Chemiluminescence Energy Transfer Processes and Micellar Effects

Energy transfer in the acridinium−hydrogen peroxide chemiluminescence ... Colloids and Surfaces A: Physicochemical and Engineering Aspects 2005 254,...
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Langmuir 1997, 13, 2675-2680

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Chemiluminescence Energy Transfer Processes and Micellar Effects M. L. Grayeski* Research Corporation, 101 North Wilmont Road, Suite 250, Tucson, Arizona 85748

P. A. Moritzen Department of Chemistry, Seton Hall University, South Orange, New Jersey Received September 30, 1996. In Final Form: March 3, 1997X The first study of intermolecular energy transfer processes in a chemiluminescence system is reported. Energy transfer in the acridinium-hydrogen peroxide chemiluminescence system, observed for the first time, is evaluated for radiative, dipole-dipole, and collisional components in the presence and absence of micelles: cetyltrimethylammonium bromide, Brij-35, and sodium lauryl sulfate. In surfactant solutions, the major components were found to be radiative and collisional. Changing the surfactant had the greatest effect on the dipole-dipole component. Effects can be attributed to ion-ion forces, the mixing process, and the path length of the chemiluminescence measurement. In contrast to photoexcited energy transfer studies, the Rhodamine 110 (acceptor) concentration is varied by almost 4 orders of magnitude and is up to 105 greater than the acridinium (donor) concentration. This can be attributed to the absence of excitation absorption in a chemiluminescence measurement and the increased solubility due to the surfactants. The semiempirical method used for evaluating energy transfer should be adaptable to other systems and requires only the spectra of the donor and acceptor and the measurement of chemiluminescence intensity as a function of acceptor concentration.

Introduction Energy transfer has been observed in several chemiluminescence systems1-8 and surfactants have been shown to enhance emission from chemiluminescence reactions,9-11 but neither has been investigated with respect to contributions of energy transfer mechanisms. Understanding the factors influencing these contributions would be useful for designing chemiluminescence measurements, optimizing sensitivity, and interpreting data. The acridinium-hydrogen peroxide chemiluminescence reaction was chosen for study for several reasons. A wide concentration range can be studied due to the high efficiency achieved. It can be a very fast reaction, permitting the entire chemiluminescence emission to be measured in a reasonable time, thereby eliminating reaction kinetics from consideration. Acridinium esters react with hydrogen peroxide in the presence of base to yield excited state N-methylacridone (NMA), which then fluoresces. Emission occurs around 460 nm and is detectable at picomolar acridinium concentrations. The reaction has been extensively studied12,13 X

Abstract published in Advance ACS Abstracts, April 15, 1997.

(1) (1)Kalkar, C. D.; Doshi, S. V.; Ranade, M. Indian J. Pure Appl. Phys. 1989, 27, 813. (2) Lechtken, P.; Turro, N. J. Mol. Photochem. 1974, 6, 95. (3) Roswell, D. F.; Paul, V.; White, E. H. J. Am. Chem. Soc. 1970, 92, 4855. (4) Roberts, D. R.; White, E. H. J. Am. Chem. Soc. 1970, 92, 4861. (5) Garner, T. W.; Yeung, E. S. Anal. Chem. 1990, 62, 2193. (6) Adam, W.; Cueto, O. J. Am. Chem. Soc. 1979, 101, 6511. (7) Campbell, A. K.; Patel; A. Biochem. J. 1983, 216, 185. (8) Hinze, W. L.; Srinivasan, N.; Smith, T. K.; Igarashi, S.; Hoshino, H. In Advances in Multidimensional Luminescence; Warner, I. M., McGowen, L. B., Eds.; JAI Press: Greenwich, CT, 1990; Vol. 1, pp 149206. (9) Hinze, W. L.; Riehl, T. E.; Singh, H. N.; Baba, Y. Anal. Chem. 1984, 56, 2180. (10) Nikokavouras, J.; Vassilopoulos, G. Z. Phys. Chem. 1984, 265, 618. (11) Gundermann, K. D.; McCapra, F. Chemiluminescence in Organic Chemistry; Springer-Verlag: New York, 1987. (12) McCapra, F. In Progress in Organic Chemistry; Carruthers, W., Sutherland, J. K., Eds.; Wiley: New York, 1973; Vol. 8, pp 231-237. (13) McCapra, F. M. Acc. Chem. Res. 1976, 9, 201.

