J . Phys. Chem. 1989, 93, 7694-7698
7694
The absence of significant hydrocarbon density at the 12-ring window site in the benzene-loaded material contrasts with the benzene distribution observed in sodium zeolite Y.i9,20 As even simple atom-atom potential calculations demonstrate, several factors may contribute to this distinction between the two systems. The configurations of the 0 and T atoms that define the 12-ring windows are a little different. In zeolite L, these species are coplanar, whereas in zeolite Y, the aperture is slightly puckered. The environments of benzene molecules in capping positions adjacent to the window would clearly have different interactions with a benzene molecule located in the window site compared to the analogous configuration in sodium zeolite Y. Such benzenebenzene interactions, supplementing the van der Waals interactions between the benzene molecule and the oxygen atoms of the 12-ring window, are apparently responsible for the observed occupancy of the window site in the latter system. As Figure 5 illustrates, of the six type D K+ cation sites in any given unit cell section (on average 4.5 of which are occupied), only one has on average a benzene molecule in the capping position. The diffraction data (Figure 4) show no evidence for coherence to the distribution of benzene (trial PND pattern simulations and refinements were made based on a 2c supercell of P6/mmm symmetry), arguing against sequential aggregation of pairs of benzene molecules followed by vacant unit cells. Steric constraints are such that any two opposite benzene sites could be occupied at one time (and, likewise, that diffusion of benzene at these loading levels does not require a cooperative process of site exchange). The maximal benzene sorption capacity of LTLframework materials is 12.5 molecules uc-l The apparent absence of pairing at the loading level studied here suggests that possible benzene-benzene interactions are not a major factor in determining the sorbate configuration. The type of distribution observed here is therefore expected to be maintained up to a level n = 2.0. The stability of the benzene site in which the ring n-electron density can interact with the K+ cation is consistent with earlier .233
observation^'^*^^ and calc~lations.~~ The environment of the site (Figure 5) is such that rotation of the molecule about the K+ cation-ring center vector is not greatly restricted (Figure 6 ) . Deuterium NMR data for this system for T I 150 K and n 5 2.0 show a reduction in the effective quadrupolar coupling constant of almost exactly 1/2, indicating rotation of the benzene molecules about an axis perpendicular (90 f l o ) to the C-D b ~ n d , consistent ’ ~ ~ ~ ~with the picture afforded by the structural results.
-
Conclusion
The complete structures of dehydrated potassium zeolite L at 298 and 78 K and of the same zeolite at 78 K containing 1 molecule of perdeuteriobenzene/unit cell have been determined by neutron powder diffraction. The results have enabled a discussion of the variation of the framework geometry on dehydration, on benzene sorption, and with temperature change 78-298 K. The use of powder neutron diffraction has enabled a direct measure of the aluminum partitioning between the fwo inequivalent T sites in the structure that, consistent with the complementary measure from bond length arguments, demonstrates a distinct aluminum preference for the 12-ring, Si1 (T4) sites. The nonframework K+ configuration shows only subtle changes over the range of conditions studied. Perdeuteriobenzene a t an average loading level of 1 molecule/unit cell is observed at 78 K in “capping” positions above the channel wall (type D) K+ cations, a location that permits facile reorientation of the molecules in the molecular plane. Acknowledgment. I would like to thank K. R. Poeppelmeier, W. T. Koetsier, J. P. Verduijn, B. G. Silbernagel, A. R. Garcia, R. Hulme, J. J. Steger, T. H. Vanderspurt, and A. J. Jacobson for assistance, advice, and helpful discussions during the course of this work. Registry No. K9A19Si27072, 1335-30-4; C6D6, 1076-43-3. (38) Sauer, J.; Deininger, D. Zeolites 1982, 2, 114-120.
Rotational Dlffuslon of Acridine Orange Attached to SDS Mlcelles Shiow-Hwa Chou and Mary J. Wirth* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 (Received: February 21, 1989; In Final Form: May 2, 1989)
The reorientation behavior of acridine orange in SDS micellar solution at neutral pH is studied by time-resolved fluorescence spectroscopy. A combination of lifetime and reorientation measurements for varying SDS concentrations shows that the motions of acridine orange are anisotropic and that there is only one type of acridine orange species above the critical micelle concentration. Acridine orange is shown to reorient as a noncylindrical hindered rotor. Below the critical micelle concentration, acridine orange partitions between a premicellar aggregate and the surrounding solution. The solute is more rigidly attached to the premicellar aggregate and again reorients as a noncylindrical hindered rotor.
