Effects of Subzero Temperatures on Fluorescent Probes Sequestered

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Effects of Subzero Temperatures on Fluorescent Probes Sequestered within Aerosol-OT Reverse Micelles Chase A. Munson, Gary A. Baker,† Sheila N. Baker,‡ and Frank V. Bright* Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000 Received July 7, 2003. In Final Form: December 1, 2003 We report on the effects of temperature (+30 to -100 °C) and water loading on the steady-state fluorescence from four fluorescent probe molecules when they are sequestered within the interior of sodium bis(2ethyl-1-hexyl) sulfosuccinate (Aerosol OT, AOT) reverse micelles. Each probe locates itself to different average microenvironments within the reverse micelle, and these individual microenvironments behave differently to changes in the molar ratio of water-to-AOT (R) and temperature. Changes in probe steadystate fluorescence emission spectra or fluorescence anisotropy indicate that “freezing” occurs within the water pool at temperatures between -10 and -60 °C. The lowest freezing points are generally observed at the lowest R values.

Introduction Sodium bis(2-ethyl-1-hexyl) sulfosuccinate (Aerosol OT, AOT) surfactant molecules can aggregate in apolar solvents and solubilize substantial quantities of water.1 Mixtures of AOT and H2O in an alkane can form thermodynamically stable solutions of reverse micelles or microemulsions at water loadings (defined as the molar ratio of water-to-surfactant, [H2O]/[surfactant] ) R) up to ∼60.2 Over the years, a variety of techniques (e.g., nuclear magnetic resonance (NMR),3 electron spin resonance,4 infrared and Raman,5 absorbance and fluorescence,6 differential scanning calorimetry (DSC),3b,7 high-frequency dielectric spectroscopy,8 laser-induced optoacoustic spectroscopy,9 and ultrasound velocity measurements10) have * To whom correspondence should be addressed. Voice: 716-645-6800 ext 2162. Fax: 716-645-6963. E-mail: chefvb@ acsu.buffalo.edu. † Michelson Resource, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545. ‡ Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545. (1) (a) Eicke, H. F. Top. Curr. Chem. 1980, 87, 85-145. (b) Robb, I. D. Microemulsions; Plenum Press: New York, 1984. (c) Reverse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1984. (d) Luisi, P. L.; Magrid, J. CRC Crit. Rev. Biochem. 1986, 20, 409-474. (e) Structure and Reactivity in Reverse Micelles; Pileni, M. P., Ed.; Elsevier: Amsterdam, 1989. (f) Nandi, N.; Bhattacharyya, K.; Bagchi, B. Chem. Rev. 2000, 100, 2013-2046. (2) (a) Menger, F. M.; Donohue, J. A.; Williams, R. A. J. Am. Chem. Soc. 1973, 95, 286-288. (b) Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979, 83, 480-486. (c) Eicke, H. F.; Markovic, Z. J. Colloid Interface Sci. 1981, 79, 151-158. (d) Robinson, B. H.; Toprakcioglu, C.; Dore, J. C.; Chieux, P. J. Chem. Soc., Faraday Trans. 1 1984, 80, 13-27. (e) Kotlarchyk, M.; Chen, S. H.; Huang, J. S.; Kim, M. W. Phys. Rev. Lett. 1984, 53, 941-944. (f) Chen, S. H. Annu. Rev. Phys. Chem. 1986, 37, 35-3991. (g) Teubner, M.; Strey, R. J. J. Chem. Phys. 1987, 87, 31953200. (h) Ricˇka, J.; Borkovec, M.; Hofmeier, U. J. Chem. Phys. 1991, 94, 8503-8509. (h) Silber, J. J.; Biasetti, M. A.; Aubin, E.; Lissi, E. Adv. Colloid Interface Sci. 1999, 82, 189-252. (3) (a) Quist, P. O.; Halle, B. J. Chem. Soc., Faraday Trans. 1 1988, 84, 1033-1046. (b) Hauser, H.; Haering, G.; Pande, A.; Luisi, P. L. J. Phys. Chem. 1989, 93, 7869-7876. (c) Gradzielski, M.; Hoffmann, H.; Panitz, J.; Wokaun, A. J. Colloid Interface Sci. 1995, 169, 103-118. (4) Yoshioka, H. J. Colloid Interface Sci. 1981, 83, 214-220. (5) (a) Christopher, D. J.; Yarwood, J.; Belton, P. S.; Hills, B. P. J. Colloid Interface Sci. 1992, 152, 465-472. (b) Li, Q.; Weng, S.; Wu, J.; Zhou, N. J. Phys. Chem. B 1998, 102, 3168-3174. (c) Cheng, G. X.; Shen, F.; Yang, L. F.; Ma, L. R.; Tang, Y.; Yao, K. D.; Sun, P. C. Mater. Chem. Phys. 1998, 56, 97-101. (d) Zhou, N.; Li, Q.; Wu, J.; Chen, J.; Weng, S.; Xu, G. Langmuir 2001, 17, 4505-4509. (e) Zhou, G. W.; Li, G. Z.; Chen, W. J. Langmuir 2002, 18, 4566-4571.