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and used in analytical and biological studies. Surfactant micelles increased acridinium chemiluminescence efficiency by the presence of either the cationic surfactant cetyltrimethylammonium bromide (CTAB), the nonionic surfactant Brij-35, or the anionic surfactant sodium lauryl sulfate (SLS).14-16 Proposed mechanisms attribute the micellar-enhanced chemiluminescence to the microenvironment of the micelle. However, the influence of micelles on the energy transfer processes has not been investigated. The xanthene dye rhodamine 110 was chosen as the energy transfer acceptor since its properties allow all three of the energy transfer processes. There is a large overlap of the NMA emission and the rhodamine 110 absorbance spectra which should enhance energy transfer. A relatively large separation of the acceptor emission maxima and absorbance maxima (approximately 35 nm) allows the measurement of the acceptor emission. In addition, rhodamine 110 has a high fluorescence efficiency,17,18 has been an analyte in biological studies,19 and has appreciable water solubility. This paper investigates the influence of micelles on energy transfer processes in the acridinium-H2O2 chemiluminescence reaction. Chemiluminescence energy transfer is evaluated by developing a model to predict the chemiluminescence donor emission in the presence of radiative, dipole-dipole, and collisional energy transfer. Micellar effects are demonstrated by a comparison of the predicted chemiluminescence emission to the experimental data obtained in the presence and absence of surfactant micelles. This is the first study of energy transfer in the (14) Howie, C. L.; Grayeski, M. L. In Bioluminescence and Chemiluminescence, New Perspectives; Scholmerich, J., Andreesen, R., Kapp, A., Ernst, M., Woods, W. G., Eds.; John Wiley: New York, 1987; pp 415-418. (15) McCapra, F.; Roth, M.; Hysert, D.; Zaklika, K. A. In Chemiluminescence and Bioluminescence; Cormier, M. J., Hercules, D. M., Lee, J., Eds.; Plenum Press: New York, 1973; p 313. (16) Bagazgoitia, F. J.; Garcia, J. L.; Diequez, C.; Weeks, I.; Woodhead, J. S. J. Biolumin. Chemilumin. 1988, 2, 121. (17) Drexhage, K. H. In Dye Lasers, 2nd ed.; Scha¨fer, F. P., Ed.; Springer-Verlag: New York, 1977; Chapter 4. (18) Kubin, R. F.; Fletcher; A. N. J. Lumin. 1982, 27, 455. (19) Vera, M. M. J. Liq. Chromatogr. 1989, 12, 583.

© 1997 American Chemical Society

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Table 1. Contrast of Photexcitation and Chemiexcitation Methods for Evaluating Energy Transfer feature

photoexcitation

chemiexcitation

excitation source mixing acceptor concentration emission

external none limited by coabsorption of excitation source energy most intense from the excitation source side

internal required to initiate the reaction restricted only by the acceptor solubility equal in all directions

acridinium-H2O2 chemiluminescence reaction, the first study of simultaneous intermolecular energy transfer processes in a chemiluminescence system, and the first report of micellar effects on chemiluminescence-initiated intermolecular energy transfer. Theory The interpretation of energy transfer data is made difficult by the simultaneous contributions of several energy transfer processes. Photoexcitation studies frequently simplify data interpretation by designing experiments where the measured emission is dominated by a single energy transfer process (Table 1). This may involve limiting the concentration range to less than 1 order of magnitude with the acceptor concentration lower than the donor concentration to reduce coabsorption of the excitation radiation, measuring the emission from the same side of the cell as the excitation to reduce the contribution of radiative energy transfer, and measuring the emission from a static system (no mixing) to minimize collisional energy transfer. These techniques used in photoexcitation studies to favor a single dominant energy transfer process are not applicable to chemiluminescence for two reasons. Chemiluminescence emission originates throughout the solution rather than at one face of the cell, making it difficult to reduce the contribution of radiative energy transfer experimentally. Chemiluminescence reactions are frequently initiated by rapidly mixing two or more reagents, increasing the collisional energy transfer efficiency. A model is developed here to evaluate the contributions of dipole-dipole, radiative, and collisional energy transfer to the measured chemiluminescence emission. Energy transfer efficiencies Φrad (radiative), Φdd (dipole-dipole), and Φcol (collisional) are combined in eq 1 to obtain a theoretical chemiluminescence signal, ITheory. Application

Itheory ) (1 - Φrad)(1 - Φdd)(1 - Φcol)

(1)

of this model to chemiluminescence data assumes (a) the molecule absorbing the radiation does not reemit that energy within the wavelength range being detected, (b) the absorbing species concentration and absorbance spectra are known, (c) the emission spectrum of the emitting species is known, (d) the acceptor is homogeneously distributed.