The fluorescence properties of dyes attached to micelles have been shown to provide information about micellization. For example, energy transfer between dyes and quenchers attached to micelles has been shown to sense the diameter of the micelle,’ assuming negligible perturbation of the micelle upon attachment. Also, an increase in fluorescence also occurs upon attachment of dyes to micelles, allowing an estimate of the critical micelle concentration (cmc).2 In addition to these studies of the micelle
itself, there is an increasing interest in solute-micelle interactions. This has been stimulated by the development of a new separation technique called micellar electrokinetic capillary ~hromatography.~ The entire basis of this separation technique is the differential interactions of solutes with micelles, which are presently poorly understood. Further studies are needed. Several fluorescence properties are affected when a solute interacts with a micelle. Lifetimes are well-known to increase upon micellar attachment, giving rise to the higher fluorescence quantum yields. Fluorescence spectra provide information about the polarity
( I ) Choi, K.-J.; Turkevich, L.A.; L o a , R. J. Phys. Chem. 1988,92,2248. (2) Samsonoff, C.; Daily, J.; Almog, R.; Berns, D. S. J . Colloid Inreflace Sci. 1986, 109, 325.
(3) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsychiya, A,; Ando, T. Anal. Chem. 1984, 56, 11 1 .
Introduction
0022-3654/89/2093-7694$01.50/0
0 1989 American Chemical Society
Rotational Diffusion of Acridine Orange of the solute environment. The decay of the fluorescence anisotropy, which provides a measurement of solute reorientation dynamics, is changed upon attachment to the micelle. These three properties, lifetime, spectrum, and anisotropy decay, can be applied in concert to the study of dye attachment. In fluorescence spectroscopy, these properties can be determined for very low concentrations of solutes to avoid problems with solutesurfactant comicellization. There are two previous studies of the reorientation of cationic dyes attached to SDS micelles. In the first study, phase fluorometry was used to measure the reorientation of rhodamine 6G in SDS.4 The experimental results revealed a double-exponential decay of the anisotropy, in addition to a component having an infinite time constant. The interpretation was that the solute was free to reorient about only one molecular axis. The magnitude of the infinite component was not reported, although it is estimated from the data to be only a few percent, In the second study, transient dichroism measurements were used to study the reorientation of this same rhodamine 6G/SDS system, in addition to cresyl violet/SDS and acridine orange/SDS? Contradictory to the previous work, no infinite component in the anisotropy decay was observed in the transient dichroism measurements. The authors disagree with the interpretation in the phase-resolved work, attributing the fast decay component to unbound rhodamine 6G in water. There was insufficient time resolution to observe the fast component directly, but the concentration dependence of the anisotropy decay showed an increased contribution of the fast component at low micelle concentrations. Acridine orange and cresyl violet were shown to behave similarly to rhodamine 6G. Since these papers appeared, there has been significant progress in the development of models describing hindered rotation, Le., the rotation of species whose motions are hindered by the envir ~ n m e n t .The ~ ~purpose ~ of this work is to study the reorientation of cationic dyes attached to micelles, in light of these recently developed models, to determine whether the solute freely rotates or is hindered by its attachment to the micelle. Rhodamine 6G is a difficult candidate for study because its lack of symmetry complicates the interpretation. Acridine orange is chosen instead because it is symmetric (C,) and it is protonated symmetrically at neutral pH. A detailed study of acridine orange is performed to determine how its reorientation behavior is influenced by micelle attachment. A model for the hindered rotation of acridine orange is described. The fluorescence lifetimes and anisotropy decays of the solute are measured for a range of SDS concentrations from zero, through the submicellar region, and above the cmc.