been used to investigate reverse micelle systems. The results of these experiments have shown that the water molecules that comprise the “water pool” within the reverse micelle/microemulsion possess physical properties unlike those of bulk water. For example, the water pools within AOT reverse micelles are reported to exhibit “freezing” temperatures as low as -40 °C depending on the AOT concentration and R.3a,7d,11a,c,d Zulaf and Eicke12 performed one of the earliest lowtemperature studies on AOT reverse micelles. In the AOT/ water/iso-octane system, the authors reported spontaneous solution demixing at +18 °C when R was greater than 24. For R < 24, no turbidity was indicated over the temperature range studied (+5 to +60 °C).12 After sedimentation, Zulaf and Eicke described their system as having a lower, water-rich phase associated with AOT and an upper phase consisting of iso-octane. However, Balny et al.11d demonstrated nearly complete recovery of (6) (a) Keh, E.; Valeur, B. J. Colloid Interface Sci. 1981, 79, 465478. (b) Zhang, J.; Bright, F. V. J. Phys. Chem. 1991, 95, 7900-7907. (c) Correa, N. M.; Biasutti, M. A.; Silber, J. J. J. Colloid Interface Sci. 1995, 172, 71-76. (d) Heitz, M. P.; Bright, F. V. Appl. Spectrosc. 1995, 49, 20-30. (e) Hasegawa, M.; Sugimura, T.; Shindo, Y.; Kitahara, A. Colloids Surf., A 1996, 109, 305-318. (f) Riter, R. E.; Willard, D. M.; Levinger, N. E. J. Phys. Chem. B 1998, 102, 2705-2714. (g) Pal, S. K.; Mandal, D.; Sukul, D.; Bhattacharyya, K. Chem. Phys. Lett. 1999, 312, 178-184. (h) Lissi, E. A.; Abuin, E. B.; Rubio, M. A.; Cero´n, A. Langmuir 2000, 16, 178-181. (i) Falcone, R. D.; Correa, N. M.; Biasutti, M. A.; Silber, J. J. Langmuir 2000, 16, 3070-3076. (j) Laia, C. A. T.; Costa, S. M. B. Langmuir 2002, 18, 1494-1504. (7) (a) Hauser, H.; Haering, G.; Pande, A.; Luisi, P. L. J. Phys. Chem. 1989, 93, 7869-7876. (b) Czarniecki, K.; Jaich, A.; Janik, J. M.; Rachwalska, M.; Janik, J. A.; Krawczyk, J.; Otnes, K.; Volino, F.; Ramasseul, R. J. Colloid Interface Sci. 1983, 92, 358-366. (c) Senatra, D.; Zhou, Z.; Pieraccini, L. Prog. Colloid Polym. Sci. 1987, 73, 66-75. (d) Schulz, P. C. J. Therm. Anal. Calorim. 1998, 51, 135-149. (e) Garti, N.; Aserin, A.; Tiunova, I.; Fanun, M. Colloids Surf., A 2000, 170, 1-18. (8) D’Angelo, M.; Fioretto, D.; Onori, G.; Palmieri, L.; Santucci, A. Phys. Rev. E 1996, 54, 993-996. (9) Borsarelli, C. D.; Braslavsky, S. E. J. Phys. Chem. B 1997, 101, 6036-6042. (10) Amararene, A.; Gindre, M.; Le Hue´rou, J.-Y.; Nicot, C.; Urbach, W.; Waks, M. J. Phys. Chem. B 1997, 101, 10751-10756. (11) (a) Douzou, P. Cryobiochemistry: An Introduction; Academic Press: London, 1977. (b) Douzou, P.; Balny, C.; Franks, F. Biochimie 1978, 60, 151-158. (c) Balny, C.; Douzou, P. Biochimie 1979, 61, 445452. (d) Balny, C.; Hui, B. H. G.; Douzou, P. Catal. Chem. Biochem.: Theory Exp. 1979, 12, 37-50. (e) Douzou, P.; Keh, E.; Balny, C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 681-684. (f) Thompson, J. S.; Gehring, H.; Vallee, B. L. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 132-136. (12) Zulaf, M.; Eicke, H.-F. J. Phys. Chem. 1979, 83, 480-486.

