In the Laboratory
Molecular Photophysics of Acridine Yellow Studied by Phosphorescence and Delayed Fluorescence An Undergraduate Physical Chemistry Experiment Julius C. Fister III and Joel M. Harris* Department of Chemistry, University of Utah, Salt Lake City, UT 84112 Diana Rank and William Wacholtz Department of Chemistry, University of Wisconsin–Oshkosh, Oshkosh, WI 54901 Fluorescence and phosphorescence spectra yield information about the structures and relative energies of molecular electronic excited states. Lifetime measurements allow one to explore the kinetics of competing excited-state decay pathways. However, sophisticated equipment and sample preparation are generally needed to acquire fluorescence lifetime data. Most common fluorophores exhibit nanosecond excited-state lifetimes demanding short pulse excitation sources, fast detectors and digitizers, and deconvolution techniques to interpret the data. Phosphorescence, although of longer duration than fluorescence, is difficult to achieve at room temperature because rapid quenching by dissolved oxygen limits triplet state lifetimes to < 100 µs. Therefore, most laboratory experiments designed for undergraduates utilize steady-state fluorescence measurements to determine analyte concentrations or to study bimolecular quenching reactions (1–6). Even with sophisticated equipment, excited-state photophysics usually occurs on such short time scales as to render direct observation very challenging. Here we describe a dynamic demonstration of excited-state molecular photophysics that can be performed with modest equipment suitable for an undergraduate laboratory. When the organic dye acridine yellow is dissolved in a rigid glass composed of trehalose (a disaccharide) and glucose, triplet state lifetimes exceed 150 ms at room temperature. Over the temperature range from 35 to {75 °C the primary route of triplet state decay changes from thermally activated delayed fluorescence to phosphorescence. This is reflected by a 10-fold increase in lifetime and a shift from yellow-green fluorescence to an orange phosphorescence. Lifetime- and wavelength-dependent emission spectroscopies may be used to investigate parameters such as excited-state energy splittings and the rate of reverse intersystem crossing from the excited triplet to the excited singlet state (7–9). Depending upon the desired level of sophistication, data acquisition may be accomplished with light sources ranging from a camera flash to a low-power laser used in conjunction with a photomultiplier tube and low-speed digitizing oscilloscope. Alternatively, a commercial spectrofluorometer having ≈ 5 ms or better time resolution may be used. Theory Photophysical processes relevant to the formation and decay of acridine yellow excited states in a rigid solvent are shown in the energy level diagram in Figure 1. During the *Corresponding author.
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Figure 1. Three-level photophysical model for acridine yellow excited states. Absorption produces the first excited singlet state of the dye followed by relaxation to the lowest vibrational level of this electronic state. Prompt fluorescence at a rate kf occurs on a nanosecond time scale. Triplet states are produced by forward intersystem crossing, kfisc. Equilibration between vibrational levels of the triplet state occurs on a picosecond time scale, kvib. Direct decay of the triplet state occurs at a rate kt. Reverse intersystem crossing to the excited singlet manifold occurs at a rate k° risc, from triplet vibrational levels having energy that exceeds the S1-T1 energy gap, ∆E.
