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Selective Analysis of Fluorene by Quenched Fluorescence in Cetylpyridinium Bromide Micelles
MICROCHEMICAL JOURNAL ARTICLE NO.
60, 101–109 (1998)
MJ981637
Selective Analysis of Fluorene by Quenched Fluorescence in Cetylpyridinium Bromide Micelles Juan H. Ayala, Ana M. Afonso, and Venerando Gonza´lez-Diaz1 Departamento de Quı´mica Analı´tica, Nutricio´n y Bromatologı´a, Campus de Ancheta, Universidad de La Laguna, E-38201 La Laguna, Spain Received February 3, 1998; accepted May 21, 1998 Cetylpyridinium bromide (CPB) acts as a quencher, provoking inhibition of the fluorescence intensity emitted by fluorene. The differences in the fluorescence quenching of several polyaromatic hydrocarbons (PAHs) allow us to develop a selective synchronous spectrofluorometric 0 method for the determination of fluorene in a CPB micellar medium at ls,ex 5 299 nm (Dl 5 21 10 nm), with a detection limit of 8.5 ng ml . The method was applied to the analysis of groundwater samples spiked with suitable amounts of fluorene, as well as to groundwater samples polluted with combustibles. © 1998 Academic Press
INTRODUCTION Fluorene, a three-ring aromatic hydrocarbon, is a common component of the hydrocarbon fractions of various fossil fuels and is known to be important component of environmental pollution (1–3). For the analysis of environmental samples containing fluorene, chromatographic separations has been used extensively in combination with mass spectrometry (3, 4), fluorometry (5, 6), and ultraviolet spectrometry (1, 2). Most of the environmental mixtures commonly encountered contain several isomeric pairs or structurally similar polyaromatic hydrocarbons (PAHs), which emit in approximately the same spectral regions. Kalman filtering and Gaussian or other curve-fitting techniques (7–10), alone or in combination with phase-resolved fluorescence spectroscopy, theoretically allow uncoupling of overlapped spectra (11–13). However, such methods become less reliable as the number of mixture components increases. The use of selective fluorescence quenching agents further simplifies observed emission spectra by eliminating signals from undesired chemical interferents having only slightly different molecular structures. There are few studies of fluorescence quenching in a micellar medium where the surfactant acts, furthermore, as quencher. Use of cetylpyridinium bromide (CPB) as a selective quencher for polycyclic aromatic was reported by Ayala et al. (14, 15). Synchronous fluorescence methods are more selective than ordinary fluorescence methods for the analysis of mixtures (16). Synchronous fluorescence has been used to characterize crude and refined oils (17–19) and to identify and determine polycyclic aromatic hydrocarbons (20 –26). In the present work the conventional and synchronous spectrofluorometric characteristics of fluorene in a micellar medium of CPB are established. The use of a micellar medium to improve the selectivity of analytical determinations, using the spectral shifts 1
To whom correspondence should be addressed. Fax: 134 22 318090. E-mail: [email protected]. 101 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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and the fluorescence quenching provoked by CPB, is studied for the first time. By using CPB micelles, a selective method for determination of fluorene by synchronous spectrofluorometry is proposed. EXPERIMENTAL Apparatus Fluorescence measurements were made with a Perkin–Elmer LS-50 luminescence spectrometer equipped with a xenon discharge lamp and connected via an RS232C interface to an Epson PCAX2e computer. The spectrometer was controlled using Fluorescence Data Manager software. Fluorescence measurements were made in standard 1 3 1-cm quartz cells, thermostated at 25 6 0.1°C with a Selecta Frigitherm S-382 ultrathermostat. Absorption measurements were made with a Hewlett–Packard HP8452A diodearray spectrophotometer, furnished with a quartz cell of 1-cm pathlength and interfaced with a Hewlett–Packard ES computer and a Think Jet printer. Contour maps were obtained by elaboration of a Basic program within the OBEY application of the Fluorescence Data Manager software. This program allows successive scanning of several emission or synchronous spectra with different excitation wavelengths or Dl, respectively, and transforms them into a matrix of experimental data. The file containing this matrix is used as input in the commercial program SURFER to obtain contour maps. Reagents and Solutions A stock solution (1023 M) of fluorene (Aldrich) was prepared in ethanol (Merck). Working solutions were prepared by appropriate dilution with ethanol. A stock solution (5 3 1022 M) of CPB (Sigma Chemical Co.) was prepared in deionized water. All chemicals used were of analytical reagent grade. General Procedure for the Determination of Fluorene To an aliquot containing 0.33–13.30 mg of fluorene in a 25-ml calibrated flask, add 1.3 ml of 5 3 1022 M CPB solution and the necessary volume of ethanol so that the final solution contains 0.5% (v/v) organic solvent, and dilute to volume with deionized water. The fluorescence intensity measurements are made at synchronous maximum of fluorene, l0s,ex 5 299 nm (Dl 5 10 nm). Calibration graphs are obtained from standard solutions prepared under the same experimental conditions. Recovery of Fluorene in Groundwater and in Polluted Groundwater Water samples were spiked with suitable amounts of fluorene and analyzed according to the above method. RESULTS AND DISCUSSION Conventional Fluorescence To determine the influence of the micellar system on the excitation and emission spectra of fluorene, the following solutions were used: 0.5% (v/v) ethanol/water solutions, which are referred to as aqueous solutions, and 0.5% (v/v) ethanol/water solutions containing 2.6 3 1023 M CPB to ensure a surfactant concentration greater than its critical micellar
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FIG. 1. Excitation (1,3) and emission (2,4) spectra of fluorene in the absence (1,2) and presence (3,4) of 2.6 3 1023 M CPB. Cfluorene 5 2 3 1027 M (1,2) and 4 3 1026 M (3,4). Slits 5 2.5 nm.