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Experimental Section

Figure 1. Chemical structures of the fluorescent probes used in this study.

cytochrome c from the alkane-rich, upper phase of AOT/ water/heptane solutions after cooling to subzero temperatures. Given that cytochrome c exhibits negligible solubility in pure heptane, these authors concluded that “an unknown amount of surfactant and water certainly precipitated but sufficient concentrations of both remain in solutions to accommodate the protein.” Several more recent studies3a,3b indicate the presence of 2-6 molecules of “unfreezable” water per molecule of AOT in supercooled AOT solutions. Further, despite the complications of AOT precipitating at low temperatures, several research groups have used spectroscopic methods to monitor enzymatic reactions in AOT reverse micelle solutions below 0 °C.11c-e Thus, the alkane-rich portion of AOT solutions below 0 °C can solubilize solutes as large as proteins and these systems can be studied by optical spectroscopy. In this paper, we explore the behavior of the AOT reverse micelles that remain dissolved in the alkane phase over a wide temperature range. Toward this end, we report on the steady-state fluorescence intensity or anisotropy of four fluorescent probe molecules (Figure 1) sequestered individually within the water pool of AOT reverse micelles that have been formed in n-heptane as a function of R and temperature (+30 to -100 °C). These particular probes preferentially distribute themselves into the aqueous phase and/or do not fluoresce significantly from the alkane phase. Thus, despite the inevitability of some AOT/water precipitation,3,11,12 our optical configuration and the nature of the probe molecules provide information only on the interior of those AOT reverse micelles that remain dissolved in the alkane phase. These studies impact the field of cryoenzymology.11 This work is also timely given the recent use of AOT reverse micelles to increase the protein spin-spin relaxation times in multipulse NMR experiments13 and the use of pure supercooled water to reduce protein internal dynamics and reduce NOE (nuclear Overhauser effect) quenching, chemical exchange of labile protons, and conformational exchange including the flipping of aromatic rings.14 (13) Wand, A. J.; Ehrhardt, M. R.; Flynn, P. F. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15299-15302. (14) (a) Skalicky, J. J.; Sukumaran, D. K.; Mills, J. L.; Szyperski, T. J. Am. Chem. Soc. 2000, 122, 3230-3231. (b) Skalicky, J. J.; Mills, J. L.; Sharma, S.; Szyperski, T. J. Am. Chem. Soc. 2001, 123, 388-397. (c) Mills, J. L.; Szyperski, T. J. Biomol. NMR 2002, 23, 63-67.

