Anal. Chem. 1997, 69, 1946-1951
Phosphorescence Properties of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Benzo[f]quinoline in Glucose Glasses via Temperature Variation and Spectral Characterization Jiangshan Wang and Robert J. Hurtubise*
Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071
Phosphorescence intensities and lifetimes of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and benzo[f]quinoline (B[f]Q) in glucose glasses were measured over a wide range of temperatures. As the temperature decreased, both the phosphorescence lifetimes and the phosphorescence intensities for PhIP and B[f]Q in glucose glasses increased. However, the phosphorescence intensity-to-lifetime ratio was not constant at different temperatures, which indicated that the triplet state formation efficiency was a function of temperature. Also, the ln(1/τp - 1/τp0) vs 1/T plots for both PhIP and B[f]Q in glucose glasses with and without a heavy-atom salt gave nonlinear plots. From the plots for the samples with and without a heavy-atom salt, activation energies were obtained. The activation energies were related to lowfrequency vibrational modes in the solid matrix, rotational relaxation of functional groups, and diffusion of water in the solid matrix. Infrared spectrometry revealed water in the glasses, and phosphorescence decay curves and confocal laser scanning microscopy showed the heterogeneity of the distribution of NaI in the glucose glasses. Room-temperature fluorescence (RTF) and room-temperature phosphorescence (RTP) have become important methods for characterizing and determining trace amount of organic compounds.1,2 Solid-matrix luminescence is one of the most widely used techniques in obtaining strong RTF and RTP.2-4 Although many solid matrices have been used, such as filter paper and cyclodextrin/sodium chloride powders, they have relatively high background signals compared with those of solution luminescence, and the sample preparation procedures are very critical in obtaining reproducible data. Therefore, it is important to develop new solid matrices with lower background signals and with simpler sample preparation techniques. Recently, details on the preparation of samples, experimental conditions, and analytical figures of merit were reported for several lumiphors in glucose and trehalose that formed clear glasses at (1) Vo-Dinh, T. Room Temperature Phosphorimetry for Chemical Analysis; John Wiley & Sons: New York, 1984. (2) Hurtubise, R. J. Phosphorimetry, Theory, Instrumentation, and Applications; VCH Publishers, Inc.: New York, 1990. (3) Hurtubise R. J. Solid-Surface Luminescence Analysis; Marcel Dekker, Inc.: New York, 1981. (4) Gunshefski, M.; Santana, J.; Stephenson, J.; Winefordner, J. D. Appl. Spectrosc. Rev. 1992, 27 (2), 143-149.
1946 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
room temperature.5,6 The RTF and the RTP signals of several model compounds obtained in the glucose glasses and trehalose glasses were very strong, and the glasses had low blank signals.5 For the glasses without a heavy atom present, the limits of detection were in the subnanogram range,5 and for the glasses with a heavy atom present, the limits of detection were in the picogram range.6 Also, the RTF and the RTP signals of all the compounds studied in the sugar glasses were stable under ambient conditions; that is, they were not susceptible to ambient oxygen and moisture quenching as with other kinds of solid matrices, such as filter paper.5 In addition, the sample preparation procedure was simple.5 In related work, Fister et al.7 used acridine yellow dissolved in a rigid saccharide glass as a sensor material for optical thermometry. Also, time-resolved and wavelengthresolved delayed fluorescence have been studied for acridine yellow in a saccharide glass.8 Experimentally, glucose forms a glass much more easily than trehalose.5 This was especially true with a heavy-atom salt present.6 Although trehalose can be used as a solid matrix in trace organic analysis, because of the ease of preparing glucose glasses, glucose glasses were used in the experiments reported in this work.5,6 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and benzo[f]quinoline (B[f]Q) were used as model compounds. PhIP is a recently found toxic compound in foods, and it is suspected of causing cancer.9-13 B[f]Q was previously studied in other solid matrices13-18 and was used in this work to compare its solid-matrix luminescence properties from the glucose glasses with earlier solid-matrix luminescence data. (5) Wang, J.; Hurtubise, R. J. Appl. Spectrosc. 