation, allowed for greaterprecision in solution concentrations, acquired a largenumber of ¯ uorescence power ratios in each quenchingexperiment, and increased the overall speed of thequenching measurements. However, this method was lim-ited to the analysis of one ¯ uorophore±quencher pair ata single wavelength, and acquired only one ¯ uorescencepower ratio at each quencher concentration. Moreover, itwas reliant on the precision of the linear gradient programto calculate the quencher concentration and did not makecorrections for absorbance effects.

A different approach was taken by Chen et al., whomeasured quenching constants by using a laser for exci-tation in a capillary ¯ ow cell.4 The short pathlength ofthe capillary minimized absorption by the quencher atboth the excitation and emission wavelengths. However,this system required premixed solutions of the ¯ uoro-phore and quencher and was also limited to a singlewavelength. A ® nal approach for determining thequenching constants of a number of ¯ uorophores in-volved separating a complex mixture by using capillaryliquid chromatography with laser-induced ¯ uorescencedetection.4,5 The observed peak heights both with andwithout quencher dissolved in the mobile phase wereused to calculate conditional quenching constants at thedetected wavelength. While this approach obviated theneed for purifying the ¯ uorophores as well as allowedfor the analysis of complex samples, it required the useof lengthy chromatographic separations. Furthermore, itwas limited to a single wavelength for all ¯ uorophores,and only one ¯ uorescence power ratio was used to cal-culate each quenching constant.4,5

In the present study, novel instrumentation for evalu-ating Stern±Volmer constants has been developed thatcombines the advances of previous studies while over-coming many of their limitations. In this system, solutionpreparation is automated through the use of ¯ ow injectiontechniques. This approach allows the measurement ofquenching constants for many ¯ uorophores in a singleexperiment with precise control over the quencher con-centration. Moreover, a large number of replicate mea-surements can be made at each quencher concentration,leading to greater accuracy and precision. A capillary¯ ow cell is used to eliminate absorbance effects, and acharge-coupled device (CCD) detector allows for the cal-culation of Stern±Volmer constants on the basis of either

single wavelengths or integrated emission spectra. Theperformance of this system is evaluated and the accuracyand precision are compared with traditional methods forthe determination of Stern±Volmer constants.

EXPERIMENTAL

Reagents. Quinine sulfate (Baker) is used as received.Stock solutions of quinine sulfate are prepared in 0.05 MH2SO4 in deionized, distilled water (Corning GlassWorks, Model MP-3A). Pyrene (MCB), ¯ uoranthene (Al-drich), benzo(a)pyrene (Aldrich), and benzo(b)¯ uoranthene(Aldrich) are puri® ed by vacuum sublimation. Reagent-grade nitromethane (EM Science) and triethylamine(Spectrum) are used as received. Stock solutions of thepolycyclic aromatic hydrocarbons and quenchers are pre-pared in high-purity, spectroscopic-grade methanol (Bax-ter Healthcare, Burdick and Jackson Division).

Flow Injection System. In order to allow for the rapidevaluation of quenching constants, a ¯ ow injection ap-paratus that combines solutions of ¯ uorophores andquenchers has been developed (Fig. 1). The apparatusconsists of a dual syringe pump (Brownlee Labs, ModelG) that is programmed to deliver a step gradient of thequencher in a suitable solvent at a ¯ ow rate of 35m L/min. A second syringe pump (Applied Biosystems,Model 140) delivers the solvent at 35 m L/min to an in-jector with a 75 m L loop (Valco Instruments, ModelEC6W), which is used to introduce discrete zones of the¯ uorophore. The solutions of the quencher and ¯ uorop-hore are combined with a mixing tee (Upchurch Scien-ti ® c, Model P-727) in fused-silica capillary tubing (200m m i.d., 375 m m o.d., Polymicro Technologies). Aftermixing, the ¯ uorophore±quencher solution is directed toa ¯ uorescence detector (vide infra) followed by a UV-visible absorbance detector (Jasco, Model UVIDEC-100-V).