Materials. The following reagents were used: AOT (98%) and n-heptane (spectrophotometric grade) (Sigma); 1-heptanol (98%) and 2,2,4-trimethylpentane (iso-octane, spectrophotometric grade) (Aldrich); ethanol (200 proof, anhydrous) (Pharmco); 2-(ptoluidinyl)naphthalene-6-sulfonic acid, sodium salt (2,6- TNS) (Molecular Probes); and 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM, laser grade) and rhodamine 6G, chloride salt (R6G, laser grade) (ACROS). The “rigidochromic” Re complex [ClRe(I)(CO)3(bathophenanthroline disulfonic acid, disodium salt)] was a generous gift from Dr. Mahdi M. AbuOmar (UCLA).15 The chemical structure of each probe is given in Figure 1. All reagents were used as received. AOT was dried to constant weight in a vacuum oven at 60 °C. Deionized water was prepared to a specific resistivity of >18 MΩ cm by using a Barnstead NANOpure II system. Reverse Micelle Preparation and Probe Incorporation. A 0.3 M AOT stock solution was prepared by dissolving AOT in n-heptane under ambient conditions. Fluorophore-loaded reverse micelles were typically prepared by micropipetting aqueous solutions of 2,6-TNS, R6G, or the Re complex into aliquots of the AOT stock solution. R was adjusted by using a micropipet to add water to the sample. The DCM-loaded reverse micelles were prepared by micropipetting an aliquot of DCM dissolved in EtOH into a clean glass vial. The EtOH was removed with a dry N2 gas stream. We then added an aliquot of the AOT stock solution and adjusted the R with water as described above. The R ) “0” samples for the R6G experiments were prepared in the same way as the DCM samples, but no extrinsic water was added to this sample. All samples were vortex mixed for 1 min and allowed to stand overnight prior to any spectroscopic measurements. The final fluorescent probe concentrations (in terms of total solution volume) were 5 µM for the 2,6-TNS, R6G, and DCM samples and 25 µM for the Re complex samples. Under our experimental conditions at ambient temperatures, less than one reverse micelle in 500 contains a probe molecule and the probability of any single reverse micelle containing two or more probes is less than 1 in 104. We investigated the following R values: 0 (R6G only), 5, 10, 15, and 20. Fluorescence Measurements. An SLM-AMINCO model 48000 MHF spectrofluorimeter was used to record the fluorescence spectra and perform the steady-state fluorescence anisotropy measurements. A 450 W xenon arc lamp was used as the excitation source. The sample temperature was controlled from +30 to -100 °C by using our home-built temperature stage.16 Spectroscopic measurements were performed only in the alkanerich portion of each sample. The Re complex was excited at 400 nm, and the emission spectra were recorded between 475 and 700 nm. 2,6-TNS was excited at 320 nm, and its emission spectra were recorded between 350 and 550 nm. DCM was excited at 488 nm, and its emission spectra were recorded between 550 and 700 nm. Steady-state fluorescence anisotropy measurements were performed on the R6G-loaded reverse micelles by exciting at 488 nm while monitoring the entire emission through a 515 nm long pass filter (Oriel). Data Analysis. The Re complex and DCM emission spectra were evaluated by using the emission center of gravity.17 The 2,6-TNS emission spectra were deconvoluted into the sum of two spectral features by using PeakFit (Jandel). (15) (a) Giordano, P. J.; Wrighton, M. S. J. Am. Chem. Soc. 1979, 101, 2888-2897. (b) Salman, O. A.; Drickamer, H. G. J. Chem. Phys. 1982, 77, 3337-3343. (c) McKiernan, J.; Pouxviel, J. C.; Dunn, B.; Zink, J. I. J. Phys. Chem. 1989, 93, 2129-2133. (d) Lang, J. M.; Dreger, Z. A.; Drickamer, H. G. Chem. Phys. Lett. 1992, 192, 299-302. (e) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I.; Snyder, R. W. Inorg. Chem. 1993, 32, 2570-2575. (f) Hanna, S. D.; Dunn, B.; Zink, J. I. J. Non-Cryst. Solids 1994, 167, 239-246. (g) Lin, R.-J.; Lin, K.-S.; Chang, I. J. Inorg. Chim. Acta 1996, 242, 179-183. (h) Lees, A. J. Coord. Chem. Rev. 1998, 177, 3-35. (i) Wu, J.; Abu-Omar, M. M.; Tolbert, S. H. Nano Lett. 2001, 1, 27-31. (16) Baker, S. N.; Baker, G. A.; Munson, C. A.; Bright, F. V. Appl. Spectrosc. 2001, 55, 1273-1277. (17) Lakowicz, J. R.; Hogen, D. Biochemistry 1981, 20, 1366-1373.

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Figure 2. Photographs of 0.08 M AOT reverse micellar solutions at R ) 0, 5, 10, 15, 20, and 30 (left to right in each panel) at +4 °C. (Panel A) Iso-octane after 1 h. (Panel B) Iso-octane after 3 h. (Panel C) Iso-octane after 7 h. (Panel D) n-Heptane after 1 h. (Panel E) n-Heptane after 3 h. (Panel F) n-Heptane after 7 h. The steady-state fluorescence anisotropy, r, was calculated by using the following expression:18

r ) [IVV - GIVH]/[IVV + 2GIVH]

(1)

Here, IVV and IVH represent the parallel (VV) and perpendicular (VH) intensity components of the polarized fluorescence when the sample is excited with vertically (V) polarized light. The G term is an instrument-specific “grating” correction factor that accounts for the preferential transmission of one polarization through the cuvette, temperature stage windows, optical filters, optics, and detector envelope; G ) IHV/IHH. All experiments were performed on at least three separate occasions using separate reagent batches. The average results from all experiments are reported along with the corresponding standard deviations.