Theory 1 . Anisotropy Decay. The fluorescence anisotropy, r(t), is a quantity that is directly measurable from the time dependence of the emission of polarized light. r(t) is well-known to be described by the correlation function for the orientations of the excitation, q, and emission, 7 , dipoles. r(t) = (2/5)(P~(dO)--dt))) (1) P, is the second Legendre polynomial. Under the expected conditions of strong damping of rotational motions, r ( t ) for a symmetric solute, whose transition moment lies along a symmetry axis, is a double e~ponential'~ 2A)t] + r(t) = (3/10)(p a ) exp[-(6D (3/10)(8 - a ) exp[-(6D - 2A)t] (2)
+
+
(4) Klein, U. K. A.; Haar, H.-P. Chem. Phys. Lett. 1978, 58, 531. (5) Lasing, H. E.; von Jena, A. Chem. Phys. 1979, 41, 395. (6) K!nosita, K.; Kawato, S.; Ikegami, A. Biophys. J . 1977, 20, 289. (7) Lipari, G.; Szabo, A. Biophys. J . 1980, 30, 489. (8) Szabo, A. J . Chem. Phys. 1984.81, 150. (9) Kinosita, K.; Ikegami, A.; Kawato, S. Biophys. J . 1982, 37, 461. (10) Lakowicz, J. R.; Cherek, H.; Maliwal, B. P.; Gratton, E. Biochemistry 1985, 24, 376. (1 1) Fujiwara, T.;Nagayama, K. J . Chem. Phys. 1985,83, 3110. (1 2) Van der Meer,B. W.; Kooyman, R. P. H.; Levine, Y . K. Chem. Phys. 1982, 66, 39. (13) Lipari, G.;Szabo,A. J . Chem. Phys. 1981, 75, 2971. (14) Chuang, T. J.; Eisenthal, K. B. J . Chem. Phys. 1972, 57, 5094.
The Journal of Physical Chemistry, Vof. 93, No. 22, 1989 7695 z
/n\
/
X
Figure 1. Structure of protonated acridine orange with reference to the system of coordinates.
where the parameters CY, /3, D, and A have their usual meaning. Equation 2 describes the anisotropy decay of a freely diffusing solute. The anisotropy function has been derived for the hindered rotation model, more descriptively referred to as the wobblingin-cone modeL6 In spherical polar coordinates, a cylindrically symmetric solute is allowed to rotate through the polar angles 0 I6 I,e and the azimuth angles 0 I 4 5 2 ~ Thus, . the solute diffuses freely within a cone of semiangle 6,. The potential energy function for the cone boundary is not critical: a wall at 80 suffices? Relatively simple behavior is predicted for the anisotropy decay. From the decay of the spherical harmonic correlation function, Lipari and Szabo have derived closed-form approximations for r(t) with respect to Bo, which are virtually identical with the numerical solutions over a wide range of angles.' These approximations are, in turn, nearly single exponential, with an additive constant: r ( t ) = r(0)((1 - F) exp(-t/Tw) + F J (3) T~ is the time constant for randomizing the orientation within the cone. F is calculated by integrating the second Legendre polynomial over all accessible angles and is related to the cone angle? If the transition dipole is parallel to the axis of attachment F = (0.5 COS o0(i COS e,))z (4)
+
F varies between zero and unity. If the transition dipole is perpendicular to the axis of attachment F = 10.25 sin4 0$ (5) In this case, F varies between zero and 0.25. The smaller limit of F arises because the dipoles populate the inverse of the cone. If the cone itself is able to reorient on a time scale longer than the motions within the cone, a double-exponential decay of the anisotropy is predicted,' where T , is the time constant for overall reorientation of the cone, as determined by the medium. The structure of protonated acridine orange in the spherical polar coordinate system is shown in Figure 1. The Cartesian coordinates of the solute axes are also shown. The y axis is the axis of attachment. Acridine orange is far from a good approximation to a cylinder; therefore, the existing hindered rotor model cannot be applied reliably. The problem of describing the motions of hindered acridine orange simplifies if the time scales of the rotational motions can be separated. The solute tilts through angle 0 by rotation about both the x and y axes. The time scales for these two motions can be separated if the correlation time for free rotation about they axis, T ~is, long compared to that for hindered rotation about the x axis, T*. The dimensions of the solute favor this circumstance, as does the fact that the solute must rotate through 180° about they axis but only 0, about the x axis. From the dimensions of acridine orange and the numerically determined relation between 19, and T,' it is predicted that T , / T ~ 1 6 for Bo ~
~~
(15) Matsuoka, Y.; Kamaoka, K. Bull. Chem. SOC.Jpn. 1979, 52, 3163. (16) Youngren, G. Y.; Acrivos, A. J . Chem. Phys. 1975, 63, 3846.