1996, 50, 53-58. (6) Wang, J.; Hurtubise, R. J. Anal. Chim. Acta 1996, 332, 299-305. (7) Fister, J. C.; Harris, J. M. Anal. Chem. 1995, 67, 4269-4275. (8) Fister, J. C.; Harris, J. M. Anal. Chem. 1996, 68, 639-646. (9) Felton, J. S.; Knize, M. G.; Shen, N. H.; Lewis, P. R.; Andresen, B. D.; Happe, J.; Hatch, F. T. Carcinogenesis 1986, 7, 1081-1086. (10) Manabe, S.; Tohyama, K.; Wada, O.; Aramaki, T. Carcinogenesis 1991, 12, 1945-1947. (11) Manabe, S.; Kurihara, N.; Wada, O.; Izumikawa, S.; Asakuno, K.; Morita, M. Environ. Pollut. 1993, 80, 281-286. (12) Manabe, S.; Susuki, H.; Wada, O.; Ueki, A. Carcinogenesis 1993, 14, 899901. (13) Wakabayashi, K.; Nagao, M.; Esumi, H.; Sugimura, T. Cancer Res. (Suppl.) 1992, 52, 2092s-2098s. (14) Ramasamy, S. M.; Hurtubise, R. J. Appl. Spectrosc. 1989, 43, 616-620. (15) Richmond, M. D.; Hurtubise, R. J. Anal. Chem. 1991, 63, 169-173. (16) Hurtubise, R. J.; Ramasamy, S. M. Appl. Spectrosc. 1991, 45, 555-559. (17) Burrell, G. J.; Hurtubise, R. J. Anal. Chem. 1987, 59, 965-970. (18) Ramasamy, S. M.; Hurtubise, R. J. Anal. Chem. 1987, 59, 2144-2148. S0003-2700(96)01160-2 CCC: $14.00
© 1997 American Chemical Society
Because the use of glucose glasses in solid-matrix luminescence is a significant departure from previous solid matrices, it is very important to investigate the interactions between the phosphors and glucose glasses. In this work, phosphorescence intensities and lifetimes of the phosphors were studied as a function of temperature. Also, confocal laser scanning microscopy was employed to determine the distribution of a fluorophor in the glucose glasses, and infrared spectroscopy was used to detect water in the glucose glasses. EXPERIMENTAL SECTION Apparatus. A Perkin-Elmer LS-50B luminescence spectrometer (Norwalk, CT) was used to obtain phosphorescence intensities and phosphorescence decay curves at different temperatures ranging from 296 to 93 K. An experimental setup to change the temperature in increments from room temperature to low temperature with cooled nitrogen gas was attached to the LS-50B sample compartment when measuring the phosphorescence intensities and phosphorescence decay curves. The experimental details were described previously.19 The sample holder used was similar to the one described earlier5 with a few modification so that it would fit into the low-temperature setup for LS-50B. Phosphorescence lifetimes were calculated from the phosphorescence decay data by using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). A Laitz LEICA TCS 4D confocal scanning fluorescence microscope system was used to obtain the fluorescence images of fluorescein in glucose glasses. A Kr/Ar laser in the confocal microscope system employed a 488 nm wavelength to excite the fluorescence of fluorescein in the glucose glasses. A Perkin-Elmer 1600 Fourier transform infrared spectrometer was used to obtain infrared spectra from KBr pellets of glucose powder and from KBr pellets of glucose glass material. Reagents. B[f]Q (Golden Label, Aldrich Chemical Co., St. Louis, MO) was used as received. PhIP was obtained from Toronto Research Chemicals Inc. (Downsview, Ontario, Canada) and was used as received. Methanol (HPLC) and water (HPLC) were purchased from J. T. Baker Inc. (Phillipburg, NJ). Glucose was purchased from Sigma Chemical Co. (St. Louis, MO), and sodium iodide (99.999%) was purchased from Johnson Matthey Catalog Co., Inc. (Wayne, PA). Fluorescein reference standard was purchased from Molecular Probes (Eugene, OR). Procedures. Glucose glasses were produced by using the procedures previously described.5,6 The glucose glass samples were prepared on a 1 cm × 2 cm quartz plate with a circular depression with a diameter of 6 mm and a depth of 1 mm. The quartz plate sample holder was put into a semicylinder-shaped Teflon sample holder which was attached to a fiberglass rod. The sample holder and fiberglass rod fit into a quartz dewar, which was placed in the sample compartment of the instrument.19 The sample compartment was constantly purged with cold, dry nitrogen gas. By adjusting the flow rate of the cold nitrogen gas, the temperature of the sample was controlled. The temperature was kept at the same level for at least 25 min before any measurements were taken. The delay time and the gate time for the LS-50B were 0.1 and 10 ms, respectively, when recording the phosphorescence intensity values and lifetime decay curves. The integration time for measuring the phosphorescence intensity values of PhIP and (19) Hurtubise, R. J.; Ramasamy, S. M. Appl. Spectrosc. 1993, 47, 116-121.