Fluorescence System. A ray tracing program (StellarSoftware, BEAM4 Optical Ray Tracer) has been used toaid in the design and optimization of the spectroscopicsystem. In this system, a helium±cadmium laser (MellesGriot, Model 3074-40M, 325 nm, 40 mW) is utilized asthe excitation source, and all optics are enclosed withinan aluminum housing to minimize light leaks and other

1002 Volume 53, Number 8, 1999

sources of stray light. The ¯ ow cell is constructed offused-silica capillary tubing (75±200 m m i.d., 375 m mo.d., Polymicro Technologies). Fluorescence emission iscollected and collimated by using either two f /2 fused-silica lenses (Melles Griot, Model 01 LQP 001/066) or asingle f /1 concave mirror (Oriel, Model 44351). Scat-tered light originating from the laser is attenuated bypassing the collimated emission through either a long-pass glass ® lter (Andover Corporation , ModelANDV4922) or a liquid ® lter consisting of an aqueoussolution of 2 M KNO3 contained in a 1 cm cylindricalfused-silica cell (Perfector Scienti® c). The ® ltered emis-sion is refocused with an f /6 fused-silica lens (MellesGriot, Model 01 LQP 013/066) onto the entrance slit ofa 0.34 m Czerny±Turner monochromator (InstrumentsSA, Model 340E, 300 grooves/mm grating), which isequipped with a photomultiplier tube (PMT) (Hamama-tsu, Model R-106) and a charge-coupled device detector[Instruments SA, Model (A)TECCD-2000 3 800-7]. TheCCD detector is thermoelectrically cooled and is main-tained at a temperature of 2 40 8 C. Instrument control anddata acquisition are provided by commercially availableelectronic interfaces (Instruments SA, Model DS1010and Model CCD 2000) and the associated software (In-struments SA, Spectramax for Windows, Version 2.76).

For purposes of validation and performance evaluation,the system described above is compared with a commer-cially available scanning ¯ uorimeter (Hitachi, Model F-4500) with a 1 cm cuvette cell. This ¯ uorimeter containsa 150 W xenon lamp, excitation and emission mono-chromators (900 grooves/mm grating, 2.5 nm bandpass),and a PMT detector, which is operated at 700 V.

Calculations. For the characterization studies, the ¯ uo-rescence spectra of quinine sulfate are integrated from370 to 540 nm, and the integrated power is plotted vs.the variable of interest. For calculations of the signal-to-noise (S/N) ratio, the signal is de® ned as the height ofthe quinine sulfate emission spectrum at its wavelengthof maximum intensity (450 nm). The noise is calculatedby ® rst smoothing the spectrum by using a Savitsky±Golay routine9 with a second-order polynomial and 213/n points, where n is the number of pixels that are hori-zontally binned on the CCD chip. The smoothed spec-trum is then subtracted from the original spectrum andthe standard deviation of the remaining noise from 445to 455 nm is calculated. The logarithm of the calculatedS/N ratio is then plotted vs. the logarithm of the signal.

Stern±Volmer plots are prepared by calculating the¯ uorescence power at a single wavelength or an inte-grated range of wavelengths as a function of the quencherconcentration. The wavelength range for each ¯ uoro-phore is as follows: pyrene (350±500 nm), ¯ uoranthene(380±560 nm), benzo(a)pyrene (370±510 nm), and ben-zo(b)¯ uoranthene (373±560 nm). The ratio of the ¯ uo-rescence power in the absence and presence of quencheris then graphed as a function of the quencher concentra-tion. The slope of this graph is determined by linear re-gression in order to obtain the Stern±Volmer constant ac-cording to Eq. 1.

RESULTS AND DISCUSSION

Fluorescence System Characterization. Ray tracingsimulations have been used to evaluate the performance

of various con® gurations of the optical components.These studies have identi® ed a number of factors as im-portant to our optical design. For example, simulationsdemonstrate that a properly oriented plano-convex lensyields higher throughput than a biconvex lens. Moreover,two f /2 objective lenses in series provide sharper focusthan a single f /1 collimating lens. Finally, a concave con-denser mirror decreases spherical and chromatic aberra-tions as well as increases the overall throughput relativeto the lens systems.