excitation pulse, absorption populates the first excited singlet state of the dye. Since acridine yellow has a large extinction coefficient in the visible light range, high excitation rates can be achieved even with modest light sources such as a camera flash. Within picoseconds of photon absorption, essentially all of the molecules undergo vibrational relaxation to the lowest vibrational level of the first excited singlet state. Several pathways contribute to the excited singlet state decay. A significant fraction decays within several nanoseconds, kf, via an intense yellow-green prompt fluorescence (10). Nonradiative internal conversion to the ground state, conversely, proceeds slowly in molecules like acridine yellow because the large energy gap and rigid molecular geometry prevent efficient coupling between the two potential energy surfaces (11). Finally, a large fraction of the excited singlet states undergo forward intersystem crossing to the triplet state manifold at a rate kfisc. The quantum yield of fluorescence, therefore, is given by φf = 1 – φfisc under the assumption that the contributions of other nonradiative processes to the decay of the excited singlet state are small. After intersystem crossing, relaxation to the lowest vibrational level of the first excited
Journal of Chemical Education • Vol. 74 No. 10 October 1997
In the Laboratory triplet state occurs within tens of picoseconds. In fluid solutions, quenching by molecular oxygen and impurities generally limit the triplet lifetimes of organic molecules to microseconds. However, rigid glassy matrices dramatically reduce diffusion rates, which, as a consequence, prevent bimolecular quenching from competing with the intrinsic decay processes of the excited triplet state. Internal decay routes of the triplet state that lead directly to the ground singlet state include nonradiative intersystem crossing and phosphorescence. However, an alternative decay route becomes available near ambient temperatures for triplet states of many organic dyes in rigid solution, where the first excited singlet state is repopulated following reverse intersystem crossing from the triplet state (7–9). A fraction of the singlet states decay via delayed fluorescence with a spectrum indistinguishable from the prompt fluorescence observed during excitation (7–9). Reverse intersystem crossing back to the excited singlet state can occur only from thermally excited vibrational levels of the triplet state that have energies greater than or equal to the S 1-T1 energy splitting ∆E (7–9). This thermally excited fraction of the triplet population undergoes reverse intersystem crossing at a rate, k°risc. Thermal equilibration between triplet vibrational levels occurs on a picosecond time scale, which is much faster than depopulation of the triplet state by reverse intersystem crossing, kvib >> k°risc . Therefore, the vibrationally excited triplet state populations exist in a dynamic equilibrium described by a Boltzmann distribution (8). The fact that only triplet states having vibrational energy equal to or exceeding ∆E can undergo reverse intersystem crossing reduces the actual reverse intersystem crossing rate to krisc as compared to k°risc: krisc = k°risc exp({∆E / kT)
(1)
where k is the Boltzmann constant and T is the absolute temperature. The repopulated singlet states undergo the same decay processes as those formed directly after photoexcitation. A fraction φfisc returns to the triplet state through forward intersystem crossing. The remaining fraction, 1 – φfisc = φf , fluoresces to the ground state at a rate kf. The combined rate of intersystem crossing and fluorescence is much larger than the effective rate of reverse intersystem crossing, (kf + kfisc) >> krisc. Therefore, the rate at which delayed fluorescence depopulates the triplet state, kdf, is given by kdf = φf k°risc exp({∆E/ RT) (2) In the absence of bimolecular quenching, the observed tripletstate decay rate, kobs , is determined by the sum of the rates of direct triplet decay, kt , and delayed fluorescence: kobs = kt + φf k°risc exp({∆E / RT)
(3)
Since the rates of phosphorescence and direct intersystem crossing to the ground state are not strong functions of temperature above 77 K (11), variation in the delayed fluorescence rate determines the temperature dependence of the triplet state lifetime. Subtracting the phosphorescence rate from both sides of eq 3 and taking the natural log results in a linear expression relating the rate of delayed fluorescence to the S1-T1 energy splitting and the temperature (8): ln (kobs – kt) = ln (φ fkrisc) – ∆E / RT
(4)
Analysis of eq 4 allows the singlet triplet energy splitting, ∆E, and the limiting rate of reverse intersystem crossing, k°risc , to be determined.
Figure 2. Experimental diagram for flash excitation of glass solutions. A camera flash is the excitation source. Glass filters to isolate excitation and emission wavelengths are described in the text. Sample is a Pyrex test tube containing a rigid glass solution. Photodiode is used to trigger data acquisition.