B[f]Q in glucose glasses at different temperatures was 10 s. In measuring the phosphorescence intensity values at different temperatures, the excitation and the emission slits and the PMT voltage were the same at different temperatures for a given sample. In recording phosphorescence lifetime decay curves, however, the excitation and the emission slits and the PMT voltage were adjusted at different temperatures for the same sample so that the initial intensities of the phosphorescence decay curves were relatively strong. The excitation and emission monochromators were set at 321 and 478 nm, respectively, for PhIP and at 268 and 497 nm, respectively, for B[f]Q. For the confocal scanning fluorescence microscopy experiments, the glucose glasses contained 20 ng/mg fluorescein, and they were prepared on microscope slides. The glucose glasses had a diameter of about 1 mm and a thickness of about 50 µm. The samples were excited by a Kr/Ar laser at 488 nm, and the fluorescence images of fluorescein were obtained at several depths in the glucose glasses. The samples for Fourier transform infrared spectroscopy (FTIR) experiments were prepared in KBr pellets. Glucose powder was put into a drying oven at 110-120 °C for 1 h or put into a container attached to a vacuum system below 5 mmHg for 24 h. About 20 mg of dried glucose powder was mixed with about 200 mg of dried KBr in a capped metal container with a steel ball inside. The metal container was fastened to a Crescent WIG-LBUG sample mixer. After 20-30 s of shaking, 30-50 mg of the glucose and KBr mixture was used to make a KBr/glucose pellet. The glucose glass employed for the IR experiments was produced without analyte on a microscope slide instead of the quartz plate sample holder. The glucose glass was removed from the microscope slide with a knife and was put into the capped metal container with a steel ball. After KBr was added, the metal container was shaken as described earlier in this section. The mixture was used to make a KBr pellet. In the process of making a KBr pellet, in order to keep the sample from absorbing moisture from air, efforts were made to keep the time period in which the samples were exposed to the open air as short as possible for both glucose powder and glucose glass KBr pellets. RESULTS AND DISCUSSION Relative Changes in Phosphorescence Intensities and Phosphorescence Lifetimes with Temperature. The phosphorescence intensity (Ip) and phosphorescence lifetime (τp) of PhIP and B[f]Q in glucose glasses were plotted against temperature as shown in Figures 1 and 2, respectively. It is clear that, as the temperature decreased, both the phosphorescence intensity and the lifetime of PhIP and B[f]Q in the glucose glasses increased. However, the plots for both phosphorescence intensity and lifetime tended toward constant values as the temperature decreased. Also, by comparing the plots in Figures 1 and 2, it is clear that the general shapes of the intensity and lifetime plots are different. In addition, the errors associated with the phosphorescence intensity plots are greater than those for the phosphorescence lifetime plots. This is most likely related to the cracks that were formed in the glass as the temperature was lowered from 185 to 178 K. Note that the error for the roomtemperature intensity data is very low. This results because clear, uncracked glasses are formed at room temperature. Figure 3 shows a plot of Ip/τp vs temperature. It can be seen that Ip/τp changed significantly as the temperature decreased in the range of 193-296 K, but Ip/τp was almost constant in the Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
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Figure 1. Relative phosphorescence intensity of 40 ng/mg PhIP and 40 ng/mg B[f]Q in glucose glasses vs temperature. The error bars represent relative standard deviation.