concentration (CMC), 7.32 3 1024 M. This CMC value was determined conductimetrically under experimental conditions similar to those used in fluorescence measurements. Figure 1 shows the fluorescence spectra of fluorene in aqueous and micellar media. The micellar medium causes, in relation to the aqueous medium, the following changes: bathochromic shifts in the excitation and emission maxima; disappearance of the signal corresponding to wavelengths lower than 275 nm in the excitation spectrum; significant inhibition of the fluorescence intensity emitted by fluorene. To quantify the fluorescence inhibition that fluorene experiences in the micellar medium, we used the term fluorescence inhibition by action of micellar medium (I ), I~%! 5 ~1 2 B m/B a! z 100, where Bm and Ba represent the slopes of the calibration graphs obtained in the presence and absence, respectively, of 2.6 3 1023 M CPB. The values of Bm and Ba were obtained
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FIG. 2. Influence of CPB concentration on the fluorescence intensity of 2 3 1027 M fluorene. lexc/lem 5 209/302 nm. Slits 5 2.5 nm.
using the wavelengths lem 5 311 nm (lexc 5 298 nm) and lem 5 302 nm (lexc 5 209 nm), respectively. The value of I found for fluorene is 98.50%. The variation of the fluorescence intensity emitted by fluorene as a function of CPB concentration is illustrated in Fig. 2. At concentrations below the CMC, the fluorescence of fluorene decreases suddenly on increasing the surfactant concentration, whereas at concentrations above the CMC, the fluorescence does not vary significantly. The exponential decreases in fluorescence intensity, which vary in a regular way with the quencher concentration (see Fig. 2), can be considered typical of quenching processes. The degree of fluorescence quenching (27), EQ, defined as 1 2 F/F0, was measured as a function of CPB concentration, where F0 and F are the fluorescence intensities of fluorene in the absence and presence of CPB, respectively. The CPB concentration necessary to reach an inhibition of 90% is 5.0 3 1024 M, around the CMC. To determine the interactions involved in the quenching of fluorene by CPB, Stern– Volmer plots were constructed. Figure 3A is a plot of F0/F versus CPB concentration, in which we can select three different regions: AB, BC, and CD. Region AB. At [CPB] ,, CMC, there is no micellar phase and CPB is present as monomers or partially aggregated as premicellar aggregates. For CPB concentrations below 1.50 3 1024 M, we obtained good relationships (r 5 0.999) between F0/F and CPB concentration (see Fig. 3B), with a Ksv value of (11.27 6 0.18) 3 103 M21. A linear Stern–Volmer plot with an intercept near one (0.95) indicates that a single mechanism is involved in the quenching process, which could be either dynamic or static in nature. Region BC. At 1.50 3 1024 M , [CPB] , 4.95 3 1024 M. At CPB concentrations higher than 1.50 3 1024 M, positive deviation from linearity was observed, which suggests that quenching is a combination of both dynamic and static quenching processes (28, 29). The possible coexistence of both types of quenching requires the utilization of
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FIG. 3. Fluorescence quenching of fluorene by cetylpyridinium bromide. (A,B) Stern–Volmer plots. (C) Plot of F0/FeV[Q] and (D) plot of ln(F0/F) versus CPB concentration.
models different from that used. The loss of the Stern–Volmer equation linearity occurs because only part of the fluorene molecules is deactivated by collisional mechanisms, while the remainder forms the complex fluorenepCPB in the ground state. The different models developed to explain this instantaneous phenomenon (static quenching) lead to a modified form of the Stern–Volmer equation (28), F 0 /Fe V@Q# 5 ~1 1 K D@Q#!,
(1)
in which V is the constant of static quenching, KD represents the dynamic aspects of the deactivation of the fluorene excited state, and [Q] is the CPB concentration. For the treatment of experimental data, values of V are given until a linear plot is obtained (r 5 0.999) of F0/FeV[Q] versus CPB concentration. The value of V that satisfies linearity is considered the static quenching constant, while the slope of the straight lines obtained represents the dynamic quenching constant. The curve obtained on applying Eq. [1] is shown in Fig. 3C. The model is accurate for CPB concentrations lower than 4.95 3 1024 M and intercept near one (0.96). The values obtained for KD and V are (8.74 6 0.09) 3 103 and 1.31 3 103 M21, respectively. The value of V obtained should be taken as an association constant between fluorene and premicellar or pseudomicellar aggregates of CPB.