Φrad ) 1 - (I′/I°)

(2)

Radiative energy transfer efficiency is defined by eq 2 where I° and I′ are the total donor emissions excluding and including radiative energy transfer, respectively. I° can be assumed as unity since the transfer efficiency is defined by the relative decrease in emission. I′ is the emission leaving the reaction cell and can be calculated from Beer’s law when the absorbtion spectrum of the energy transfer acceptor and the emission spectrum of the donor are known. This is expressed as I′ ) ∑{I°(Ifl(λ)/ Ifl)10-b(λ)c} dλ, where Ifl(λ) ) fluorescence emission intensity at wavelength λ, Ifl ) total fluorescence emission integrated over the wavelength range, b ) average absorption path length,  ) molar absorptivity of the energy transfer acceptor, and c ) molar concentration of

the acceptor.

Φdd ) R06/(R06 + r6)

(3)

Dipole-dipole energy transfer efficiency, first described by Fo¨rster,20-24 is approximated by eq 313,25 where “r” is the donor-acceptor intermolecular distance and “R0” is the critical transfer distance. Distance “r” has been estimated for homogeneous systems from the acceptor concentration where the acceptor is present in a large excess over the donor.5 Critical transfer distance is estimated from the equation R0 ) [(9000(ln 10)K2Φd)/ (128π5n4N)]J, where K is the relative spatial orientation factor of the donor and acceptor transition dipoles (assuming a random orientation, K2 ) 2/3), Φd is the donor fluorescence quantum yield, J is the overlap integral, n is the refractive index of the medium, and N is Avogadro’s number. The overlap integral, J, describes the degree of overlap between the donor fluorescence and the acceptor absorbance properties. Fo¨rster defined J as J ) [∫(λ) Ifl(λ) λ4 dλ]/[∫Ifl(λ) dλ], where (λ) is the acceptor molar absorptivity and Ifl(λ) is the donor fluorescence intensity at wavelength λ. Collisional energy transfer efficiency is calculated from eq 4 where KQ is the Stern-Volmer constant and [A] is the acceptor concentration. The Stern-Volmer constant is obtained from Stern-Volmer plots of the emission data after removing the effects of the filter, radiative energy transfer, and dipole-dipole energy transfer.

Φcol ≡ 1 - (1/(1 + KQ[A])

(4)

Coabsorption of the excitation radiation is not included in the model. Although an important consideration in fluorescence studies, it is not relevant to chemiluminescence since the donor is the only excitation source. This feature also explains why the acceptor concentration can be much greater than the donor concentration and energy transfer can be examined over wide acceptor concentration ranges. Procedure/Experimental Instrumentation. Chemiluminescent measurements were made on a dual photomultiplier (PMT) system with PMTs positioned 180° to each other (Figure 1). Optical low-pass and high-pass filters were included to measure the NMA and rhodamine 110 emissions, respectively. Filters, PMTs, and power supplies were combined as: [550 nm high pass filter (Corion Corp., Holliston, MA, #LL-550-F), Hamamatsu R268 PMT, model 227 power supply], and [460 nm low pass filter (Ealing ElectroOptics, Holliston, MA, #35-5248), EMI 9824B PMT, model 20403L power supply]. PMT housing assemblies were from Products for Research, Danvors, MA, Model PR1401, rf and power supplies were from Pacific Precision Instruments, Concord, CA. Signals were integrated (Spectra-Physics, Piscataway, NJ, #SP4290) with 422 Ω resistors across the input terminals of each integrator. (20) Fo¨rster, Th. Discuss. Faraday Soc. 1959, 27, 7. (21) Fo¨rster, Th. Z. Electrochem. 1969, 64, 157. (22) Fo¨rster, Th. Naturwissenschaften 1946, 33, 166. (23) Fo¨rster, Th. Ann. Phys. 1948, 2, 55. (24) Fo¨rster, Th. In Comparative Effects of Radiation; Burton, M., Kirby-Smith, J. S., Magee, J. I., Eds.; John Wiley: New York, 1960. (25) Niu, E. P.; Ghiggino, K. P.; Smith, T. A.; Mau, A. W. H. J. Lumin. 1990, 46, 191.