Equation 1 shows the relationship between phosphorescence quantum yield (φp), triplet state formation efficiency (φt), phosphorescence rate constant (kp), and phosphorescence lifetime (τp).2 If absorbance is constant with temperature, then (φp/τp) ∝ (Ip/τp). It has been shown that kp is constant for phosphors in essentially all the solid-matrix luminescence systems investigated.2,19,20 Thus, the change in Ip/τp in Figure 3 indicates that φt changes over a wide range for PhIP and B[f]Q. Figure 3 also shows that, at the lower temperatures, the Ip/τp ratio for PhIP approaches a constant value, which indicates that φt is tending toward a constant value at the lower temperatures. For B[f]Q, the data in Figure 3 show that φt has essentially reached a constant value at the lower temperatures. Changes in Phosphorescence Lifetime with Temperature for PhIP and B[f]Q in Glucose Glasses without a Heavy Atom. To understand the interactions responsible for solid-matrix luminescence from trace organic compounds, it is necessary to determine which fundamental luminescence parameters are important in solid-matrix luminescence. Normally, the nonradiative transition rate constant (km) from excited triplet state to ground state is considered to be composed of two terms:2
km ) kIm + k1 exp(-Ea/RT)
(2)
where kIm is a term independent of temperature and k1 exp(-Ea/ RT) is a temperature-dependent term. Ea is activation energy, T is temperature, and R is the gas constant. If km from eq 2 is substituted into the phosphorescence lifetime equation,
τp ) (kp + km)-1
Figure 2. Phosphorescence lifetime of 40 ng/mg PhIP and 40 ng/ mg B[f]Q in glucose glasses vs temperature. The error bars represent relative standard deviation.
where τp is phosphorescence lifetime at certain temperature and kp is the rate constant of phosphorescence decay, then the following equation can be obtained:
τp-1 - τp0-1 ) k1 exp(-Ea/RT)
Figure 3. Ratio of relative phosphorescence intensity to phosphorescence lifetime vs temperature.
lower temperature range. The changes in the Ip/τp with temperature can be explained by the following arguments.
φp ) φtkpτp 1948
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(1)
(3)
(4)
where τp0 is the low-temperature limit of phosphorescence lifetime.2,21,22 The τp0 values were obtained by acquiring extrapolated values of phosphorescence lifetimes at low temperature for PhIP and B[f]Q from the graphs in Figure 2. Then, by changing the numerical values of the extrapolated data in small increments, the incremented values of the lifetime were used to obtain the best linear fit for the low-temperature regions in Figure 4. Once this was achieved, the τp0 values for PhIP and B[f]Q were used to obtain the linear fits in the higher temperature regions in Figure 4 for PhIP and B[f]Q. If ln(τp-1 - τp0-1) is plotted against 1/T, a straight line should be obtained.2,19-23 Linear relationships for ln(τp-1 - τp0-1) vs 1/T have been acquired for phosphors adsorbed on other solid matrices, such as filter paper and cyclodextrin/ sodium chloride powders.14,19,21,23 However, the plots of (20) Ramasamy, S. M.; Hurtubise, R. J. Anal. Chem. 1987, 59, 432-436. (21) Ramasamy, S. M.; Hurtubise, R. J. Talanta 1989, 36, 315-320. (22) Hurtubise, R. J.; Ramasamy, S. M.; Boerio-Goates, J.; Putnam, R. J. Luminesc. 1996, 68, 55-68. (23) Ramasamy, S. M.; Hurtubise, R. J. Anal. Chem. 1990, 62, 1060-1064.
Table 1. Activation Energies and Preexponential Factors of 40 ng/mg PhIP and 40 ng/mg B[f]Q in Glucose Glasses without a Heavy-Atom Salta,b
PhIP B[f]Q
Ea1 (cm-1)
k1 (s-1)
Ea2 (cm-1)
k2 (s-1)
286 ( 24.6 209 ( 9.4
0.12 ( 0.03 0.24 ( 0.02
2536 ( 193 1597 ( 141
(4.18 ( 1.72) × 104 (3.48 ( 2.43) × 104
a The E a1 and k1 values represent the lower temperature region, while the Ea2 and k2 values represent the higher temperature region in Figure 4. b The uncertainties are given as standard deviations.