These simulation results provided the impetus for ex-perimental studies of the optical components and howthey affect the system performance. Figures 2A and 2Bshow the contrasting performance of four different con-® gurations of the collection optics by using a 3.0 3 10 2 7

M solution of quinine sulfate as a means of comparison.These data indicate that using a concave mirror as thecondensing objective with a glass ® lter to remove laserscatter results in the poorest performance. Although over-all throughput is high, as predicted by the ray tracingsimulations, the resulting S/N ratio is lower than that forthe other optical con® gurations. In addition, the longestattainable integration time is lower than that for the othercon® gurations. This integration time is ultimately limitedby the dark charge from the CCD as well as by back-ground from laser scatter and other stray light, which ishighest for this optical con® guration. Using two plano-convex lenses as the condensing objective with a glass® lter yields similar throughput but higher S/N ratio. Per-formance is further enhanced by replacing the glass ® lterwith a liquid ® lter composed of aqueous 2 M KNO 3.When this ® lter is used, the S/N ratio is higher than thatfor other optical con® gurations for any given signal level,and the longest attainable integration time is increased asa result of the lower background. However, because ofthe smaller aperture of the liquid ® lter, longer integrationtimes are necessary to achieve the same signal levels asthe glass ® lter. Finally, it has been reported 10 that scat-tered laser light can be effectively reduced by immersingthe fused-silica capillary cell in a refractive-index match-ing ¯ uid, such as carbon tetrachloride. In the presentstudy, however, there is no signi® cant improvement inS/N ratio because of the larger ¯ uorescence backgroundoriginating from the cell and ¯ uid.

With the best optical con® guration determined fromthese studies, additional methods of S/N enhancementhave been explored such as varying the laser power, en-trance slit width, electronic binning of the CCD pixels,and integration time. In the ® rst study, the power of theHeCd laser has been varied from 0.40 to 35 mW. The¯ uorescence of a standard solution of quinine sulfate in-creases linearly with laser power, where the square of thecorrelation coef® cient (r 2) is 0.999 and the number ofmeasurements (n) is 11. A logarithmic graph of the S/Nratio vs. signal is linear (r 2 5 0.982, n 5 11) with a slope(m) of 0.54 6 0.02. Next, the entrance slit width of themonochromator has been varied from 100 to 1000 m m,corresponding to a spectral bandpass of 1±10 nm. The¯ uorescence of quinine sulfate increases linearly with slitwidth (r 2 5 0.997, n 5 6), and the logarithmic graph ofthe S/N ratio vs. signal is also linear (m 5 0.42 6 0.05,r2 5 0.937, n 5 6). Next, the CCD pixels have beenhorizontally binned in groups ranging from 1 to 10, cor-


FIG. 2. Effect of optical con® guration on the signal (A) and S/N ratio(B) of a 3.0 3 10 2 7 M solution of quinine sulfate at various integrationtimes. Data correspond to the following optical con® gurations: concavemirror, glass ® lter, and capillary cell (V); two plano-convex lenses, glass® lter, and capillary cell (M); two plano-convex lenses, liquid ® lter, andcapillary cell ( n ); and two plano-convex lenses, liquid ® lter, and cap-illary cell contained in refractive-index matching ¯ uid ( , ). The dashedline in B represents shot-noise-limited conditions with slope (m) of 0.5.Experimental conditions: HeCd laser at 325 nm and 40 mW, 75 m mcapillary cell, 0.34 m monochromator with 300 grooves/mm grating and1 nm bandpass, CCD detector thermoelectrically cooled at 2 40 8 C.

responding to a spectral bandpass of 0.15±1.5 nm. The¯ uorescence of quinine sulfate increases linearly with theextent of binning (r2 5 1.000, n 5 6), and the logarithmicgraph of the S/N ratio vs. signal is also linear (m 5 0.606 0.07, r 2 5 0.948, n 5 6). Finally, the integration timehas been varied from 0.01 to 30 s. The ¯ uorescence ofquinine sulfate increases linearly with integration time (r 2