Experimental Section
Formation of Disaccharide Glass Approximately 1 g of trehalose dihydrate crystals (Sigma) were ground to a powder. An equal quantity by weight of anhydrous glucose (Mallinckrodt) was mixed with the trehalose to make a 50:50 mixture. Approximately 1.5 g of this mixture was transferred to a small Pyrex test tube. This quantity produced approximately 1 mL of sugar solution. A 0.5-mL aliquot of a 200 µM aqueous solution of acridine yellow (Aldrich) and a 1.0-mL aliquot of water were then stirred into the sugars to produce a slurry. (CAUTION: acridine yellow is a possible mutagen; avoid direct contact with the dye; in case of contact with skin, wash with soap and flush with copious amounts of water.) The tube was suspended in a glycerol bath heated to 140 °C on an electric hot-plate. Heating was continued until all the water was boiled off (approximately 45 min). It was important that the glycerol bath temperature not exceed 140° in order to prevent decomposition (yellowing) of the sugar solution. A rigid glass produces intense long-lived emission following exposure to camera flash or flashlight in a darkened room. Camera Flash Excitation The experimental arrangement for acquiring excited triplet state lifetimes is shown in Figure 2. A camera flash (Vivitar Model 2800) was used to excite the sample. The FWHM of the flash output was ≈ 10 µs, which is much shorter than the millisecond lifetimes observed in the experiment. A combination of optical filters that prevented the excitation pulse from saturating the detector but allowed efficient collection of the fluorescence and phosphorescence was used. The excitation pulse was passed through a blue dichroic short-wave pass filter (Reynard Enterprises #900) and a blue glass filter (Schott Glass BG 3). This arrangement provided efficient excitation, since the extinction coefficient of acridine yellow in trehalose / glucose glass maximizes near 450 nm. Two orange Schott glass (OG 550 2–3 mm thickness) filters were placed in front of the photomultiplier tube so that only light of ≥ 550 nm reached the detector. Data acquisition was initiated by triggering a LeCroy 9410 oscilloscope from the output of a photodiode
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In the Laboratory arranged to collect scattered light from the excitation pulse. Alternatively, the oscilloscope could be triggered from a voltage spike obtained from a coaxial cable taped to the side of the camera flash. Lifetime measurements were performed over a sample temperature range of 35 to {70 °C. The temperature of the sample was regulated by immersing the tube for several minutes in magnetically stirred slurry baths composed of ice in combination with salts or dry ice with organic solvents of known melting point (12). Bath temperatures were monitored with a thermocouple or appropriate low-temperature thermometer. Lifetime measurements were performed over a sample temperature range of 35 to {70 °C. Ten lifetime measurements were averaged at each temperature. After the acquisition of three lifetime traces at each temperature, the tube was removed from the optical path and allowed to reequilibrate in the slurry bath. To determine the wavelength origin of fluorescence and phosphorescence, spectra were acquired using the same filtered flash excitation source in conjunction with a 0.25 m spectrograph (Chromex, Model 250IS) and TE-cooled CCD detector (EG&G PAR).
Data Analysis Following data collection, the lifetime traces were saved as ASCII files and imported into Quattro Pro for Windows. The nonlinear optimizer function of QuattroPro was used to adjust the amplitude, lifetime, and baseline of each decay curve in order to minimize the sum of the squared residuals between the data and a model exponential decay curve. Uncertainties were reported at the 95% confidence level and were calculated using Student’s t statistic. The standard deviation of the lifetime estimates was estimated at ± 3 ms from 12 replicate measurements at 0 °C acquired over a 2-hour period.
Emission Spectra and Fluorescence Quantum Yield Measurements To determine the fluorescence quantum yield, quantitative fluorescence emission was measured using a PTI emission spectrometer equipped with a xenon arc lamp excitation source; this spectrometer produced time-resolved emission results similar to those from the flash setup. Emitted light was collected at 90° to the excitation source, passed through a monochromator, and detected with a Hammamatsu R928 photomultiplier. Sample temperatures were maintained with an Oxford optical Dewar. The fluorescence quantum yield of acridine yellow in a rigid saccharide matrix was determined following the approach described by Parker and Rees (13), using the quantum yield of the dye in methanol, φf = 0.55 ± 0.02, as a standard. The absorptivities of three solutions of acridine yellow were matched to within ± 0.002 absorbance units to rigid glasses containing ≈100 µM acridine yellow. The refractive index of the saccharide glass was determined by pouring a molten saccharide mixture onto the plate of a refractometer. The fluorescence intensities of each sample were obtained using 436 nm excitation and integrating the area under the fluorescence curve from 450 to 650 nm. Results Flash excitation of a 100 µM solution of acridine yellow in a rigid saccharide glass produces a long-lived, bright orange phosphorescence at temperatures below ≈ {50 °C. A shorter-lived, intense green-yellow delayed fluorescence