Results and Discussion Physical Observations of Temperature Effects on the AOT Reverse Micelles. AOT reverse micellar solutions (0.08 M; R ) 0, 5, 10, 15, 20, and 30) were prepared in iso-octane and n-heptane. These samples were stored at 4 °C, and they were photographed at 1 h intervals. A representative sampling of results is presented in Figure 2 after 1 h (panels A and D), 3 h (panels B and E), and 7 h (panels C and F) in iso-octane (panels A-C) and n-heptane (panels D-F). Within each panel, R is (from left to right) 0, 5, 10, 15, 20, and 30. In both solvents, no precipitation is observed until 3 h has elapsed; however, precipitation is only observed in the R ) 30 samples, and (18) Jablonski, A. Bull. Acad. Pol. Sci., Ser. A 1960, 8, 259-264.

no precipitation is seen at longer times (i.e., studies after 35 h are identical to the 7 h results which are identical to the 3 h results). Zulaf and Eicke12 reported phase separation below +18 °C for AOT/water/iso-octane mixtures with R > 24. Our photographic evidence at +4 °C (Figure 2) is not consistent with the Zulaf and Eicke report.12 In our spectroscopic studies, we have chosen to use lower water loadings (R e 20) which yield transparent, isotropic solutions at lower temperatures.11d Probe Localization Within the Reverse Micelles. The position of a probe molecule within any microheterogeneous system (e.g., reverse micelle) dictates the information that each probe reports.6c,e,h For example, if the probe associates exclusively with the micelle palisade region, it will sense and report on a much different microenvironment in comparison to the same probe located in the water pool “center”. The four fluorescent probes that we have used (Figure 1) were intentionally selected based on two criteria: (a) preferred probe location in the classical reverse micelle architecture and (b) the type of information attainable from each probe. Specifically, 2,6TNS and the Re complex are anionic and water soluble. Thus, they should experience electrostatic repulsion by the anionic AOT headgroups and reside mostly toward the water pool center. DCM is neutral, it is not soluble in water, it fluoresces weakly in n-heptane (quantum yield ) 0.01), and it has been previously shown to reside at the water/surfactant interface.6g R6G, a water soluble and n-heptane insoluble cation, will reside in the water pool,

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Figure 3. Effects of R and temperature on the R6G steadystate fluorescence anisotropy.

and it will tend to associate with the anionic AOT headgroups.6d,e Because the Re complex, 2,6-TNS, and DCM respond to their environment through changes in their emission spectra, spectral information obtained from each of these probes is not biased by solution turbidity. R6G (Cation). Steady-state fluorescence anisotropy measurements provide information on the mobility of fluorophores in solution and within microheterogeneous media.19,20 In addition, fluorescence anisotropy measurements are based on polarization changes, making them good indicators of multiple scattering events (e.g., onset of AOT precipitation). In R6G, increases in the experimentally measured fluorescence anisotropy, in the absence of any complicating factors, are related to decreases in the probe molecule’s rotational reorientation dynamics.6d,21,22 R6G has been used previously to investigate dopant mobility within sol-gel-derived xerogels21 and the microviscosity and rotational reorientation kinetics within AOT reverse micelles formed in n-heptane6d and liquid and near critical propane.22 Balny et al.11d previously showed that lowering R results in a lower precipitation temperature. For example, in an AOT reverse micelle solution having an R of 5.5, precipitation occurred at -42 ( 1 °C. On increasing R to 24.6, the precipitation temperature shifted to +4 ( 1 °C. Figure 3 summarizes the effects of R and temperature on the R6G fluorescence anisotropy. In each case, the anisotropy initially increases as we lower the temperature from +30 °C. This is consistent with an increase in the overall solution viscosity. As we continue to lower the sample temperature, we see a sharp, R-dependent drop in the fluorescence anisotropy. We ascribe this drop in anisotropy to the precipitation3a,4,11d of AOT/water which depolarizes the fluorescence by scattering.19 The temperature at which this transition occurs is R dependent, with lower water content resulting in lower temperatures attainable before the onset of precipitation. This is in agreement with previous work.11d In the R ) 0 samples, R6G anisotropy increases gradually as temperature is decreased from 30 to -60 °C. Below -60 °C, a slow decrease is observed followed by a drastic drop at -100 °C. This behavior is consistent with (19) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum: New York, 1999. (20) Kawski, A. Crit. Rev. Anal. Chem. 1993, 23, 459-529. (21) (a) Narang, U.; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993, 47, 229-234. (b) Narang, U.; Wang, R.; Prasad, P. N.; Bright, F. V. J. Phys. Chem. 1994, 98, 17-22. (c) Baker, G. A.; Jordan, J. D.; Bright, F. V. J. Sol.-Gel Sci. Technol. 1998, 11, 43-54. (d) Baker, G. A.; Pandey, S.; Maziarz, E. P., III; Bright, F. V. J. Sol.-Gel Sci. Technol. 1999, 15, 37-48. (22) Heitz, M. P.; Bright, F. V. Appl. Spectrosc. 1996, 50, 732-739.