5 1.000, n 5 6), and the logarithmic graph of the S/Nratio vs. signal is also linear (m 5 0.59 6 0.03, r 2 50.988, n 5 6). For each of these variables, the slope ofthe logarithmic graphs indicates that the noise is propor-tional to the square root of the ¯ uorescence signal. Thisbehavior is characteristic of the quantized nature of ¯ uo-rescence emission, and implies that the spectroscopic sys-tem is quantum or shot noise limited.11±13 Although anyof these variables can be used to enhance the S/N ratioin a similar manner, they may have other detrimental ef-fects on the system performance. For example, an in-crease in these variables will increase the background,thereby decreasing the available dynamic range of theanalog-to-digital (A/D) converter and the resulting linearrange of the ¯ uorescence measurements and Stern±Vol-mer plots. Furthermore, an increase in either the slit widthor the extent of binning will decrease the spectral reso-lution, and an increase in laser power may cause photo-chemical reaction or degradation.14 The most simple andleast detrimental approach to increase the S/N ratio is bymeans of the integration time, although problems canarise at long integration times (vide infra).

The performance of the ¯ uorescence spectrometer hasbeen characterized by means of the calibration curves forquinine sulfate shown in Fig. 3A. Overall, the linearityof the calibration curves is quite good (r 2 50.994 2 1.000, n 5 3±6). Deviations from linearity nearthe limit of detection arise from blank noise in the signal,which contributes a larger proportion of the integrated¯ uorescence power at low concentrations of quinine sul-fate. With an integration time of 10 s, the detection limitfor quinine sulfate is 3.0 3 10 2 9 M (2.3 ppb) at an S/Nratio of 3.4 (n 5 3).11 At high concentrations, there is nocurvature or other evidence of absorbance effects becauseof the short pathlength of the capillary ¯ ow cell. Hence,the limit of linearity is determined solely by the shortestintegration time of the CCD detector (0.01 s). At thisintegration time, the highest concentration that does notexceed the dynamic range of the A/D converter (216) is3.0 3 10 2 4 M (230 ppm). Thus, the linear range of thecalibration curves for quinine sulfate is 1.0 3 105.

The effect of quinine sulfate concentration on the S/Nratio is shown in Fig. 3B. This logarithmic graph showstwo distinct regions of behavior. The ® rst occurs at signallevels above 103 counts, where the slopes range from m5 0.50±0.80 (r2 5 0.993±1.000, n 5 2±3) and generallyincrease with integration time. This slope implies that thenoise is proportional to the square root of the signal andthat the system is exhibiting shot-noise-limited behavior,as described above. At lower signal levels, however, theS/N ratio becomes linearly dependent on the signal, withslopes ranging from m 5 0.88±1.05 (r 2 5 0.981±1.000,n 5 2±3). This slope implies that the noise is independentof the signal and that the system is exhibiting blank-noise-limited behavior. Additional studies have indicatedthat this noise is predominantly due to dark and readnoise of the CCD, with a smaller component due to ¯ uc-tuations in the background arising from laser scatter andother stray light. The dark current is largely independentof integration time up to 1 s (e.g., 1303 6 14 counts),but increases signi® cantly thereafter. Thus, the regionwith blank-noise-limited behavior appears at higher sig-nal levels with increasing integration time in Fig. 3B.


FIG. 3. Effect of quinine sulfate concentration on the signal (A) andS/N ratio (B) using integration times of 0.01 s ( , ), 0.1 s ( n ), 1 s (M),and 10 s (V). Other experimental conditions as described in Fig. 2.

Overall, these studies have indicated that optimum per-formance of the system can be achieved by ® rst reducingthe background to its fundamental limit. This limit is de-termined by the dark and read noise of the CCD.15 Oncethis has been achieved, the integration time can then beadjusted to control the signal level and maximize theS/N ratio. Ideally, the signal level should be near theupper limit of the dynamic range of the A/D converterso that the subsequent ¯ uorescence quenching measure-ments will have the largest possible linear range.

