ANALYST. JANUARY 1992, VOL. 117 83 Quenching of Fluorescence of Polynuclear Aromatic Hydrocarbons by Chlorine Saschi A. Momin and Ramaier Narayanaswamy" Department of Instrumentation and Analytical Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 IQD, UK A number of polynuclear aromatic hydrocarbon molecules, dissolved in methanol, have been investigated for their fluorescence quenching by chlorine. Extremely efficient quenching is observed between the fluorophore and the quencher, which leads to enhanced non-radiative decay of the fluorescent state of the molecule with increasing chlorine concentration. The findings demonstrate the possibility of developing an analytical method for the determination of chlorine. Keywords: Fluorescence quenching; polynuclear aromatic hydrocarbon; chlorine The use of fluorescence-based analysis is becoming increas- ingly popular in many branches of the chemical and biological sciences.The principal advantages of this technique, which encourage its use, are its high sensitivity which allows the measurement of low analyte concentrations, its selectivity which is, in part, due to the two characteristic wavelengths (excitation and emission) of each fluorescent species, and the variety of sampling methods available. In addition, the phenomenon of fluorescence quenching provides a kinetic method for the detection and determination of quencher molecules. I Fluorescence quenching is the process by which the fluorescence intensity or quantum yield of luminescent species is decreased, or even eliminated, by interaction with other chemical species.The quenching effect of oxygen on various luminescent species has been observed since the early 1930s;' however, analytical applications for the determination of oxygen were not reported until much Although there is a growing interest in the development of analytical methods based on dynamic quenching of fluorescence, which arises from the full and rapid reversibility of the process, quenching data for chlorine are not available. The need for chlorine sensing arises from its extensive use in the chemical industry and in the sterilization of drinking water. Owing to its toxic nature,' there is a risk of over exposure to chlorine, both in its manufacture and use. Hence the danger of chlorine pollution demands a sensitive and reliable method for its determination. This paper reports the extremely efficient quenching of the fluorescence of certain polynuclear aromatic hydrocarbon (PAH) molecules by chlorine and demonstrates the possibility of developing an analytical method based on this principle for the quantitative determination of chlorine.Measurement of Spectra Fluorescence spectra were recorded on a Perkin-Elmer Model LS-5 luminescence spectrometer equipped with an 8.3 W xenon discharge lamp pulsed at line frequency (50 Hz). All experiments were conducted using 1 x 1 cm rectangular quartz cells. Excitation and emission slit-widths were fixed at 2.5 nm for all the experiments. Absorbance measurements were recorded on a Perkin- Elmer Lambda 5 ultraviolet/visible spectrophotometer.Procedure The PAHs chosen were based on a high quantum yield of fluorcscence, possibility of near visible excitation and avail- ability in pure form. Before investigating the effect of chlorine on the fluores- cence of the PAH dyes it was first necessary to determine whether the process of static quenching was taking place. This was carried out by recording the absorption spectra of the PAHs in the absence and presence of chlorine in methanol. Perturbations in the absorption spectra of PAH molecules, due to chlorine, were taken as evidence of ground state quenching. Polynuclear aromatic hydrocarbons that showed static quenching were eliminated from further investigation. The effect of chlorine dissolved in methanol on the fluorescence of those dyes that did not show signs of static quenching was investigated at room temperature (22 "C).The concentration of the PAH solutions was kept constant at about 1 x 10-7 mol dm-3 in order to avoid self-quenching or inner filter effects. Experimental Reagents Polynuclear aromatic hydrocarbons of analytical-reagent grade were used as obtained. Their spectral properties were found to be in accordance with published data.8 Methanol of spectroscopic grade was dried over molecular sieves (Merck, Type 3A) and used as the common solvent. Chlorine solutions were prepared by passing gaseous chlorine through methanol. The concentration of chlorine dissolved in methanol was determined by standard iodimetric titration. *: To whom corrcspondcncc should be addressed.Table 1 Quenching of fluoresccncc of PAH moleculcs in the presence o f 3 x 10-5 mol dm-3 chlorine Polynuclear Wavelcngth of Decrcasc in aromatic cxcitationl fluorescence due hydrocarbon emissionlnm to chlorine (%) Anthraccne 9.10-Dichloroanthracene 9.10-Diphenylanthracene 9-Methylanthraccne 9-Phcnylanthracene 9-Vinylanthracenc Pcrylene Bcnzo[ghi]pcrylene Pyrene Benz[e]pyrcne Fluoranthcne 3551397 3781426 3701426 36614 10 36414 10 386141 0 3801437 3801427 3341392 3291387 3501440 41 31 26 32 30 23 39 28 22 20 984 ANALYST, JANUARY 1992, VOL. 117 Results The per cent. decrease in fluorescence of PAH molecules that did not show any static quenching, in the presence of 3 x 10-5 mol dm-3 chlorine, is shown in Table 1. All the PAH molecules show a decrease in fluorescence due to chlorine.Anthracene shows the greatest sensitivity to chlorine but has its excitation maxima too