Figure 4. Plot of ln(1/τp - 1/τp0) vs 1/T for 40 ng/mg PhIP and 40 ng/mg B[f]Q in glucose glasses.
ln(τp-1 - τp0-1) vs 1/T from our data showed mainly two regions instead of a single straight line (Figure 4). We propose that the rate constant of nonradiative transition from the first excited triplet state to ground state consists of three terms, as shown in
km ) kIm + k1 exp(-Ea1/RT) + k2 exp(-Ea2/RT) (5) where kIm is a temperature-independent term and k1 exp(-Ea1/ RT) and k2 exp(-Ea2/RT) are the two temperature-dependent terms which contribute to km. Ea1 and Ea2 are activation energies of the temperature-dependent processes which are involved in the deactivation of the excited triplet state, and k1 and k2 are preexponential factors. Equation 5 is similar to the equations discussed by Kropp et al.24 and Yamauchi et al.25 In eq 5, it is assumed that Ea2 . Ea1. The previous assumption is supported by the large difference in the slope for the two regions of the plots in Figure 4. At low temperatures, the second term, [k2 exp(-Ea2/RT)], can be neglected. From the plot of ln(τp-1 - τp0-1) vs 1/T for the lower temperature region (the last six data points in Figure 4), the values of Ea1 and k1 can be obtained from slope and intercept of the linear relationship (the linear correlation coefficients for B[f]Q and PhIP in the lower temperature region were 0.996 and 0.992, respectively). In the higher temperature region (the first four data points in Figure 4), ln[(τp-1 - τp0-1) k1 exp(-Ea1/RT)] vs 1/T was plotted (the linear correlation coefficients for B[f]Q and PhIP in the higher temperature region were 0.987 and 0.994, respectively). The values of k1 exp(-Ea1/ RT) were calculated using the k1 and Ea1 values obtained in the lower temperature region. From the slope and the intercept of the straight line, the activation energies (Ea2’s) and the preexponential factors (k2’s) for both B[f]Q and PhIP in glucose glass were obtained. The activation energies and preexponential factors are shown in Table 1. Some have considered the activation energy and the preexponential factor as a measure of how strongly the phosphor is held to the solid matrix.2,14 However, the full meanings of the activation energies and the preexponential factors are not developed.26 Recently, Hurtubise et al.22 related heat capacities of several solid matrices to phosphorescence lifetimes over a wide (24) Kropp, J. L.; Dawson, W. R. J. Phys. Chem. 1967, 71, 4499-4506. (25) Yamauchi, S.; Mibu, K.; Komada, Y.; Hirota, N. J. Phys. Chem. 1987, 91, 6173-6177.
temperature range. Their plots of ln(1/τp - τp0) vs 1/T gave linear relationships for the phosphors adsorbed on a variety of solid matrices. The Ea values reported for the different phosphors were in the range from 332 to 488 cm-1. They proposed that the relatively small activation energies were due to the low-frequency vibrational modes of the solid matrix coupling with the triplet excited state of the adsorbed phosphor.22 The coupling mechanism provided a means to populate the nonzero vibrational levels of the excited triplet state so that intersystem crossing to the ground state potential energy surface could take place. As indicated in Table 1, the Ea1 values were in the same general range as reported by Hurtubise et al.22 The magnitude of the Ea1 values and the fact that the phosphorescence intensities and lifetimes in the lower temperature region in Figures 1 and 2 change very little indicate that a similar coupling mechanism was occurring with the glucose glasses. The k1 values in Table 1 are 0.12 and 0.24 s-1, respectively, for PhIP and B[f]Q in the low-temperature region. These small values for k1 indicate that the phosphor is held rather rigidly in the solid.14 For example, Ramasamy and Hurtubise14 have compared k1 values from the literature for phosphors absorbed on several different solid matrices. Generally, the values range from 1.1 to 15, but one value was 2 × 104. This high value was obtained by Plauschinat et al.27 for phenanthrene adsorbed on alumina, and it was explained by the fact that the phosphor was only physisorbed. Thus, it was weakly interacting with the solid matrix.14,27 All of the solid matrices that have been employed in solidmatrix RTP gave individual straight line plots for graphs of ln(τp-1 - τp0-1) vs 1/T,2,15,21,23 except for 1.4% sodium acetate/NaCl.22 Thus, the plots in Figure 4 are unusual, as are the Ea2 values given in Table 1. Also, the preexponential factors (k2) are much larger than the preexponential factors obtained with Ea1. As considered in the preceding paragraph, a relatively large preexponential factor indicates that the phosphor is not interacting strongly with the solid matrix.14,27 The k2 values in Table 1 are 4.18 × 104 and 3.48 × 104, respectively. To acquire additional data to explain the results in Figure 4 and in Table 1 for Ea2, other experiments were performed with weight measurements and with FT-IR spectrometry to detect any water that might be present in the glucose glasses. For the weight measurement experiments, the quartz sample holder was accurately weighed before a sample was added, and it was weighed again after the glucose glass was prepared. The weight difference would be the total weight of glucose plus any other material in the glucose glass. Also, the total weight of the solid in the solution used to produce the glucose glass could (26) Honnen, W.; Krabichler, G.; Uhl, S.; Oelkrug, D. J. Phys. Chem. 1983, 87, 4872-4877. (27) Plauschinat, M.; Honnen, W.; Krabichler, G.; Uhl, S.; Oelkrug, D. J. Mol. Struct. 1984, 115, 351-354.