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Figure 1. Instrumentation design for chemiluminescence measurements: syringe (S), reaction cell (X), low pass filter (L), high pass filter (H), photomultiplier tube assemblies (P1 and P2), power supplies (HV1 and HV2), output integrators (O1 and O2). Absorbance measurements were made on a Hewlett-Packard diode array spectrophotometer with a 2 nm resolution and a minimum of a 1 s integration. Fluorescence measurements were obtained on a Spex fluorometer with a 2 nm resolution and 0.5 s integration time. A micro pH electrode was used to measure solution pH in the reaction cell before and after the chemiluminescent reaction to assure pH stability. Reagents. Four aqueous solvent systems were used to prepare the rhodamine 110 solutions: water, cetyltrimethylammonium bromide (CTAB), polyoxyethylene(23)dodecanol (Brij35), and sodium lauryl sulfate (SLS). Aqueous surfactant solutions of CTAB, Brij-35, and SLS were prepared at 25 mM. This concentration is above the critical micelle concentration for all three surfactants.26 Laser grade rhodamine 110 (Kodak Inc., Rochester, NY) was used as the energy transfer acceptor (ΦFl ) 0.85-0.92 in ethanol18,27). Saturated solutions of rhodamine 110 were prepared in each of the four solvents. Final concentrations were determined spectrophotometrically as a dilute solution in HPLC grade methanol (approximately 1 µM). Hydrogen peroxide solution was made by a suitable dilution of a 30% stock solution (Fisher Scientific). A solution of 60 mM H2O2 was used for all experiments except for the SLS system where the peroxide concentration was increased to 200 mM to increase the rate of the reaction.14 The acridinium ester used was 2,4,6-trichlorophenyl-10-methylacridinium-9-carboxylate fluorosulfonate (synthesized by T. J. Novak28) and was stored as a 1 mM solution in acetonitrile. A working solution was made by dilution in water which had been adjusted to pH 3 with nitric acid to prevent hydrolysis.29 A buffer of 0.1 M phosphate pH 9.0 was prepared using potassium phosphate, dibasic, and dilute phosphoric acid. This buffer was used for all experiments except that involving SLS. Since the potassium salt of lauryl sulfate is insoluble in water,30,31 a 0.1 M pH 9 disodium hydrogen phosphate buffer was used for the SLS studies. Hydrogen peroxide, rhodamine 110, and acridinium ester solutions were made fresh daily. All other solutions were kept less than 1 week. Triply distilled water was used in the preparation of all solutions. Chemiluminescence Measurements. An appropriate amount of rhodamine 110 was added to an empty 1.6 mL polypropylene reaction tube (8 × 50 mm, Turner Designs, Sunnyvale, CA, no. 20-015) followed by enough solvent to bring the cell volume to 450 µL. Buffer (50 uL) and hydrogen peroxide (26) Fendler, J. H. Membrane Mimetic Chemistry; John Wiley: New York, 1982. (27) Drexhage, K. H. In Dye Lasers, 2nd ed.; Scha¨fer, F. P., Ed.; Springer-Verlag: New York, 1977; p 149. (28) Grayeski, M. L.; Novak, T. J.; Mohan, A. G. In Bioluminescence and Chemiluminescence, Current Status; Stanley, P. E., Kricka, L. J., Eds.; John Wiley & Sons: New York, 1990; p 131. (29) Hammond, P. W.; Wiese, W. A.; Waldrop, A. A., III; Nelson, N. C.; Arnold, L. J., Jr. J. Biolumin. Chemilumin. 1991, 6, 35. (30) Klopf, L. L.; Nieman, T. A. Anal. Chem. 1984, 56, 1539. (31) Hinze, W. L.; Riehl, T. E.; Singh, H. N.; Baba, Y. Anal. Chem. 1984, 56, 2180.