Quenching Studies. The ability of the ¯ uorescencesystem to measure quenching constants accurately hasbeen assessed by using two well-known and well-char-

acterized quenchers: nitromethane and triethylamine.Both of these compounds are effective quenchers of poly-cyclic aromatic hydrocarbons (PAHs), which are an im-portant group of pollutants found at trace levels through-out the environment. PAHs form the largest known classof chemical carcinogens and mutagens and have beenidenti® ed by the U.S. Environmental Protection Agencyas priority pollutants.16,17 Structurally, PAHs consist oftwo or more fused aromatic rings that can exist in manyisomeric arrangements. These isomers are organized intotwo main classes; alternant PAHs have a fully conjugatedaromatic system, whereas nonalternant PAHs have dis-rupted aromaticity due to the presence of ® ve-memberedrings.16,17 Sawicki et al. ® rst reported that nitromethane,an electron acceptor, selectively quenches the ¯ uores-cence of alternant PAHs compared with their nonalter-nant isomers.18 Subsequent systematic studies by Acreeand co-workers have shown that the so-called ``nitro-methane selective quenching rule’ ’ is broadly applicablewith only a few exceptions.19±23 Although triethylaminehas been identi® ed as an effective quencher of PAH ¯ uo-rescence, 24,25 only one published report to date has indi-cated any selectivity.26 As an electron donor, triethyl-amine would be expected to show selectivity for nonal-ternant PAHs over their alternant isomers.

Two representative pairs of PAHs have been selectedfor this study: pyrene and ¯ uoranthene, as well as ben-zo(a)pyrene and benzo(b)¯ uoranthene. These isomericPAHs have the same molecular formula, the same num-ber of aromatic rings, and a similar length-to-breadth ra-tio, differing only in their alternant/nonalternant ringstructure. Figures 4A and 4B show the Stern±Volmerplots for these representative PAHs with triethylamineand nitromethane, respectively, as quenchers. These plotsare constructed from ¯ uorescence power ratios derivedfrom the integrated emission spectra, as described pre-viously. Both quenchers are demonstrating the expectedselectivity for PAH isomers, with nitromethane being amore effective quencher of the alternant PAHs and tri-ethylamine being a more effective quencher of the non-alternant PAHs. The linearity of the Stern±Volmer plotsis best for those ¯ uorophore±quencher systems with largequenching constants, where the correlation coef® cientsrange from r2 5 0.998±1.000. Determination of smallerquenching constants is necessarily more dif® cult as thesmaller differences in the ¯ uorescence power ratio aremore sensitive to random errors in concentration and in-strumental response.5 As a result, these quenching con-stants have lower correlation coef® cients of r 2 5 0.889±0.995. The y intercepts for all plots fall within the rangeof 0.98±1.09, as expected from Eq. 1. Finally, there is noevidence of curvature in the Stern±Volmer plots, whichwould be indicative of either absorbance effects or staticquenching.

Table I compares the quenching constants for the PAHswith triethylamine determined from Fig. 4A with thoseobtained by using a commercially available scanning¯ uorimeter with a 1 cm cuvette cell. The agreement isquite good and, in general, the Stern±Volmer constantsdetermined by using the two systems are statistically in-distinguishable. The small discrepancies may arise fromdifferences in the spectral bandpass, which is 1 nm forthe present system and 2.5 nm for the conventional ¯ uo-


FIG. 4. Stern±Volmer plots for the quenching of 10 2 5 M pyrene (V),¯ uoranthene (M), benzo(a)pyrene ( n ), and benzo(b)¯ uoranthene ( , ) bytriethylamine (A) and nitromethane (B) in methanol. Other experimentalconditions as described in Fig. 2.

TABLE I. Stern± Volmer constants for polycyclic aromatic hydrocarbons with triethylamine in methanol.a

PAH Classi® cation Kd (M 2 1)b s(Kd) (M 2 1)b (r 2)b Kd (M 2 1)c s(Kd) (M 2 1)c (r 2)c

PyreneBenzo(a)pyreneFluorantheneBenzo(b)¯ uoranthene

AlternantAlternantNonalternantNonalternant

4.41.8

23.021.7

0.30.20.60.3

0.9860.9670.9980.999

4.62.1

20.720.6

0.20.20.10.4

0.9960.9741.0000.999

a Kd 5 Stern±Volmer constant; s(K d) 5 standard deviation of Stern±Volmer constant; r 2 5 square of correlation coef ® cient.b Use of a 1 cm cuvette cell; this work.c Use of a 75 m m capillary cell; this work.

rimeter. The linearity of the Stern±Volmer plots for thepresent system is at least comparable to and, in somecases, superior to that for the conventional ¯ uorimeterwith absorbance corrections via Eq. 3. Table II comparesthe quenching constants for the PAHs with nitromethanedetermined from Fig. 4B with previously published re-sults.4,5 Again, the agreement is reasonably good in spiteof signi® cant differences in instrumentation and experi-mental methods. Thus, the performance of the presentsystem has been validated by comparison to traditionalmethods by using premixed solutions for the determina-tion of Stern±Volmer quenching constants.