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be calculated from the concentration and the volume of the glucose solution used to prepare the glucose glass. The results showed that the weight of glucose glass measured by weighing was larger by 0.19 mg than the calculated value (18 mg), which indicated that there was still solvent present in the glucose glass. Water has major infrared bands that appear at 1630 and 3400 cm-1. Also, it has been shown that a band appearing at 1630 cm-1 indicates the amount of water adsorbed on paper.28,29 Infrared spectra of dried glucose samples from KBr pellets showed no band at 1630 cm-1. However, the glucose glasses showed a substantial band at 1642 cm-1, which indicated that water was present in the glucose glasses. In addition, the band centered at 3400 cm-1 was much broader compared to the same band for the dry glucose samples. These results were further substantiated by obtaining infrared spectra from dry glucose samples that were spiked with water. These samples showed a band for water at 1642 cm-1, and the band at 3400 cm-1 was broadened. Because it was established by infrared spectrometry that water was present, it is reasonable to state that, as the temperature of the glucose glasses increases, the probability of water diffusing to a phosphor molecule would increase, and thus quenching of some of the phosphor molecules would occur. The relatively large Ea2 values are most likely related to the diffusion of water molecules in the glucose glass. It is well known that the RTP of a phosphor can be quenched to various extents, depending on the amount of adsorbed moisture on the filter paper.30,31 However, because the glucose glasses were prepared with methanol/water as a solvent, there is the possibility that some methanol was also trapped in the glucose glasses, and thus methanol would undergo diffusion as the temperature increased and would cause some of the quenching of RTP. However, because of the overlap of the infrared band for glucose and methanol at 1450 cm-1, methanol could not be detected by infrared spectroscopy. In addition to the moisture present in glucose glasses and the possibility of methanol in the glucose glasses, it is important to consider the motions of functional groups in the glucose glasses as the temperature increases. For example, the higher activation energy (Ea2) could also be related to the rotational motion of functional groups and relaxation phenomena in the glucose glasses. When the temperature is increased, the solid matrix relaxes, and rotational motions of the functional groups in the solid matrix increase. These could cause a decrease in the RTP by direct interaction of the functional groups with the phosphor or by permitting a more effective pathway for the water molecules to diffuse to the phosphor. For example, Guillet et al.32,33 studied phosphorescence intensity in the range of 77-300 K for a wide variety of polymer films containing ketone and/or naphthalene groups. They found major changes in the phosphorescence intensities on going from 77 to 300 K. They concluded that both oxygen quenching and the motion of functional groups were involved in the phosphorescence quenching. However, oxygen quenching was largely responsible for the phosphorescence quenching in their work for the samples that were not degassed. In our work, the RTP data were acquired under dry nitrogen gas. (28) Marchessault, R. H. Pure Appl. Chem. 1962, 5, 107-129. (29) Marchessault, R. H.; Sundararajan, R. R. In The Polysaccharides; Aspinall, G. O., Ed.; Academic Press: New York, 1983; Vol. 2. (30) Purdy, B. B.; Hurtubise, R. J. Anal. Chem. 1992, 64, 1400-1404. (31) Chen, J.; Hurtubise, R. J. Appl. Spectrosc. 1995, 49, 98-104. (32) Guillet, J. Polymer Photophysics and Photochemistry; Cambridge University Press: Oxford, UK, Chapter 8. (33) Somersall, A. C.; Dan, E.; Guillet, J. E. Macromolecules 1974, 7, 233-244.