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Region CD. At [CPB] . CMC. The previously applied model presents important deviations of linearity at CPB concentrations above the CMC, possibly due to the incorporation of PAHs into the hydrophobic core of the micelles, which practically prevents dynamic quenching. From Eq. [1], substituting V with the static quenching constant (Ks) and applying Napierian logarithms, we obtain ln~F 0 /F! 5 ln~ t 0 / t ! 1 K s z @Q#. This equation shows that when the t0/t ratio becomes constant, a plot of 1n(F0/F) versus CPB concentration becomes linear and only static quenching exists. This representation allows the calculation of Ks and 1n(t0/t). From data plotted in Fig. 3D, the values obtained for Ks and the intercept are (0.66 6 0.13) 3 103 M21 and 2.05, respectively. The low fluorescence intensity of fluorene in a micellar medium result in slightly reliable F0/F values, which explains the low correlation coefficient (r 5 0.930) obtained. The value of Ks obtained should be taken as an association constant between fluorene and CPB micelles. If in the range of CPB concentrations considered there is static quenching, the t0/t relationship would equal one; consequently, the intercept would be zero. The value obtained (2.05), different from zero, must take into account that dynamic and/or static quenching processes are produced below the lowest concentration limit in this zone. The absorption spectra of fluorene in solutions with variable CPB concentrations can supply additional information to differentiate static and dynamic quenching processes. As collisional quenching affects only the excited state of the fluorene, it is to be expected that changes are not produced in its absorption spectra. On the contrary, the formation of complexes in the ground state between fluorene and CPB would explain the appearance of modifications in its absorption spectra. Figure 4 shows the absorption spectra obtained, which explain the significant bathochromic shifts at CPB concentrations higher than 2 3 1024 M and the disappearance of the signal corresponding to wavelengths lower than 220 nm at CPB concentrations higher than 6 3 1024 M. Synchronous Fluorescence The introduction of a micellar medium of CPB, which produces selective inhibition, and the correct selection of Dl in the synchronous spectra would let us analyze fluorene in the presence of other hydrocarbons. To select the optimum values of Dl, synchronous contour maps were used. These maps were obtained by plotting fluorescence intensity as a function of the excitation wavelength (horizontal axis) and the difference (Dl) between the emission and excitation wavelengths (vertical axis). Thus, a set of contour lines that connect points of equal fluorescence intensity was obtained. Although the optimum values of Dl can also be selected through contour maps obtained from conventional spectra, synchronous contour maps (30, 31) supply more selective information. Figure 5 shows the contour map of 1027 M fluorene in the presence of 2.6 3 1023 M CPB and 0.5% (v/v) ethanol. For comparative purposes, contour map of a solution of 2.6 3 1023 M CPB and 0.5% (v/v) ethanol is included in Fig. 5. It can be observed that
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FIG. 4. Absorption spectra of 2 3 1027 M fluorene in the presence of different concentrations of CPB: (1) 0 M, (2) 5 3 1025 M, (3) 1 3 1024 M, (4) 2 3 1024 M, (5) 3 3 1024 M, (6) 6 3 1024 M, (7) 1.5 3 1023 M.
the value of Dl that produces the maximum intensity peak of the synchronous spectra is 10 nm, with l0s,ex 5 299 nm. Great agreement exists between this Dl and the difference between the emission and excitation wavelength maxima of conventional fluorescence spectra. Furthermore, this Dl value almost coincides with the Stokes shift. In addition, it must be stressed that the value of l0s,ex almost coincides with the excitation wavelength maxima in the conventional spectrum. Analytical Determinations To develop a procedure for the determination of fluorene, the analytical characteristics of the synchronous spectrum recorded using Dl 5 10 nm were evaluated.
FIG. 5. Synchronous contour maps of 2.6 3 1023 M CPB and 2 3 1027 M fluorene in a micellar medium of CPB. Slits 5 5 nm.
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108
TABLE 1 Recovery of Fluorene in Groundwater Samples (1) and Groundwater Samples Polluted with Fuel Oil (2) and Diesel Oil (3) Groundwater sample
Fluorene added (ng ml21)
Recoverya (%)
1 2 3
267.2–534.4 33.4–100.2 33.4–100.2
93.2–92.4 95.4–97.5 103–108.6
a
Mean of three determinations.