A powerful modi® cation of this system is to use ¯ owinjection techniques27 to automate the measurement ofquenching constants, as shown in Fig. 1. This approachfacilitates the rapid determination of quenching constantswith minimal solution preparation, requiring only one so-lution of the ¯ uorophore and one of the quencher. A dual-piston syringe pump is used to deliver the quencher so-lution in a step gradient, which offers several advantagesover the linear gradient used in previous work.8 Each stepis programmed for a speci® ed duration, which allows forreplicate injections of a single ¯ uorophore or injectionsof several ¯ uorophores. The concentration of the quench-er is accurately known and does not vary over the widthof the ¯ uorophore zone. The ¯ uorophore zones are in-jected in suf® ciently large volume for them to form ¯ at-topped peaks. In this way, the concentration at the zonecenter is the same as that in the prepared solution and isnot dependent on the broadening processes in the ¯ owinjection system. Hence, the resulting precision of thequenching constants is independent of ¯ uctuations in¯ ow rate, injection volume, etc. Moreover, the ¯ at-toppedpro ® le enables the integration time of the CCD detectorto be varied as necessary to achieve the optimal S/N ratioand linear range. Finally, the method of merging zonesis used in order to ensure adequate mixing of the ¯ uo-rophore and quencher solutions. 27

An example of the data resulting from this ¯ ow injec-tion approach is illustrated in Fig. 5 for pyrene quenchedby nitromethane. The lower trace from the UV-visibleabsorbance detector at 254 nm shows the step gradientof the quencher ranging in concentration from 0 to 0.05M in increments of 0.01 M, together with triplicate in-jections of the ¯ uorophore at each step. This absorbancetrace provides independent veri® cation of the ¯ uorophoreand quencher concentrations and their precisions as wellas evidence for or against static complexation. The mid-dle trace shows the ¯ uorescence of pyrene with excitationat 325 nm and emission detected by the PMT detector at371 nm. The decrease in ¯ uorescence power with in-creasing nitromethane concentration is evident, and pro-


TABLE II. Stern ± Volmer constants for polycyclic aromatic hydrocarbons with nitromethane in methanol.a

PAH Classi® cation K d (M 2 1)b Kd (M 2 1)c (r 2)c Kd (M 2 1)d s(Kd) (M 2 1)d (r 2)d

PyreneBenzo(a)pyreneFluorantheneBenzo(b)¯ uoranthene

AlternantAlternantNonalternantNonalternant

125Ð

0.15Ð

1006712

0.9990.9980.9540.994

94610.070.64

0.31.60.010.02

1.0000.9980.9000.997

a Kd 5 Stern±Volmer constant; s(Kd) 5 standard deviation of Stern±Volmer constant; r 2 5 square of correlation coef® cient.b Use of a 1 cm cuvette cell, absorbance correction via Eq. 3; Ref. 4.c Use of a 1 cm cuvette cell, absorbance correction via Eq. 3; Ref. 5.d Use of a 75 m m capillary cell; this work.

FIG. 5. Experimental data obtained by using the ¯ ow injection approach to determine Stern±Volmer constants. Fluorophore: 75 m L injections of10 2 5 M pyrene in methanol, 35 m L/min. Quencher: 0.0±0.05 M nitromethane in increments of 0.01 M per 20 min step, 35 m L/min. Lower traceshows UV absorbance at 254 nm. Middle trace shows ¯ uorescence detected by PMT at 371 nm, 1 nm bandpass. Upper traces show ¯ uorescencespectra detected by CCD detector at 350±500 nm, 1 nm bandpass, 0.2 s integration time. Other experimental conditions as described in Fig. 2.

vides the information necessary for the determination ofthe Stern±Volmer quenching constant and its precision ata single wavelength. Finally, the upper traces show the¯ uorescence spectra of pyrene obtained with the CCDdetector, which are used to determine the quenching con-stant and its precision at a single wavelength (371 nm)or over an integrated range of wavelengths (350±500nm).