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Also, we performed experiments in which all the sample preparation and measurement steps were carried out under nitrogen. This did not help to enhance the RTP signals. Our results indicate that oxygen quenching is not a major factor in decreasing the RTP signals, but relaxation phenomena and rotation of functional groups could be partly responsible for the decrease in phosphorescence as the temperature increases. In the higher temperature region, it is entirely possible that at least two decay mechanisms are simultaneously active and/or that any inhomogeneity is being averaged on a time scale that is long compared to the triplet lifetime. Phosphorescence Lifetime Changes with Temperature for PhIP and B[f]Q in Glucose Glasses with 10% NaI. By adding 10% sodium iodide as a heavy-atom salt into glucose glasses, the RTF and RTP from organic compounds decrease and increase, respectively.6 This is also the case as the temperature is lowered with a heavy atom present. In addition, the phosphorescence lifetimes of phosphors decrease with a heavy atom present in the glucose glasses compared to those of glasses without a heavy atom.1,2 Phosphorescence decay curves for 5 ng/mg PhIP in glucose glasses with 10% NaI were recorded at several temperature from 93 K to room temperature (296 K). Previous work has shown that, at room temperature, clear glucose glasses were readily obtained with 10% NaI present in the glass.6 In contrast to the phosphorescence decay curves with no heavy atom present, the phosphorescence decay curves at each temperature with NaI present in the glucose glasses fit a double-exponential equation so that two lifetimes were obtained at all temperatures. This indicated that there were at least two phosphorescent components in the system with 10% NaI present. It is possible that there is a distribution of phosphorescence lifetimes at each temperature, but it is not feasible with the present data to resolve more than two lifetimes. Because the NaI is present at a relatively high level, most likely the sodium iodide did not distribute homogeneously in the glucose glass. This is supported by the fact that the decay curves fit a double-exponential equation. Also, one would expect that the phosphorescence lifetime of PhIP would be longer in the region of glucose glass with a smaller amount of NaI, while in the region of the glass with a larger amount of NaI, the lifetime would be shorter. The difference in lifetime would result because of the closer proximity of NaI to the phosphor in regions of higher amounts of NaI. For example, Ramamurthy et al.34 showed that, for aromatics included in zeolites with a heavy atom present, the external heavy-atom effect was operative only at close distances. In this work, laser scanning confocal microscopy was used to verify the nonhomogeneous distribution of NaI in the glucose glasses. In the confocal microscopy experiment, a laser beam can be focused and scanned across the sample in different depths or slices to obtain fluorescence from a fluorophor. In our work, a three-dimensional fluorescence image was obtained for fluorescein in glucose glasses. It was necessary to employ fluorescein because the laser scanning confocal microscopy system used a Kr/Ar ion laser source with the shortest excitation wavelength of 488 nm. PhIP and B[f]Q are excited in the ultraviolet region. Comparing the confocal fluorescence images of fluorescein in glucose glasses with and without 10% NaI, it was clear that, without NaI present, the visible fluorescence from fluorescein was quite (34) Ramamurthy, V.; Caspar, J. V.; Eaton, D. F.; Kuo, E. W.; Corbin, D. R. J. Am. Chem. Soc. 1992, 114, 3882-3892.
Table 2. Long-Lived and Short-Lived Phosphorescence Lifetimes of 5 ng/mg PhIP in Glucose Glasses with 10% NaI Presenta temp (K)
average of long-lived component lifetime (s)
average of short-lived component lifetime (s)
296 273 233 193 153 93
1.28 ( 0.013 1.33 ( 0.014 1.44 ( 0.019 1.57 ( 0.005 1.58 ( 0.005 1.58 ( 0.005
0.28 ( 0.006 0.29 ( 0.004 0.33 ( 0.010 0.33 ( 0.008 0.34 ( 0.006 0.34 ( 0.008
a The uncertainties are given as standard deviations from four runs at each temperature.