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dominates at higher temperatures. At intermediate temperatures a yellow-orange emission is observed, indicating that both processes contribute to the decay of the excited triplet states. The increased lifetime at reduced temperatures, coupled with the change in color, provides dramatic evidence of the underlying photophysics. Despite the high intensity of the long-lived emission, prompt fluorescence during the camera flash overwhelms delayed fluorescence and phosphorescence, since these latter processes are distributed over hundreds of milliseconds. Filters cannot be used to discriminate against the prompt fluorescence because its emission spectrum is indistinguishable from that of delayed fluorescence. Therefore, oscilloscope delay was adjusted to capture only the long-lived emission arising from the decay of the excited triplet-state population. Emission decay curves of a 25 µM acridine yellow saccharide glass at {70, {17, 0, 23, and 35 °C were acquired using the flash setup in Figure 2. The data, scaled to the same initial intensity for clarity, are shown in Figure 3. Owing to the high quantum yield of delayed emission, excellent signal-to-noise ratios can be obtained even with modest excitation and detection systems and no collection optics. Although eq 3 indicates that a single, first-order rate constant should account for the triplet state decay, the individual decay curves in Figure 3 are not well modeled by single exponential functions over their entire range. Since the local environment surrounding some of the dye molecules in the glass may differ from the average, shorter triplet lifetimes are observed for these molecules owing to site-specific interactions (14–16). To discriminate against any residual prompt fluorescence and the shorter-lived excited-state emission, the first 150 ms of each decay curve was omitted from the analysis. The decay curves beginning 150 ms after the flash were fit to single exponential functions having a characteristic lifetime τobs = 1/kobs , amplitude A, and baseline offset B: I df,p = A exp({ t / τobs) + B
(5)
The lifetimes determined from the best fits of eq 5 to the data are 1.03, 0.77, 0.46, 0.30, and 0.17 s, at { 70, { 17, 0, 23, and 35 °C, respectively. The amplitudes were not analyzed because their interpretation requires knowledge of the response of the optical system and detector as a function of wavelength. The exponential term in eq 3 predicts that the rate of delayed fluorescence decreases at low temperatures, since smaller fractions of the excited triplet population have sufficient vibrational energy to undergo reverse intersystem crossing. Since the rate of triplet decay, kobs, is given by the sum of the rates of delayed fluorescence and phosphorescence, eq 4 predicts that the triplet lifetime should increase at low temperatures, in agreement with the data in Figure 3. In order to use the measured triplet state lifetimes and eq 4 to elucidate the temperature-dependent delayed fluorescence kinetics, the natural triplet lifetime, 1/kt, must be estimated in the absence of delayed fluorescence. Two assumptions simplify the analysis. First, kt is generally not a sensitive function of temperature above {70 °C (11), and second, since the observed triplet lifetime did not increase significantly below {70 °C, the rate of delayed fluorescence must be negligible at this temperature (kt >> kdf ). Therefore, the observed lifetime at {70 °C , 1.03 s, was used as an estimate of 1/kt. The rate of delayed fluorescence, kdf = 1/τobs – kt, was then plotted as a function of 1/kT in accord with eq 4, as shown in Figure 4. The S 1-T1 energy splitting ∆E determined from the slope of the line is 2,700 ± 108 cm{1.
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Intensity (scaled)
In the Laboratory
Time (s)
ln ( 1 /τobs – 1 /τt )
Figure 3. Temperature-dependent decay of acridine yellow delayed fluorescence in a rigid saccharide glass. In order of increasing lifetime, the temperatures of the samples are 35, 23, 0, {17, {70 oC. The first 150 ms of each transient exhibit nonexponential decay; these data are omitted from fitting and are not plotted. Smooth curves represent single-exponential fits to the data.
1/kT × 103 (cm)
Intensity (photoelectrons)
Figure 4. Delayed fluorescence lifetimes from the data in Figure 3, plotted according to eq 4, as explained in the text.
25 °C
×10 {78 °C
Wavenumber × 10 {3 (cm{1 ) Figure 5. Delayed fluorescence (right) and phosphorescence (left) spectra of acridine yellow in a saccharide glass.