Table III summarizes the quenching constants deter-mined from this ¯ ow injection experiment as well as acomparison to the static system with premixed solutions,as described above. These results con® rm that quenchingconstants determined by ¯ ow injection are statisticallyequivalent to those determined by static measurements,both of which show a high degree of linearity (r 2 50.999±1.000). In addition, the quenching constants ob-


TABLE III. Comparison of experimental methods for the determ ination of Stern± Volmer constants for pyrene with nitromethane inmethanol.a

Method Detector Wavelength (nm)Pathlength

( m m) Kd (M 2 1) s(Kd) (M 2 1) b r 2 n

StaticStaticFlow injectionFlow injectionFlow injectionFlow injectionFlow injectionFlow injection

CCDCCDPMTPMTCCDCCDCCDCCD

371350±500

371371371

350±500371

350±500

7575

200200200200200200

103.093.896.195.596919792

0.990.330.350.131.41.71.11.3

1.021.090.991.000.890.920.900.93

1.0001.0001.0001.0000.9990.9991.0000.999

555

5055

5050

a Kd 5 Stern±Volmer constant; s(Kd) 5 standard deviation of Stern±Volmer constant; b 5 y-intercept; r 2 5 square of correlation coef® cient; n 5number of measurements at each quencher concentration.

tained with the PMT detector are comparable in magni-tude but higher in precision than those obtained with theCCD detector at the same wavelength. When the numberof measurements (n) at each quencher concentration isincreased from 5 to 50, the magnitude of the resultingquenching constants remains relatively constant and theprecision is improved. However, the PMT detector showsa more signi® cant improvement in precision than theCCD detector, which is limited by dark and read noise.Finally, the quenching constants calculated at a singlewavelength of 371 nm tend to be larger in magnitudethan those calculated over an integrated wavelength rangeof 350±500 nm. This observation suggests that this vi-bronic transition of pyrene interacts differently with ni-tromethane when compared to the integrated emissionspectrum. This conclusion is consistent with previousmeasurements of the quenching of pyrene by nitrometh-ane at a series of single wavelengths. 5 In cases such asthis, the integrated spectra can provide more reproducibleand reliable quenching constants. This approach is alsobene® cial for ¯ uorophores with emission spectra that arehighly structured or that vary with the concentration ofthe quencher or solvent.4,5

CONCLUSION

Although selective ¯ uorescence quenching is a prom-ising technique, only a few ¯ uorophore±quencher sys-tems have been characterized in suf® cient depth and de-tail to permit their use for routine analysis. The furtherdevelopment and application of this technique will re-quire more rapid and accurate methods for the determi-nation of Stern±Volmer constants. The system developedin the present work automates the preparation and mixingof ¯ uorophore and quencher solutions by means of cap-illary ¯ ow injection methods. The small diameter of thecapillary allows the ¯ uorescence measurements to bemade without interference from primary and secondaryabsorbance effects. The ¯ uorescence spectrometer isequipped with a charge-coupled device that has a detec-tion limit of 3.0 3 10 2 9 M (2.3 ppb) and a linear rangeof 105 with integration times of 0.01±10 s. This spec-trometer has a 300 nm spectral range with 1 nm resolu-tion, which allows the Stern±Volmer constants to be cal-culated at single wavelengths or over integrated wave-length ranges. This system was validated by determina-tion of the quenching constants for selected alternant andnonalternant PAHs as ¯ uorophores with nitromethaneand triethylamine as quenchers. These quenching con-

stants compare favorably with those determined by tra-ditional methods in terms of both accuracy and precision.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical support of Mr. Mi-chael Hasselhuhn of the Melles Griot Laser Group. This research wassupported by the U.S. Department of Energy, Of® ce of Basic EnergySciences, under Grant Number DE-FG02-89ER14056. In addition, thesupport of the National Science Foundation by means of a graduateresearch fellowship to J.V.G. is gratefully acknowledged.