far in the ultraviolet region to be of any use in the development of inexpensive analytical techniques. Similarly, pyrene, benzo[e]pyrene and fluoranthene are not suitable owing to their short wavelength excitation. By taking 9,10-diphenylanthracene as an example, a typical fluorescence spectrum of a PAH molecule in the presence of various chlorine concentrations is shown in Fig. 1. The fluorescence intensity, I , and quencher concentration, [ Q], are related by the Stern-Volmer equation where I,) is the fluorescence intensity in the absence of quencher and Ksv is the Stern-Volmer constant. The Stern-Volmer plot of the quenching of the fluorescence of 9,lO-diphenylanthracene by chlorine is shown in Fig.2. Within the concentration range investigated, the fluorescence quenching by chlorine does not show a linear dependence as predicted by the Stern-Volmer equation. However, the Ksv value calculated using the linear part of the plot was found to be 11 120 dm3 mol-I. Discussion The results show that the fluorescence of the PAHs investi- gated is sensitive to the presence of chlorine. Fig. 2 shows a positive curvature at higher chlorine concentrations, indicat- ing the presence of more than one quenching mechanism. The diffusion-controlled bimolecular quenching constant, ko, is related to the Stern-Volmer constant by Ksv = koYo where yo is the fluorescence decay lifetime. By taking the fluorescence decay lifetime of 9,lO-diphenyl- anthracene to be 8.41 ns,8 a bimolecular quenching constant of 1.32 X 1012 dm3 mol-1 s-* was obtained.This value is larger than that predicted by purely diffusion-controlled processes. Fluorescence quenching data, obtained by intensity measure- ments alone, cannot distinguish between dynamic and static quenching. The lifetime, temperature, viscosity dependence o r careful examination of the absorption spectrum can be used to discriminate between the two processes. Observation of static quenching implies a ground state chemical reaction and might not result in the reversibility that is inherent in dynamic quenching. For this reason PAHs that showed static quench- ing, as observed by perturbations in the absorption spectra, were eliminated from further investigation.The process of dynamic quenching is a diffusion-controlled process that only takes place during the lifetime of the excited state of the molecule. As perturbations in the absorption spectra of the dyes investigated were not observed in the presence of chlorine, it has to be assumed that there is a combination of dynamic quenching with other non-radiative processes. Further, removal of chlorine from the PAH solution by a non-interfering chlorine scavenger such as sodium thiosul- phate returned the fluorescence of the dye to its original intensity. Reaction between chlorine and the PAH molecules was not observed within the concentration range investigated (<0.03 mol dm-3) and the excitation wavelengths used. Oxygen was observed to reduce the fluorescence intensity of the PAH molecules. This does not affect the quenching data provided the concentration of oxygen, or any additional quenchers, remains constant.This was taken to be the case A 380 400 420 440 460 480 500 Wavelengthlnm Fig. 1 Emission spectra of 9,lO-diphenylanthracene in the presence of various chlorine concentrations: A, 0; B. 9.2; C, 18.3; D, 27.5; E, 36.7; F, 45.8; G. 55.0; and H, 64.2 pmol dm-3 chlorine 2.0 1 I 1.8 i I .6 I Y 1.4 1.2 1 X X X X X X I .o I I I I I I 0 10 20 30 40 50 60 70 Chlorine concentration/pmol dm-3 Fig. 2 9.10-diphenylanthracene by chlorine Stern-Volmer plot of the quenching of the fluorescence of with ambient oxygen in these experiments. Where one fluorescent species is quenched by more than one quencher, the Stern-Volmer equation can be extended to take into account the contribution of the second quencher.9 With the extremely efficient quenching of the PAH molecules by chlorine, interference caused by oxygen or any other diffu- sion-controlled quencher is not thought to be significant.Conclusion The fluorescence of certain PAHs has been found to be quenched extremely efficiently by chlorine. The findings demonstrate that the PAH molecules can be applied in measurements of chlorine concentration. This might be adopted in the development of optical fibre based sensors for chlorine. Removal of the chlorine from the dye with a suitable agent will facilitate the development of a reusable sensor in which the reagent is continuously replenished. One of us (S. A. M.) acknowledges the financial support of the Science and Engineering Research Council (UK).ANALYST, JANUARY 1992, VOL. 117 85 References 1 Guilbault. G. G., Practical Fluorescence-Tlteory, Methods, and Techniques, Marcel Dekker. New York, 1973. 2 Kautsky, H.. and de Brujin, H . , Nuturwissenschufren. 1931,19, 1043. 3 Bergman, I.. Nurure (London), 1968, 218, 396. 4 Kroneis, H. W., and Marsoner, H. J., Sens. Actuators, 1983.4. 587. 5 Lubbers. D. W., and Opitz, N.. Sens. Actuators, 1983, 4, 641. 6 Li, P. Y. F., and Narayanaswamy, R., Analyst. 1989, 114,663. 7 Turner, R. M., and Fairhurst, S . , Toxicology of Substances in Relation to Mujor- Hazards. Chlorine, Health and Safety Executive, HM Stationery Office, London. 1990. 8 Berlman, I. B., Handbook of Fluorescence Spectra of Aromatic Molecules, Academic Press, New York, 1971. 9 Wolfbeis, 0. S.. Posch, H . E . , and Kroneis, H. W.. Anal. Chem., 1985.57, 2556. Paper 1 I001 31 K Received January 11, 1991 Accepted August 5, 1991