Figure 2. Experimental chemiluminescence emission before (0) and after stepwise removal of the energy transfer components radiative (3), and dipole-dipole (O), and collisional ([). Solvents are as follows: (A) no surfactant, (B) CTAB, (C) Brij35, (D) SLS. Conditions: pH 9, 8.9 mM phosphate buffer, 0.2 mM surfactant, 8.9 µM acridinium, 1.1 mM hydrogen peroxide except 3.6 mM for the SLS solvent system. (10 µL) were added, the mixture was vortexed, and the cell was placed in the sample holder of the detector. The reaction was initiated by injecting 50 µL of 0.1 µM acridinium solution into the sample cell. The emission was integrated until the emission returned to near zero. Experiments were performed in duplicate for each rhodamine concentration. Absorbance Spectra. Spectra of aqueous rhodamine 110 solutions were used to calculate the overlap integral, J, and determine rhodamine 110 concentrations. A molar absorptivity of 84000 M-1 cm-1 at 500 nm was obtained from a spectrum of a rhodamine 110 standard solution in alkaline HPLC grade methanol. A dilute methanol solution was chosen to assure the absence of rhodamine dimers.17 Methanol was made alkaline by the addition of 2 drops of 2.5 N aqueous NaOH to 250 mL of methanol. An alkaline solution was required to assure that all of the rhodamine 110 was present in a single form.17 Concentrations of the aqueous rhodamine 110 solutions used for the chemiluminescence measurements were determined spectrophotometrically after diluting a 100 µL aliquot of the test rhodamine 110 solutions to 5 mL with alkaline HPLC grade methanol. Concentrations were determined to be 40 µM in H2O, 310 µM in CTAB, 910 µM in Brij-35, and 420 µM in SLS. Fluorescence Spectra. The fluorescence emission spectrum of the spent reaction mixture in the absence of rhodamine 110 was acquired. The reaction solution was made identically to those described previously for the chemiluminescence measurements. Fluorescence excitation and emission spectra of the rhodamine 110 solutions were also obtained. General Procedure. The total chemiluminescence emission of the donor was measured as described above. All conditions were kept constant except the acceptor concentration, which was varied to include a wide concentration range. The emission spectrum of the donor and absorbance spectrum of the acceptor were acquired under the same solvent conditions as the chemiluminescence experiments. Energy transfer efficiencies were calculated using eqs 2, 3, and 4. The Stern-Volmer constant was obtained from SternVolmer plots of the emission signals after removing the influence

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Figure 3. Calculated energy transfer efficiencies. Efficiencies: radiative + filter (O); radiative (0); collisional (s); dipoledipole (×). Solvents are as follows: no surfactant, CTAB, Brij35, SLS. of radiative and dipole-dipole energy transfer, as well as the filter. Emission loss by the filter was accounted for by including the filter transmission spectrum, f(λ), in the radiative energy transfer efficiency calculations: Φrad ) 1 - ∑{f(λ) I′(λ)} dλ. Theoretical chemiluminescence signals were calculated using eq 1.

Figure 4. Fractional energy transfer contributions. Efficiencies: radiative (0); collisional (O); dipole-dipole (×). Solvents are as follows: no surfactant, CTAB, Brij-35, SLS.

Results Verification of Energy Transfer. Energy transfer was shown for the first time to occur in acridinium chemiluminescence by observation of a decreasing donor emission and increasing acceptor emission as the acceptor concentration was increased. Acceptor emission decreased at the higher rhodamine concentrations due to selfquenching processes. Donor emission is shown in Figure 2 (0). Energy Transfer Efficiency. Theoretical energy transfer efficiencies are shown in Figure 3. The radiative energy transfer efficiency shown is the theoretical efficiency (eq 2) in the absence of other energy transfer rather than the effective contribution to the total energy transfer efficiency. As nonradiative energy transfer efficiency increases, the effective contribution of radiative energy transfer decreases. If the nonradiative transfer efficiency equaled unity, then radiative energy transfer would have no contribution to the total transfer efficiency irrespective of the theoretical radiative transfer efficiency. The fractional contribution of each energy transfer process for the surfactant containing systems is shown in Figure 4. Dipole-dipole energy transfer efficiency was calculated from eq 3. Critical transfer distance, R0, was obtained where K2 ) 2/3 and Φd ) 0.85.31 The results given in nanometers are as follows: no surfactant ) 5.0; CTAB ) 5.0; Brij-35 ) 5.0; SLS ) 4.9. The overlap integral expressed as cm6 mol-1 was calculated as follows: no surfactant ) 8.4 × 10-14, CTAB ) 8.3 × 10-14, Brij-35 ) 8.9 × 10-14, SLS ) 8.2 × 10-14. The spectral overlap for