Figure 4. Emission results for DCM in various solvent systems. (Panel A) DCM emission spectra in (spectrum a) AOT reverse micelles (R ) 20), (spectrum b) pure n-heptane, and (spectrum c) pure water. (Panel B) Effects of temperature on the DCM emission in AOT reverse micelles (R ) 20). Each spectrum was recorded at 10 °C intervals between + 30 and -100 °C. The inset shows the effects of temperature on the DCM emission intensity. A typical error bar is shown. (Panel C) Effects of R and temperature on the DCM emission center of gravity in AOT reverse micelles. The lines are aids for the eye. A typical error bar is shown.

the previously described increase in solvent viscosity followed by precipitation of AOT/water (recall R ) 0 samples have no added water, but there is likely intrinsic water1e present). (Note: One would expect the observed R6G anisotropy to approach the limiting anisotropy when the solution freezes,19 ca. -90 °C. The turbidity of the solution, however, depolarizes the fluorescence completely, and this is not the case here. The drastic drop in anisotropy observed at -100 °C is caused by depolarization of the emission due to freezing of the n-heptane.) DCM (Neutral). DCM is strongly solvatochromatic,23 and changes in the local solvent dipolarity are manifest in its emission spectra. Figure 4A presents the steadystate emission spectra for equal molar concentrations of DCM: (spectrum a) sequestered within an AOT reverse micelle (R ) 20), (spectrum b) dissolved in pure n-heptane,

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Figure 5. Effects of R and temperature on the 2,6-TNS fluorescence in AOT reverse micelles. (Panel A) Temperature-dependent emission spectra at R ) 20. The direction of the arrow indicates decreasing temperature. Each spectrum was recorded at 10 °C intervals between + 10 and -100 °C. (Panel B) Emission maxima for the LE and TICT spectra. (Panel C) Full widths at halfmaximum for the LE and TICT spectra. (Panel D) Fractional contribution of the LE and TICT species. In panels B-D, the 2,6-TNS TICT and LE states are indicated by closed and open symbols, respectively. The R values are 5 (b,O), 10 (9,0), 15 (2,4), and 20 ([,]).

and (spectrum c) “dissolved” in pure water, all at +20 °C. For all practical purposes, DCM does not emit in pure water. The emission in n-heptane is weak and appears near 535 nm. The DCM emission in the reverse micelle is significantly stronger than the emission in water or n-heptane, and it appears at 611 nm. Pal et al.6g reported that DCM emits at 625 nm within an AOT reverse micelle at the same water loading ([AOT] ) 0.09 M) and there was a 40-fold intensity enhancement over DCM in pure n-heptane. Given that DCM is essentially nonfluorescent in water, weakly fluorescent in n-heptane, and strongly fluorescent in the AOT reverse micelle, we conclude that the DCM resides at the water/surfactant interface and reports exclusively from this site. Figure 4B presents the DCM emission spectra in AOT (R ) 20) between +30 and -100 °C. Control experiments in pure n-heptane and water over the same temperature range yielded spectra (not shown) that were at least 50fold less intense than the DCM/AOT spectra, indicating that no significant report arises from the DCM in n-heptane or water at any R or temperature. The Figure 4B inset shows that the emission intensity increases by 10-fold with decreasing temperature between +30 and ca. -40 °C. Below -40 °C, the DCM emission intensity remains constant. At these low temperatures, increases in DCM emission intensity are apparently compensated by intensity losses due to scatter from precipitating AOT/ water and/or the decrease in the amount of dissolved DCM. (23) (a) Meyer, M.; Mialocq, J. C.; Rougee, M. Chem. Phys. Lett. 1988, 150, 484-490. (b) Mialocq, J. C.; Meyer, M. Laser Chem. 1990, 10, 277-296. (c) Birch, D. J. S.; Hungerford, G.; Imhof, R. E.; Holmes, A. S. Chem. Phys. Lett. 1991, 178, 177-184. (d) Pommeret, S.; Gustavsson, T.; Naskrecki, R.; Baldacchino, G.; Mialocq, J. C. J. Mol. Liq. 1995, 64, 101-112.