uniform at different depths in the glucose glasses, which indicated that the fluorescein was distributed homogeneously. However, the visible fluorescence images of fluorescein in the glucose glasses with 10% NaI showed a dimmer region in the center of the sample and a much brighter region in the outer part of the sample at different depths in the glass, indicating the nonhomogeneous distribution of NaI in the glass. Thus, the two phosphorescence lifetimes obtained with NaI present in the glucose glasses and the results from the confocal microscopy experiments showed that the NaI was not distributed homogeneously in the glucose glasses. Table 2 lists the two phosphorescence lifetimes as a function of temperature for 5 ng/mg PhIP in glucose glasses with 10% NaI present which were obtained from two-component analysis of ln(intensity) vs time plots. The longer-lived component would represents the PhIP phosphor molecules that are farther removed from NaI compared to other phosphor molecules that would be closer to NaI and would give a shorter phosphorescence lifetime. The Ip/τp1 and Ip/τp2 values at different temperatures for both PhIP and B[f]Q in glucose glasses with 10% NaI were also obtained, where τp1 and τp2 are long-lived and short-lived phosphorescence lifetimes, respectively, and Ip is the phosphorescence intensity. The results showed that both the Ip/τp1 and Ip/τp2 values for PhIP and B[f]Q changed with temperature over the temperature range investigated. This indicated that the triplet state formation efficiencies (φt’s) changed with temperature over the temperature range for both long-lived and short-lived components of PhIP and B[f]Q in glucose glasses with 10% NaI. Plots of ln(τp-1 - τp0-1) vs 1/T were obtained for the longlived component and the short-lived component over the temperature range investigated for PhIP in a glucose glass with 10% NaI. The plots gave two linear ranges for both long- and short-lived phosphorescence components (the linear correlation coefficients for the data from the lower temperature region for the long-lived component and the short-lived component were 0.997 and 0.994, respectively; for the data from the higher temperature region, the linear correlation coefficients were 0.996 and 0.986, respectively). The same method of data analysis discussed earlier in the text for the samples without NaI was used to calculate the activation energies and preexponential factors for higher and lower temperature regions for both long and short lifetimes. These values are listed in Table 3. The lower temperature activation energy
Table 3. Activation Energies and Preexponential Factors for Long-Lived and Short-Lived Components of 5 ng/mg PhIP in Glucose Glasses with 10% NaI, Long and Short Lifetimea,b Ea1 (cm-1)
k1 (s-1)
Ea2 (cm-1)
k2 (s-1)
long-lived 258 ( 18.8 0.032 ( 0.007 689 ( 59.5 4.21 ( 1.36 component short-lived 255 ( 21.0 0.57 ( 0.12 2034 ( 404 (1.18 ( 2.61) component × 103 a The E a1 and k1 values represent the lower temperature region, while the Ea2 and k2 values represent the higher temperature region. b The uncertainties are given as standard deviations.
values in Table 3 (258 and 255 cm-1) for both long- and shortlived components are essentially the same, and they are comparable with the lower temperature activation energy values obtained without a heavy atom present given in Table 1. Also, the preexponential factors (k1) are approximately the same as the ones in Table 1, which indicates that these phosphors are held rigidly in the solid matrix. As previously stated, the activation energies obtained with the low-temperature data are most likely due to the low-frequency vibrational modes of solid material coupling with the excited triplet state of the analyte. The activation energies and preexponential factors in the higher temperature region had quite different values, as shown in Table 3. This is related to the nonhomogeneity of the glucose glasses. In the higher temperature region, both the diffusion of water, motion of functional groups, and movement of NaI could be contributing to the Ea2 values and preexponential factors. Because the Ea2 and k2 values are so different for the higher temperature region for the shortlived and long-lived components, different mechanisms would be represented by the dissimilar Ea2 and k2 values. The Ea1, Ea2, k1, and k2 values for the long-lived component of B[f]Q in a glucose glass with 10% NaI were 304 ( 10.7 cm-1, 692 ( 55.1 cm-1, 0.269 ( 0.074 s-1, and 3.54 ( 1.06 s-1, respectively (the linear correlation coefficients for the ln(τp-1 - τp0-1) vs 1/T plots for the lower temperature region and the higher temperature region were 0.998 and 0.997, respectively). These results are similar to the results for the long-lived component of PhIP in a glucose glass with 10% NaI. Similar data analysis could not be carried out for the short-lived component of B[f]Q in a glucose glass with 10% NaI, because the phosphorescence lifetime vs temperature plots for the short-lived component did not reach a plateau at the low temperatures investigated. ACKNOWLEDGMENT Financial support for this project was provided by grants from the Department of Energy, Division of Chemical Sciences, Grants No. DE-FG02-86ER13547 and DE-FG06-95ER14551. Special thanks are given to Dr. Robert A. Jenkins and his research group for helping us to obtain the laser scanning confocal microscopy data. Received for review November 15, 1996. Accepted March 6, 1997.X AC961160P X
Abstract published in Advance ACS Abstracts, April 15, 1997.
Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
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