In the presence of CPB, a linear calibration graph with a high correlation coefficient (0.999) was constructed by plotting the fluorescence intensity measurements on the synchronous spectrum (l0s,ex 5 299 nm) against fluorene concentration. The linear concentration range is 13.3– 667.9 ng ml21 and the detection limit (32) is 8.5 ng ml21. When the method was applied to two series of 11 samples containing 49.9 and 498.6 ng ml21 fluorene, relative errors of 64.85 and 61.10% and relative standard deviations of 7.21 and 1.64% were obtained, respectively. To study the selectivity of the method, analyses of fluorene were carried out on synthetic mixtures with other PAHs. The criterion for interference was a variation of more than 65% in the fluorene concentration found. The presence of other PAHs did not interfere with the determination of 19.94 ng ml21 fluorene up to the following ratios: 20/1 for benz[a]anthracene; 10/1 for naphthalene, anthracene, and 9-methylanthracene; 5/1 for phenanthrene, 2-methylphenanthrene, pyrene, perylene, dibenz[a,h]anthracene, acenaphthylene, and fluoranthene; 2/1 for benzo[a]pyrene, acenaphthene, and benzo[b]fluoranthene. The method was applied to the determination of fluorene in groundwater samples spiked with suitable amounts of fluorene, as well as groundwater samples polluted with fuel oil and diesel oil. The absence of fluorene in the sample, was confirmed chromatographically. Determinations were carried out in triplicate, and mean values obtained for two different concentrations show good recovery indices. Table 1 summarizes these values. CONCLUSIONS As are other pyridinium salts, CPB is an effective quencher in the deactivation of the excited state of PAHs. Furthermore, CPB is a surfactant that can be found in micelles in aqueous medium. In the presence of CPB, the fluorescence spectra of fluorene experience important modifications. One is a significant decrease in fluorescence intensity, which is associated with quenching processes. The nature of these processes depends on the CPB concentration in the solution. At low surfactant concentrations, where CPB can be considered a monomer, dynamic quenching is the prevailing mechanism in the inhibition of the fluorescence of fluorene. When CPB exists as premicellar aggregates in solutions, the mechanisms of dynamic and static quenching coexist. In a micellar CPB medium, static quenching is responsible for the great decrease in the fluorescence intensity of fluorene. Solute–micelle interactions can stabilize the singlet excited state of many fluorophores,
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with a consequent increase in the intensity of the radiation emitted. The inhibition of fluorescence by CPB can be considered opposed to that stabilization of the singlet excited state. In this way, the confrontation between these opposing effects produced by CPB can be used analytically to improve the selectivity of some determinations. In the case of fluorene, the simultaneous action of a micellar CPB medium and an analytical technique such as synchronous spectrofluorometry allows analytical determination of this hydrocarbon with a significant degree of selectivity, while maintaining an acceptable sensitivity. In comparison with chromatographic separation, the proposed determination is rapid and simple. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Davies, I. L.; Bartle, K. D.; Williama, P. T.; Andrews, G. E. Anal. Chem., 1988, 60, 204. Lebo, J. A.; Smith, L. M. J. Assoc. Anal. Chem., 1986, 69, 944. Wozniak, T. J.; Hites, R. A. Anal. Chem., 1983, 55, 1791. Requero, A. G.; Sassen, R.; McDonald, T.; Denoux, G.; Kennicutt, M. C.; Brooks, J. M. Org. Geochem., 1996, 24, 1017. Beltran, J. L.; Ferrer, R.; Guiteras, J. J. Liq. Chromatogr. Relat. Technol., 1996, 19, 477. Brouwer, E. R.; Hermans, A. N. J.; Lingeman, H.; Brinkman, U. A. J. Chromatogr., 1994, 669, 45. Rutan, S. C. J. Chemom., 1987, 82, 34. Brown, S. D. Anal. Chim. Acta, 1986, 181, 1. Gampp, H.; Maeder, M.; Meyer, C. J.; Zuberbu¨hler, A. D. Talanta, 1985, 32, 1133. Maeder, M. Anal. Chem., 1987, 59, 527. Millican, D. W.; McGown, L. B. Appl. Spectrosc., 1992, 46, 28. Millican, D. W.; McGown, L. B. Anal. Chem., 1989, 61, 580. Millican, D. W.; McGown, L. B. Anal. Chem., 1990, 62, 2242. Ayala, J. H.; Afonso, A. M.; Gonza´lez, V. Talanta, 1997, 44, 257. Ayala, J. H.; Afonso, A. M.; Gonza´lez, V. Appl. Spectrosc., 1997, 51, 380. Lloyd, J. B. F. Nature, 1971, 231, 64. Lloyd, J. B. F. Forensic Sci. Soc., 1971, 11, 235. Lloyd, J. B. F. Analyst, 1980, 105, 97. John, P.; Soutar, I. Anal. Chem., 1976, 48, 520. Vo-Dinh, T.; Gammage, R. B.; Hawthorne, A. R.; Thorngate, J. H. Environ. Sci. Technol., 1978, 12, 1297. Inman, E. L.; Winefordner, J. D. Anal. Chem., 1982, 54, 2018. Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chem., 1981, 53, 253. Kerkhoff, M. J.; Lee, T. M.; Allen, E. R.; Lundgren, D. A.; Winefordner, J. D. Environ. Sci. Technol., 1985, 19, 695. Files, L. A.; Jones, B. T.; Hanamura, S.; Winefordner, J. D. Anal. Chem., 1988, 58, 1440. Santana, J. J.; Sosa, Z.; Afonso, A. M.; Gonza´lez, V. Anal. Chim. Acta, 1991, 255, 107. Santana, J. J.; Sosa, Z.; Afonso, A. M.; Gonza´lez, V. Talanta, 1992, 39, 1611. Kusumoto, Y.; Shizuka, M.; Satake, I. Chem. Lett., 1986, 529. Eftink, M. R.; Ghiron, C. A. J. Phys. Chem., 1976, 80, 486. Vaughan, W. M.; Weber, G. Biochemistry, 1970, 9, 464. Lloyd, J. B. F. Analyst, 1980, 105, 97. Oms, M. T.; Forteza, R.; Cerda´, V.; Garcı´a, F.; Ramos, A. L. Int. J. Environ. Anal. Chem., 1990, 42, 1. Long, G. L.; Winefordner, J. D. Anal. Chem., 1983, 55, 712A.
60, 101–109 (1998)
MJ981637
Selective Analysis of Fluorene by Quenched Fluorescence in Cetylpyridinium Bromide Micelles Juan H. Ayala, Ana M. Afonso, and Venerando Gonza´lez-Diaz1 Departamento de Quı´mica Analı´tica, Nutricio´n y Bromatologı´a, Campus de Ancheta, Universidad de La Laguna, E-38201 La Laguna, Spain Received February 3, 1998; accepted May 21, 1998 Cetylpyridinium bromide (CPB) acts as a quencher, provoking inhibition of the fluorescence intensity emitted by fluorene. The differences in the fluorescence quenching of several polyaromatic hydrocarbons (PAHs) allow us to develop a selective synchronous spectrofluorometric 0 method for the determination of fluorene in a CPB micellar medium at ls,ex 5 299 nm (Dl 5 21 10 nm), with a detection limit of 8.5 ng ml . The method was applied to the analysis of groundwater samples spiked with suitable amounts of fluorene, as well as to groundwater samples polluted with combustibles. © 1998 Academic Press
INTRODUCTION Fluorene, a three-ring aromatic hydrocarbon, is a common component of the hydrocarbon fractions of various fossil fuels and is known to be important component of environmental pollution (1–3). For the analysis of environmental samples containing fluorene, chromatographic separations has been used extensively in combination with mass spectrometry (3, 4), fluorometry (5, 6), and ultraviolet spectrometry (1, 2). Most of the environmental mixtures commonly encountered contain several isomeric pairs or structurally similar polyaromatic hydrocarbons (PAHs), which emit in approximately the same spectral regions. Kalman filtering and Gaussian or other curve-fitting techniques (7–10), alone or in combination with phase-resolved fluorescence spectroscopy, theoretically allow uncoupling of overlapped spectra (11–13). However, such methods become less reliable as the number of mixture components increases. The use of selective fluorescence quenching agents further simplifies observed emission spectra by eliminating signals from undesired chemical interferents having only slightly different molecular structures. There are few studies of fluorescence quenching in a micellar medium where the surfactant acts, furthermore, as quencher. Use of cetylpyridinium bromide (CPB) as a selective quencher for polycyclic aromatic was reported by Ayala et al. (14, 15). Synchronous fluorescence methods are more selective than ordinary fluorescence methods for the analysis of mixtures (16). Synchronous fluorescence has been used to characterize crude and refined oils (17–19) and to identify and determine polycyclic aromatic hydrocarbons (20 –26). In the present work the conventional and synchronous spectrofluorometric characteristics of fluorene in a micellar medium of CPB are established. The use of a micellar medium to improve the selectivity of analytical determinations, using the spectral shifts 1