Figure 4C summarizes the effects of temperature and R on the DCM emission center of gravity. As we decrease the temperature from +30 to -100 °C, we see that the DCM spectra shift blue by ∼40 nm and the shift is R independent. The inflection point occurs at ca. -20 °C. These R-independent spectral shifts are consistent with the DCM not reporting on the freezing of the water pool. The origin of this blue shift may be better understood by looking closely at the DCM photophysics. The DCM emission originates from a locally excited (LE) state and a twisted intramolecular charge transfer (TICT) state.6g The LE state emits at higher energy in comparison to the TICT state. Conversion from the LE to TICT state depends on the physicochemical properties of the solvent/surroundings. Studies on DCM dissolved in AOT reverse micelles at ambient temperature by Pal and co-workers6g showed that the LE to TICT dynamics is slowed within the reverse micelle water pool in comparison to the observed dynamics in pure water. Under the subzero temperatures used in the current studies, formation of the TICT may be further slowed. Under these circumstances, the resulting emission arises more so from the LE state, resulting in an observed blue shift with decreasing temperature. 2,6-TNS (Monoanion). 2,6-TNS exhibits spectra that depend on the local solvent dipolarity.24 These spectral shifts arise from dipolar solvent relaxation and interconversion of a LE state to a TICT state. These solvent effects have been explained and discussed in detail elsewhere.19,24 Figure 5A presents the 2,6-TNS steady-state emission spectra in AOT reverse micelles (R ) 20) as a function of (24) Seliskar, C. J.; Brand, L. J. Am. Chem. Soc. 1971, 93, 54145420.

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temperature. As the temperature decreases from +10 to -100 °C, the average emission shifts from ∼440 to ∼410 nm and the fluorescence intensity increases by ∼20-fold. Spectra recorded at intermediate temperatures (-30 to -50 °C) clearly exhibit profiles that suggest the presence of two emitting states (e.g., LE and TICT). All 2,6-TNS spectra in AOT at all R and temperatures can be reasonably well deconvolved (r2 > 0.99) into two spectra (Gaussians). Figure 5B summarizes the effects of temperature and R on the recovered LE and TICT peak maxima. The TICT (closed symbols) and LE (open symbols) spectra both blue shift as the temperature decreases. The inflection points in the LE and TICT spectra also track each other. At R e 10, the inflection points in the TICT and LE features occur at ca. -35 °C. At R g 15, the inflection point shifts to higher temperatures, occurring at ca. -20 °C. These results are consistent with the 2,6TNS interacting more with the interfacial water at low R and this interfacial water freezing at lower temperatures. The associated full width at half-maximum (fwhm) values are shown as functions of temperature and R in Figure 5C. At low temperatures (below -40 °C), the TICT and LE exhibit fwhm values that are independent of water content. As the temperature is increased above -40 °C, the fwhm values increase, suggesting that 2,6-TNS is experiencing a more heterogeneous environment or that the LE-to-TICT twisting rate is occurring on a time scale similar to the LE excited-state lifetime. For the TICT state, the temperature at which the fwhm narrows is independent of R. For the LE state fwhm, there is an R dependence that parallels the emission maxima (Figure 5B). Figure 5D presents the effects of R and temperature on the relative LE and TICT contribution to the total emission. The first observation is that R has little effect on the percent contributions. Second, the TICT species is the dominant emitting species at all temperatures, ∼80% at +10 °C and above and ∼55% at -100 °C. Thus, the TICT process is clearly slowed within the reverse micelle as we lower the temperature; however, even at -100 °C, 2,6-TNS appears to still undergo an LE-to-TICT transition within the micelle water pool on a time scale that is on the order of the 2,6-TNS excited-state fluorescence lifetime. Re Complex (Dianion). Organometallic complexes consisting of Re(I) coupled to R-R′-diimine ligands exhibit spectral shifts arising from changes in local environment rigidity (rigidochromism).15a The effects of temperature and pressure on these species have been studied in detail.15b,d,g Rigidochromic Re(I) species have been used previously to investigate the gelation/densification kinetics of sol-gel-derived materials,15c,f to determine the mechanical stability within surfactant-mesostructured siliceous composites,15i and to monitor industrially important polymerization reactions such as epoxy curing.15e,h To test the Re complex’s suitability for the current studies, we recorded its temperature-dependent emission spectra between +30 and -60 °C in pure 1-heptanol. 1-Heptanol was selected as our test solvent because the Re complex is soluble (it is not soluble in n-heptane), the carbon chain length is identical to that of n-heptane, and its freezing point, -34 °C,25 occurs near the middle of the temperature range attainable with our temperature stage and the observed “freezing points” we observe in the AOT/ water/n-heptane system. Together, these features ensure that the complex remains dissolved during the experiments and that spectral analysis can be performed before and after the freezing of 1-heptanol. Figure 6A summarizes (25) CRC Handbook of Chemistry and Physics, 76th ed.; Lide, D. R., Ed.; CRC Press: New York, 1995.