Figure 5. NMA fluorescence emission (‚‚‚) and Rhodamine 110 absorbance spectra (s).

one solvent system is graphically shown in Figure 5. The overlap integral was not changed significantly by adding

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Figure 6. Stern-Volmer plots of the measured chemiluminescence emission after removing the radiative and dipoledipole energy transfer components. Solvents are as follows: (A) no surfactant; (B) CTAB; (C) Brij-35; (D) SLS.

Figure 7. Comparison of the experimentally obtained chemiluminescence emission (O) with that calculated from the model (s) (eq 1). Solvents are as follows: no surfactant, CTAB, Brij35, SLS.

surfactant although the molar absorptivity increased almost to the level obtained in alcohol. This was due to the spectral shifts of the NMA emission to shorter wavelengths and the rhodamine 110 absorption spectrum to longer wavelengths, decreasing the degree of spectral overlap. Collisional energy transfer efficiency was obtained from eq 4. The Stern-Volmer constant, KQ, was obtained from Stern-Volmer plots of the chemiluminescence signal after removing the filter loss, radiative energy transfer, and dipole-dipole energy transfer. The plots are shown in Figure 6. In the absence of surfactant, no linear relationship is found, indicating that the collisional mechanism is not a major contributor to the total energy transfer efficiency. The plot of the CTAB data shows the best fit to the SternVolmer equation (r2 ) 0.94), with a Stern-Volmer constant of 6598 M-1. The Brij-35 and SLS data show some positive curvature at the highest rhodamine 110 concentrations, and the deviation at comparable rhodamine concentrations is greatest for the SLS system. Also, the deviation appears at lower concentrations in the SLS system than in the Brij-35 system. Deviation from linearity can be explained by errors in the calculation of the theoretical dipole-dipole efficiency. The slope of the dipole-dipole energy transfer efficiency curve in Figure 3 rapidly increases at high acceptor concentration due to the r6 dependence of the efficiency equation (eq 3). If the effective rhodamine concentration differs from the bulk solution concentration, then the estimate of the donor-acceptor intermolecular distance, r, is incorrect. When the effective concentration is greater than the bulk concentration, the actual dipole-dipole efficiency is greater than that estimated and the predicted emission will be too high. This is seen as a positive deviation in the Brij-35 (nonionic surfactant) and SLS (anionic surfactant) Stern-Volmer plots. The increased effective rhodamine concentration is presumably due to the solubilization of the dye in the micelle, decreasing the

donor-acceptor distance. The order of increasing deviation is CTAB (cationic), Brij-35 (nonionic), and then SLS (anionic). This is attributed to ionic interaction between cationic rhodamine and surfactant. Since any error in the dipole-dipole energy transfer efficiency is reduced at low concentration, the SternVolmer constants for the Brij-35 and SLS systems were obtained from a linear fit of the data at low rhodamine concentration in order to minimize the effect of any error in the dipole-dipole efficiency. They were 6400 and 4400 M-1, respectively. Combination of Energy Transfer Processes. Equation 1 combines the theoretical energy transfer efficiencies to predict the dependence of the chemiluminescence emission on acceptor concentration and to compare the theoretical dependence with the experimental data. Comparison to experimental data is made possible by multiplying ITheory by the experimentally measured emission in the absence of rhodamine 110 (no energy transfer), thereby scaling the theoretical data to the experimental. Results are shown in Figure 7. Discussion Chemiluminescence energy transfer was evaluated in terms of radiative, dipole-dipole, and collisional energy transfer components over an acceptor concentration range of 1 to 800 µM, the solubility limit of rhodamine 110. Acceptor concentration was able to greatly exceed the donor concentration (9 nM) since the donor was the only excitation source. The major energy transfer components were found to be radiative and collisional with no significant difference among the three surfactant systems. In the absence of surfactant, the emission after removing the effects of the filter and radiative energy transfer, I°, is virtually constant and independent of rhodamine concentration (Figure 2A, 3), indicating that radiative energy transfer is the only significant energy transfer process. However, the surfactant containing systems (Figure 2B,C,D) are still highly dependent upon acceptor