Equation 5 indicates that the intercept of the Arrhenius plot, 14.1 ± 0.6, contains the natural log of the product of the fluorescence quantum yield, φf, and the reverse intersystem crossing rate, k°risc. A small φf reduces the effective rate of delayed fluorescence because, with the assumption of a negligible nonradiative decay rate (11), the remaining fraction of repopulated singlet states return to the triplet state, φfisc = 1 – φf. Olmsted reported that the quantum yield of acridine yellow fluorescence in methanol was 0.55 ± 0.02 (17). Using acridine yellow in methanol as a standard (17), the fluorescence quantum yield of acridine yellow in a trehalose / glucose glass was measured to be φf = 0.33 ± 0.05, and the quantum yield of triplet state formation, φfisc = 0.67 ± 0.05. Using φf of acridine yellow in a saccharide glass, the estimated reverse intersystem crossing rate is determined to be 4.0 ± 0.7 × 106 s{1 . These results provide insight into how a balance of energetics and dynamics determines the rates of physical processes. Based upon the singlet–triplet energy splitting of 2,700 ± 108 cm{1 and the available thermal energy at room temperature (kT = 207 cm{1 at 25 °C), the Boltzmann equation predicts that only ≈ 2 out of 106 triplet states possess sufficient vibrational energy to sample the intersection of the S1 and T1 potential energy surfaces at any time. This small fraction undergoes reverse intersystem crossing on a submicrosecond time scale on the order of 1 / k°risc. In agreement with the observed tens of milliseconds triplet lifetimes, therefore, the rate at which delayed fluorescence depletes the triplet population must be limited by the energy barrier to the rapid reverse intersystem crossing event. The S1 -T1 energy splitting can also be estimated from the wavelength dependence of the fluorescence and phosphorescence emission spectra. A delayed fluorescence spectrum acquired at room temperature and a phosphorescence spectrum acquired at {70 °C are shown in Figure 5. Kasha’s rule predicts that the majority of the fluorescence and phosphorescence occurs from the lowest vibrational level of the corresponding excited state. Consequently, extrapolating the high-frequency edge of each curve to the baseline allows the excited singlet and triplet state energies relative to the ground state to be estimated as 21,200 ± 200 cm{1 and 18,300 ± 150 cm{1, respectively. The energy splitting determined from the difference of these values, ∆E = 2,900 ± 250 cm{1 , is indistinguishable from the activation energy determined from the slope of the Arrhenius plot. As described above, the limiting rate of reverse intersystem crossing can be determined from the intercept of the Arrhenius plot, k°risc = 4.0 ± 0.7 × 106 s{1. In the absence of large conformational differences between the excited states, electron spin statistics determine the relative rates of forward and reverse intersystem crossing. In such a case, the forward rate would be only slightly faster given the larger number (3 to 1) of electronic configurations in the triplet state versus the singlet state. The actual forward intersystem crossing rate can be estimated from the relationship between φf, φfisc, and the observed fluorescence lifetime. Since the quantum yield of fluorescence is ≈ 0.33 and the rate of nonradiative relaxation to the ground state is small, the forward intersystem crossing rate should be on the order of the prompt fluorescence decay rate. From φf and the observed fluorescence lifetime in ethanol of 5.1 × 10{9 s, the forward intersystem crossing rate can be estimated as kfisc ≈ 1 × 108 s{1, which is ≈ 25 times larger than the reverse intersystem crossing rate. Accounting for degeneracy of the triplet state, the 8-fold slower reverse intersystem crossing
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In the Laboratory rate is consistent with two different factors. First, nonradiative transition rates between states are proportional to the density of vibronic states in the final state that are isoenergetic with the initial state (18). Since the density of states in the vibrationally excited triplet manifold is much greater than the density of states near the lowest vibrational level of the excited singlet state, the forward intersystem crossing rate should be larger than the rate of the corresponding reverse transition. In addition, conformational differences also exist between the excited singlet and triplet states. For example, Wagner proposed