To whom correspondence should be addressed. Fax: 134 22 318090. E-mail: [email protected]. 101 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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and the fluorescence quenching provoked by CPB, is studied for the first time. By using CPB micelles, a selective method for determination of fluorene by synchronous spectrofluorometry is proposed. EXPERIMENTAL Apparatus Fluorescence measurements were made with a Perkin–Elmer LS-50 luminescence spectrometer equipped with a xenon discharge lamp and connected via an RS232C interface to an Epson PCAX2e computer. The spectrometer was controlled using Fluorescence Data Manager software. Fluorescence measurements were made in standard 1 3 1-cm quartz cells, thermostated at 25 6 0.1°C with a Selecta Frigitherm S-382 ultrathermostat. Absorption measurements were made with a Hewlett–Packard HP8452A diodearray spectrophotometer, furnished with a quartz cell of 1-cm pathlength and interfaced with a Hewlett–Packard ES computer and a Think Jet printer. Contour maps were obtained by elaboration of a Basic program within the OBEY application of the Fluorescence Data Manager software. This program allows successive scanning of several emission or synchronous spectra with different excitation wavelengths or Dl, respectively, and transforms them into a matrix of experimental data. The file containing this matrix is used as input in the commercial program SURFER to obtain contour maps. Reagents and Solutions A stock solution (1023 M) of fluorene (Aldrich) was prepared in ethanol (Merck). Working solutions were prepared by appropriate dilution with ethanol. A stock solution (5 3 1022 M) of CPB (Sigma Chemical Co.) was prepared in deionized water. All chemicals used were of analytical reagent grade. General Procedure for the Determination of Fluorene To an aliquot containing 0.33–13.30 mg of fluorene in a 25-ml calibrated flask, add 1.3 ml of 5 3 1022 M CPB solution and the necessary volume of ethanol so that the final solution contains 0.5% (v/v) organic solvent, and dilute to volume with deionized water. The fluorescence intensity measurements are made at synchronous maximum of fluorene, l0s,ex 5 299 nm (Dl 5 10 nm). Calibration graphs are obtained from standard solutions prepared under the same experimental conditions. Recovery of Fluorene in Groundwater and in Polluted Groundwater Water samples were spiked with suitable amounts of fluorene and analyzed according to the above method. RESULTS AND DISCUSSION Conventional Fluorescence To determine the influence of the micellar system on the excitation and emission spectra of fluorene, the following solutions were used: 0.5% (v/v) ethanol/water solutions, which are referred to as aqueous solutions, and 0.5% (v/v) ethanol/water solutions containing 2.6 3 1023 M CPB to ensure a surfactant concentration greater than its critical micellar
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FIG. 1. Excitation (1,3) and emission (2,4) spectra of fluorene in the absence (1,2) and presence (3,4) of 2.6 3 1023 M CPB. Cfluorene 5 2 3 1027 M (1,2) and 4 3 1026 M (3,4). Slits 5 2.5 nm.
concentration (CMC), 7.32 3 1024 M. This CMC value was determined conductimetrically under experimental conditions similar to those used in fluorescence measurements. Figure 1 shows the fluorescence spectra of fluorene in aqueous and micellar media. The micellar medium causes, in relation to the aqueous medium, the following changes: bathochromic shifts in the excitation and emission maxima; disappearance of the signal corresponding to wavelengths lower than 275 nm in the excitation spectrum; significant inhibition of the fluorescence intensity emitted by fluorene. To quantify the fluorescence inhibition that fluorene experiences in the micellar medium, we used the term fluorescence inhibition by action of micellar medium (I ), I~%! 5 ~1 2 B m/B a! z 100, where Bm and Ba represent the slopes of the calibration graphs obtained in the presence and absence, respectively, of 2.6 3 1023 M CPB. The values of Bm and Ba were obtained
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FIG. 2. Influence of CPB concentration on the fluorescence intensity of 2 3 1027 M fluorene. lexc/lem 5 209/302 nm. Slits 5 2.5 nm.