1. V. L. McGuf® n and J. V. Goodpaster, `̀ Polycyclic Aromatic Com-pounds, Fluorescence Quenching’ ’ , Encyclopedia of EnvironmentalAnalysis and Remediation, R. A. Meyers, Ed. (John Wiley andSons, New York, 1998), pp. 3814±3831.

2. J. Lakowicz, Principles of Fluorescence Spectroscopy (PlenumPress, New York, 1983).

3. R. Badley, Fluorescence Spectroscopy (Plenum Press, New York,1983).

4. S. H. Chen, C. E. Evans, and V. L. McGuf® n, Anal. Chim. Acta246, 65 (1991).

5. F. K. Ogasawara, Y. Wang, and V. L. McGuf® n, Appl. Spectrosc.49, 1 (1995).

6. C. E. Bunker, Y. Sun, and J. R. Gord, J. Phys. Chem. A 101, 9233(1997).

7. P. K. Behera, T. Mukherjee, and A. K. Mishra, J. Lumin. 65, 131(1995).

8. D. J. Desilets, P. T. Kissinger, and F. E. Lytle, Anal. Chem. 59, 1244(1987).

9. A. Savitsky and M. J. E. Golay, Anal. Chem. 36, 1627 (1964).10. X. Lu and E. S. Yeung, Appl. Spectrosc. 49, 605 (1995).11. J. J. Cetorelli, W. J. McCarthy, and J. D. Winefordner, J. Chem.

Educ. 45, 98 (1968).12. C. Th. J. Alkemade, W. Snelleman, G. D. Boutilier, B. D. Pollard,

J. D. Winefordner, T. L. Chester, and N. Omenetto, Spectrochim.Acta, Part B 33, 383 (1978).

13. G. D. Boutilier, B. D. Pollard, J. D. Winefordner, T. L. Chester, andN. Omenetto, Spectrochim. Acta, Part B 33, 401 (1978).

14. Y. P. Sun, B. Ma, G. E. Lawson, C. E. Bunker, and H. W. Rollins,Anal. Chim. Acta 319, 379 (1996).

15. J. V. Sweedler, Crit. Rev. Anal. Chem. 24, 59 (1993).16. M. L. Lee, M. V. Novotny, and K. D. Bartle, Analytical Chemistry

of Polycyclic Aromatic Compounds (Academic Press, New York,1981).

17. T. Vo-Dinh, Chemical Analysis of Polycyclic Aromatic Compounds(John Wiley and Sons, New York, 1989).

18. E. Sawicki, T. W. Stanley, and W. C. Elbert, Talanta 11, 1433(1964).

19. W. E. Acree, Jr., S. Pandey, S. A. Tucker, and J. C. Fetzer, Poly-cyclic Aromat. Compd. 12, 71 (1997).

20. S. A. Tucker, H. C. Bates, V. L. Amszi, W. E. Acree, Jr., H. Lee,P. Di Raddo, R. G. Harvey, J. C. Fetzer, and G. Dyker, Anal. Chim.Acta 278, 269 (1993).

21. S. A. Tucker, J. M. Grif ® n, W. E. Acree, Jr., P. J. Mulder, J. Lug-


tenburg, and J. Cornelisse, Analyst 119, 2129 (1994).22. S. A. Tucker, H. C. Bates, W. E. Acree, Jr., and J. C. Fetzer, Appl.

Spectrosc. 47, 1775 (1993).23. S. A. Tucker, J. M. Grif® n, W. E. Acree, Jr., J. C. Fetzer, M. Zander,

O. Reiser, A. De Meijere, and I. Murata, Polycyclic Aromat.Compd. 4, 141 (1994).

24. J. Mai, J. Cheng, and T. Ho, J. Photochem. Photobiol. A 66, 53(1992).

25. M. Vasilescu , Rev. Roum. Chim. 34, 1819 (1989).26. M. P. Fogarty and I. M. Warner, Appl. Spectrosc. 36, 460 (1982).27. J. Ruzicka and E. H. Hansen, Flow Injection Analysis (John Wiley

and Sons, New York, 1988).

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Rapid and Accurate Determination of Stern-Volmer Quenching Constants