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concentration, indicating the presence of an additional energy transfer process. Removing the dipole-dipole energy transfer component from I° has a relatively small effect on the calculated emission, even at high acceptor concentration. This is expected based on the small dipole-dipole energy transfer efficiency shown in Figure 3 but is in contrast to a similar system which used photoexcitation to study energy transfer from acridine orange to rhodamine B in alcohol.25 Acridine was present at approximately 1 mM, and rhodamine B ranged from 0.7 to 2.9 mM. Dipole-dipole energy transfer was reported as the energy transfer process. The overlap integral was reported to be about 4.2 × 10-13 cm6 mol-1 compared to the 8.2 to 8.9 × 10-14 cm6 mol-1 reported here. A lower dipole-dipole transfer efficiency is therefore expected for the chemiluminescent systems. A more significant difference between the two studies are the concentrations. Dipole-dipole energy transfer is highly dependent upon the concentrations due to the r6 dependence shown in eq 3. The lower dipoledipole transfer efficiencies reported here are therefore understandable. The use of K2 ) 2/3 in the dipole-dipole efficiency calculations assumes a random orientation which is unlikely in a micellar medium. However, several theoretical and experimental studies have shown K2 ) 2/3 may be a valid approximation even in a highly rigid system.32-34 Another source of error is the calculation of the donor-acceptor distance, r, which assumes the donor and acceptor to be randomly and homogeneously distributed throughout the solution. This may be true in the absence of surfactant but is unlikely in the presence of micelles. Stern-Volmer plots of the surfactant-containing systems had some deviation from linearity, particularly at the high rhodamine 110 concentrations. The degree of deviation increases in the order CTAB (cationic), Brij-35 (nonionic), and then SLS (anionic). This order is attributed to ionic interactions between cationic rhodamine and surfactant, creating a nonrandom distribution and thereby producing errors in the calculated distance “r”. Changing the surfactant most affected the dipole-dipole (32) Lindig, B. A.; Rodgers, M. A. J. Photochem. Photobiol. 1980, 31, 617. (33) Marsh, D. J.; Lowey, S. Biochemistry 1980, 19, 774. (34) Haas, E.; Katchalski-Katzir, E.; Steinberg, I. Z. Biochem. 1978, 17, 5064.

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energy transfer efficiency, observed as deviations in the Stern-Volmer plots and in the plots of chemiluminescence emission after removing all of the calculated components. The order of deviation, CTAB < Brij-35 < SLS, is attributed to ion-ion interactions which reduce the actual donor-acceptor distance relative to the theoretical distance. Rhodamine is a cationic dye which may concentrate within the anionic surfactant micelle (SLS) and be repelled by the cationic surfactant micelle (CTAB). This effectively increases the rhodamine concentration relative to the bulk concentration in the presence of SLS or Brij-35, increasing the dipole-dipole energy transfer efficiency above the theoretical value. In the absence of surfactant, the Stern-Volmer plot shows a scatter of data (Figure 6A) indicating that collisional energy transfer is not a major contributor to the total energy transfer efficiency. In the presence of surfactants, the Stern-Volmer plots yielded SternVolmer constants which are much greater than the theoretical maximum of 10 M-1 expected for a diffusioncontrolled aqueous solution.35 This estimated maximum suggests that diffusion-controlled collisional energy transfer is negligible at acceptor concentrations below 1 mM. This is in contrast to the transfer efficiencies reported here. The Stern-Volmer constants are relatively large due to the vigorous mixing process and a rapid chemiluminescent reaction, increasing the collision rate above that of diffusion control. Combining the theoretical transfer efficiencies to predict the emission signal showed good agreement with the experimental data in all four solvent systems over the full acceptor concentration range (Figure 7). Errors in the dipole-dipole energy transfer efficiency are not observed since the proportionate contribution of dipole-dipole energy transfer to the total energy transfer efficiency is small. This procedure may be applied to other chemiluminescence energy transfer systems provided the assumptions are true. Advantages of this approach which have been demonstrated are the wide acceptor concentration range, few experiments are required, and the ability to factor out energy transfer information. LA9609480 (35) Ingle, J. D.; Crouch, S. R. Spectrochemical Analysis; Prentice Hall: Englewood Cliffs, NJ, 1988; p 343.