using the wavelengths lem 5 311 nm (lexc 5 298 nm) and lem 5 302 nm (lexc 5 209 nm), respectively. The value of I found for fluorene is 98.50%. The variation of the fluorescence intensity emitted by fluorene as a function of CPB concentration is illustrated in Fig. 2. At concentrations below the CMC, the fluorescence of fluorene decreases suddenly on increasing the surfactant concentration, whereas at concentrations above the CMC, the fluorescence does not vary significantly. The exponential decreases in fluorescence intensity, which vary in a regular way with the quencher concentration (see Fig. 2), can be considered typical of quenching processes. The degree of fluorescence quenching (27), EQ, defined as 1 2 F/F0, was measured as a function of CPB concentration, where F0 and F are the fluorescence intensities of fluorene in the absence and presence of CPB, respectively. The CPB concentration necessary to reach an inhibition of 90% is 5.0 3 1024 M, around the CMC. To determine the interactions involved in the quenching of fluorene by CPB, Stern– Volmer plots were constructed. Figure 3A is a plot of F0/F versus CPB concentration, in which we can select three different regions: AB, BC, and CD. Region AB. At [CPB] ,, CMC, there is no micellar phase and CPB is present as monomers or partially aggregated as premicellar aggregates. For CPB concentrations below 1.50 3 1024 M, we obtained good relationships (r 5 0.999) between F0/F and CPB concentration (see Fig. 3B), with a Ksv value of (11.27 6 0.18) 3 103 M21. A linear Stern–Volmer plot with an intercept near one (0.95) indicates that a single mechanism is involved in the quenching process, which could be either dynamic or static in nature. Region BC. At 1.50 3 1024 M , [CPB] , 4.95 3 1024 M. At CPB concentrations higher than 1.50 3 1024 M, positive deviation from linearity was observed, which suggests that quenching is a combination of both dynamic and static quenching processes (28, 29). The possible coexistence of both types of quenching requires the utilization of
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105
FIG. 3. Fluorescence quenching of fluorene by cetylpyridinium bromide. (A,B) Stern–Volmer plots. (C) Plot of F0/FeV[Q] and (D) plot of ln(F0/F) versus CPB concentration.
models different from that used. The loss of the Stern–Volmer equation linearity occurs because only part of the fluorene molecules is deactivated by collisional mechanisms, while the remainder forms the complex fluorenepCPB in the ground state. The different models developed to explain this instantaneous phenomenon (static quenching) lead to a modified form of the Stern–Volmer equation (28), F 0 /Fe V@Q# 5 ~1 1 K D@Q#!,
(1)
in which V is the constant of static quenching, KD represents the dynamic aspects of the deactivation of the fluorene excited state, and [Q] is the CPB concentration. For the treatment of experimental data, values of V are given until a linear plot is obtained (r 5 0.999) of F0/FeV[Q] versus CPB concentration. The value of V that satisfies linearity is considered the static quenching constant, while the slope of the straight lines obtained represents the dynamic quenching constant. The curve obtained on applying Eq. [1] is shown in Fig. 3C. The model is accurate for CPB concentrations lower than 4.95 3 1024 M and intercept near one (0.96). The values obtained for KD and V are (8.74 6 0.09) 3 103 and 1.31 3 103 M21, respectively. The value of V obtained should be taken as an association constant between fluorene and premicellar or pseudomicellar aggregates of CPB.
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Region CD. At [CPB] . CMC. The previously applied model presents important deviations of linearity at CPB concentrations above the CMC, possibly due to the incorporation of PAHs into the hydrophobic core of the micelles, which practically prevents dynamic quenching. From Eq. [1], substituting V with the static quenching constant (Ks) and applying Napierian logarithms, we obtain ln~F 0 /F! 5 ln~ t 0 / t ! 1 K s z @Q#. This equation shows that when the t0/t ratio becomes constant, a plot of 1n(F0/F) versus CPB concentration becomes linear and only static quenching exists. This representation allows the calculation of Ks and 1n(t0/t). From data plotted in Fig. 3D, the values obtained for Ks and the intercept are (0.66 6 0.13) 3 103 M21 and 2.05, respectively. The low fluorescence intensity of fluorene in a micellar medium result in slightly reliable F0/F values, which explains the low correlation coefficient (r 5 0.930) obtained. The value of Ks obtained should be taken as an association constant between fluorene and CPB micelles. If in the range of CPB concentrations considered there is static quenching, the t0/t relationship would equal one; consequently, the intercept would be zero. The value obtained (2.05), different from zero, must take into account that dynamic and/or static quenching processes are produced below the lowest concentration limit in this zone. The absorption spectra of fluorene in solutions with variable CPB concentrations can supply additional information to differentiate static and dynamic quenching processes. As collisional quenching affects only the excited state of the fluorene, it is to be expected that changes are not produced in its absorption spectra. On the contrary, the formation of complexes in the ground state between fluorene and CPB would explain the appearance of modifications in its absorption spectra. Figure 4 shows the absorption spectra obtained, which explain the significant bathochromic shifts at CPB concentrations higher than 2 3 1024 M and the disappearance of the signal corresponding to wavelengths lower than 220 nm at CPB concentrations higher than 6 3 1024 M. Synchronous Fluorescence The introduction of a micellar medium of CPB, which produces selective inhibition, and the correct selection of Dl in the synchronous spectra would let us analyze fluorene in the presence of other hydrocarbons. To select the optimum values of Dl, synchronous contour maps were used. These maps were obtained by plotting fluorescence intensity as a function of the excitation wavelength (horizontal axis) and the difference (Dl) between the emission and excitation wavelengths (vertical axis). Thus, a set of contour lines that connect points of equal fluorescence intensity was obtained. Although the optimum values of Dl can also be selected through contour maps obtained from conventional spectra, synchronous contour maps (30, 31) supply more selective information. Figure 5 shows the contour map of 1027 M fluorene in the presence of 2.6 3 1023 M CPB and 0.5% (v/v) ethanol. For comparative purposes, contour map of a solution of 2.6 3 1023 M CPB and 0.5% (v/v) ethanol is included in Fig. 5. It can be observed that
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FIG. 4. Absorption spectra of 2 3 1027 M fluorene in the presence of different concentrations of CPB: (1) 0 M, (2) 5 3 1025 M, (3) 1 3 1024 M, (4) 2 3 1024 M, (5) 3 3 1024 M, (6) 6 3 1024 M, (7) 1.5 3 1023 M.