Munson et al.

Figure 6. Temperature-dependent emission results for the Re complex dissolved in 1-heptanol and AOT reverse micelles. (Panel A) Effects of temperature on the emission center of gravity and intensity in pure 1-heptanol. The dotted vertical line denotes freezing of the 1-heptanol. A typical error bar is shown. (Panel B) Effects of R and temperature on the emission center of gravity within AOT reverse micelles.

the results of these experiments in a plot of the spectral position and luminescence intensity versus temperature. There are small changes in the emission maximum and intensity as we progress from +30 down to -40 °C (a small red shift of -0.06 nm/°C). Near -40 °C (dotted vertical line), there is a dramatic blue shift in the Re complex emission spectrum that is accompanied by a precipitous increase in the fluorescence intensity. There is no additional shift when we continue to cool the sample from -40 to -60 °C. These results argue that we can use the Re complex to estimate the AOT water pool freezing. Figure 6B presents the emission center of gravity for the Re complex within an AOT reverse micelle as a function of R and temperature. In the reverse micelles, the Re complex exhibits an R-dependent blue shift as we decrease the solution temperature. Based on the results in Figure 6A, we use the temperature that marks the beginning of the shift to estimate the freezing of the average local microenvironment that surrounds the Re complex within the AOT micelle water pool. The position of this freezing point clearly decreases with decreasing R. Note also that the spectral shifts all occur well before the n-heptane freezing point (-90 °C). As R decreases, we suggest that the Re complex begins to sense the more constrained water molecules at the surfactant interface. Because these water molecules freeze at lower temperature,26 an R-dependent “freezing” is observed. Similar dependencies have been observed for AOT reverse micellar systems studied using DSC.7b-d For example, AOT/water/hexadecane reverse (26) (a) Agnell, C. A. Annu. Rev. Phys. Chem. 1983, 34, 593-630. (b) Luedemann, H. D. Pol. J. Chem. 1994, 68, 1-22. (c) Bergman, R.; Swenson, J. Nature 2000, 403, 283-286.

Effects of Temperature on Fluorescent Probes

micelles with potassium oleate and heptanol stabilizers were studied.7b At low water percentages (10-12%), freezing of water occurred at -39 °C. Higher water percentages (27-36%) lead to an increase in the water freezing temperature to -19 °C.7b Conclusions The interiors of AOT reverse micelles formed in nheptane “freeze” over a wide range of temperatures that depends on R and the environment in which the probe resides. No two of the fluorescent probes used in these studies report on the same microenvironment. The cationic R6G probe senses the onset of precipitation. Specifically, at low R (5) the onset of precipitation occurs at ca. -55 °C. Precipitation systematically shifts to ca. -10 °C at higher R (20). The neutral probe DCM reports only on the micelle interfacial region.6g The apparent freezing point of this domain occurs at ca. -25 °C, and it is R independent.

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The dianionic Re complex senses the water pool core more so than any of the other probes. The apparent freezing point of this environment depends on R. At low R (5), freezing occurs at ca. -35 °C and the apparent freezing point shifts to ca. -10 °C at higher R (20). The monoanion 2,6-TNS behaves in a binary manner. When R e 10, the apparent freezing point that it senses occurs at ca. -35 °C. If R g 15, the apparent freezing point increases to ca. -22 °C. Taken together, these results show how solutes may encounter significantly different microenvironments within reverse micelles that contain supercooled water. Acknowledgment. The authors thank Dr. Mahdi M. Abu-Omar (UCLA) for providing the Re complex. This work was generously supported by the NSF, DOE, and the University at Buffalo. LA0302753