that conformational differences between the excited triplet and ground state of biphenyl result in an anomalous slow rate of energy transfer from triplet state benzophenone to biphenyl (19). These and similar studies point out the importance of conformational effects in determining the rate and efficiency of excited-state photophysics and photochemistry. Discussion The investigation of temperature-dependent photophysics of acridine yellow excited states provides a simple undergraduate physical chemistry experiment that illustrates a number of fundamental concepts in unimolecular reaction kinetics. Triplet state lifetimes of acridine yellow exceed 150 ms when dissolved in a glassy saccharide host composed of trehalose and glucose, so that the excited-state decay kinetics are simple to measure. The samples are stable for 6 months or more, so that a new sample need not be prepared for each student in a lab. The temperature dependence of the color and duration of delayed emission provide dramatic, tangible evidence of the underlying photophysics. Yellow-green delayed fluorescence is the primary decay route at room temperature, whereas an orange phosphorescence predominates at lower temperatures. A linear least-squares analysis of the temperature dependence of the triple state lifetime allows both the rate of reverse intersystem crossing to the singlet state and the energy gap between the excited singlet and triplet states to be determined. The S1-T1 energy splitting determined from the wavelength dependence of the fluorescence and phosphorescence spectra was indistinguishable from the Arrhenius activation energy. The rate of reverse intersystem crossing was found to be ≈ 8 times slower than the forward intersystem crossing rate after accounting for degeneracy of the triplet state. The experiment illustrates the importance of energetic barriers in determining the rates of photophysical processes. Although the limiting rate of reverse intersystem crossing is quite large, an energy barrier that allows only a small fraction of the population to sample the intersection of S1 -T1 potential energy surfaces signifi-
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cantly reduces the effective rate. Not only does the current experiment demonstrate a number of important photophysical principles, one can envision a variety of senior research projects based on an extension of this work. Acknowledgments This work was supported in part by National Science Foundation under grants CHE90-10319 and CHE95-10312. Summer support for D.R. was provided by the a National Science Foundation REU grant CHE93-00381 and the University of Utah chemistry department. Fellowship support for J.C.F. was provided by the American Chemical Society Division of Analytical Chemistry and Dupont. We thank Lydia Olsen for assisting with acquisition of the emission spectra. Literature Cited 1. Fraiji, L. K.; Hayes, D. M.; Werner, T. C. J. Chem. Educ. 1992, 69, 424–428. 2. Byron, M. C.; Werner, T. C. J. Chem. Educ. 1991, 68, 432– 436. 3. Legenza, M. W.; Marzzacco, C. J. J. Chem. Educ. 1977, 54, 183–184. 4. Ebeid, El-Z. M. J. Chem. Educ. 1985, 62, 165–166. 5. Marciniak, B. J. Chem. Educ. 1986, 63, 998–1000. 6. Sacksteder, L.; Ballew, R. M.; Brown, E. A.; Demas, J. N.; Nesselrodt, D.; DeGraff, B. A. J. Chem. Educ. 1990, 67, 1065–1067. 7. Gilbert, A.; Baggott, J.; Essentials of Molecular Photochemistry; Blackwell Scientific: Cambridge, 1991; Chapter 4. 8. Parker, C. A.; Hatchard, C. G. Trans. Faraday Soc. 1961, 57, 1894. 9. Perrin, F. Ann. Phys. (Paris) 1929, 12, 169–275. 10. Berlman, I. B. Handbook of Fluorescence Spectra of Aromatic Molecules; Academic: New York, 1971. 11. Turro, N. J. Modern Molecular Photochemistry; Benjamin/ Cummings: Menlo Park, CA, 1978. 12. Perrin, D. D.; Armarego, W. L. F.; Perrin, D. P. Purification of Laboratory Chemicals, 2nd ed.; Pergamon: New York, 1980; p 46. 13. Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587–600. 14. Fister, J. C.; Rank, D.; Harris, J. M. Anal. Chem. 1995, 67, 4269–4275; Fister, J. C.; Harris, J. M. Anal. Chem. 1996, 68, 639–646. 15. Rosenberg, J. L.; Shombert, D. J. J. Am. Chem. Soc. 1960, 82, 3252–3257. 16. Tick, P. A.; Hall, D. W. Diffus. Defect Data 1987, 53/54, 179– 188. 17. Olmsted, J. O., III. J. Phys. Chem. 1979, 83, 2581–2584. 18. Avouris, P.; Gelbart, W. M.; El-Sayed, M. A. Chem. Rev. 1977, 77, 793–833. 19. Wagner, P. J. J. Am. Chem. Soc. 1967, 89, 2820–2825.
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