the value of Dl that produces the maximum intensity peak of the synchronous spectra is 10 nm, with l0s,ex 5 299 nm. Great agreement exists between this Dl and the difference between the emission and excitation wavelength maxima of conventional fluorescence spectra. Furthermore, this Dl value almost coincides with the Stokes shift. In addition, it must be stressed that the value of l0s,ex almost coincides with the excitation wavelength maxima in the conventional spectrum. Analytical Determinations To develop a procedure for the determination of fluorene, the analytical characteristics of the synchronous spectrum recorded using Dl 5 10 nm were evaluated.
FIG. 5. Synchronous contour maps of 2.6 3 1023 M CPB and 2 3 1027 M fluorene in a micellar medium of CPB. Slits 5 5 nm.
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TABLE 1 Recovery of Fluorene in Groundwater Samples (1) and Groundwater Samples Polluted with Fuel Oil (2) and Diesel Oil (3) Groundwater sample
Fluorene added (ng ml21)
Recoverya (%)
1 2 3
267.2–534.4 33.4–100.2 33.4–100.2
93.2–92.4 95.4–97.5 103–108.6
a
Mean of three determinations.
In the presence of CPB, a linear calibration graph with a high correlation coefficient (0.999) was constructed by plotting the fluorescence intensity measurements on the synchronous spectrum (l0s,ex 5 299 nm) against fluorene concentration. The linear concentration range is 13.3– 667.9 ng ml21 and the detection limit (32) is 8.5 ng ml21. When the method was applied to two series of 11 samples containing 49.9 and 498.6 ng ml21 fluorene, relative errors of 64.85 and 61.10% and relative standard deviations of 7.21 and 1.64% were obtained, respectively. To study the selectivity of the method, analyses of fluorene were carried out on synthetic mixtures with other PAHs. The criterion for interference was a variation of more than 65% in the fluorene concentration found. The presence of other PAHs did not interfere with the determination of 19.94 ng ml21 fluorene up to the following ratios: 20/1 for benz[a]anthracene; 10/1 for naphthalene, anthracene, and 9-methylanthracene; 5/1 for phenanthrene, 2-methylphenanthrene, pyrene, perylene, dibenz[a,h]anthracene, acenaphthylene, and fluoranthene; 2/1 for benzo[a]pyrene, acenaphthene, and benzo[b]fluoranthene. The method was applied to the determination of fluorene in groundwater samples spiked with suitable amounts of fluorene, as well as groundwater samples polluted with fuel oil and diesel oil. The absence of fluorene in the sample, was confirmed chromatographically. Determinations were carried out in triplicate, and mean values obtained for two different concentrations show good recovery indices. Table 1 summarizes these values. CONCLUSIONS As are other pyridinium salts, CPB is an effective quencher in the deactivation of the excited state of PAHs. Furthermore, CPB is a surfactant that can be found in micelles in aqueous medium. In the presence of CPB, the fluorescence spectra of fluorene experience important modifications. One is a significant decrease in fluorescence intensity, which is associated with quenching processes. The nature of these processes depends on the CPB concentration in the solution. At low surfactant concentrations, where CPB can be considered a monomer, dynamic quenching is the prevailing mechanism in the inhibition of the fluorescence of fluorene. When CPB exists as premicellar aggregates in solutions, the mechanisms of dynamic and static quenching coexist. In a micellar CPB medium, static quenching is responsible for the great decrease in the fluorescence intensity of fluorene. Solute–micelle interactions can stabilize the singlet excited state of many fluorophores,
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with a consequent increase in the intensity of the radiation emitted. The inhibition of fluorescence by CPB can be considered opposed to that stabilization of the singlet excited state. In this way, the confrontation between these opposing effects produced by CPB can be used analytically to improve the selectivity of some determinations. In the case of fluorene, the simultaneous action of a micellar CPB medium and an analytical technique such as synchronous spectrofluorometry allows analytical determination of this hydrocarbon with a significant degree of selectivity, while maintaining an acceptable sensitivity. In comparison with chromatographic separation, the proposed determination is rapid and simple. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
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