7696 The Journal of Physical Chemistry, Vol. 93, No. 22, 1989
Chou and Wirth decay functions are all assumed to be exponentials, which have simple analytical formulas for their Fourier transforms. By the additive nature of Fourier transforms, it is convenient to analyze the multiple-exponential decays expected for these data. The relations between the measured parameters of phase and amplitude and the decay parameters have previously been d e r i ~ e d . ~ A ,'~ nonlinear regression is required to calculate the decay parameters from the experimental data. Experimental Section 1. Chemiculs. For all experiments, the concentration of acridine
Figure 2. Orientational distributions of acridine orange. (a, left) After subnanosecond reorientation about the x axis, the directions of the transition dipoles are randomized within the cone of semiangle 0,. (b, right) After nanosecond reorientation about they axis, the directions of the transition dipoles are randomized within the volume described by the inverse of the double cone.
I 45'. It can therefore reasonably be expected that the time scales are separable for these two rotational motions. Three separate time scales of rotation are used to describe the anisotropy decay of acridine orange: subnanosecond (T+),nanosecond ( T ~ )and , many nanoseconds (T,,,),where T, is the reorientation time of the micelle. (1) On the shortest time scale, acridine orange wobbles through angles 0 to *Bo, with a decay constant of T ~ .Due to the three-dimensional geometry of excitation, the distribution of orientations is randomized in the cone shown in Figure 2a. (2) On the nanosecond time scale, acridine orange reorients about they axis with the continued restriction of 8 within *Bo. The orientations of the solute relax into the inverse-cone geometry shown in Figure 2b with a decay constant of fy. (3) The remaining anisotropy relaxes on the longest time scale with time constant T,, due to micelle rotation. The model for acridine orange as a noncylindrical hindered rotor thus predicts an approximate triple-exponential decay:
r(t)/r(O) = K 1 - F d ( 1 - F l ) exp(-t(l/7x + 1 / +~ 1/7m)) ~ + (1 - Fz)FIexp(-t(l/~y + l / ~ m ) )+ FZ ex~(-t/7m)I (7) Each of the orientational correlation functions has been approximated as an exponential decay. FI and Fz are described by eq 4 and 5, respectively. For cone angles less than 50°,F2 is small and eq 7 is approximately a double exponential: the long time constant due to micelle reorientation would not be observable. 2. Measurement of Anisotropy Decay. The fluorescence intensity as a function of time, I(t), for a species obeys the relation I(t) = I(0) exp(-t/rf){l
+ (3 cosz 0 - I)r(t)l
(8)
where ff is the fluorescence lifetime, 8 is the angle of polarization between the excitation and the emission, and r(t) is the fluorescence anisotropy. Equation 8 is valid for species having a population decay described by the single-exponential decay with time fI. The fluorescence lifetime can be determined by measuring I(t) at 54.7O. The anisotropy can be determined by measuring I(t) at Oo and 90' polarizations, as shown in eq 8. Alternatively, as is done in this work, r(t) can be measured by frequency domain spectroscopy, where the excitation is sinusoidally modulated and the amplitude and phase shift of the modulated emission are detected. The difference in phase shift for Oo and 90° polarizations, #o - $90, is
orange was 2.0 X M and the pH was 6.0. Sodium dodecyl sulfate was obtained from Aldrich (99+%) and was further purified by passage through a column packed with octadecylsilanized silica, as described in a published proced~re.'~The concentrations of the purified solutions were determined by conductivity measurements. Acridine orange (90+%) was purified by bulk extraction between methanol and silica gel, and the concentration of the resulting solution was determined by UV-visible absorption spectroscopy. For each measurement, freshly made solutions of acridine orange were prepared from a stock solution in methanol. The temperatures of the solutions were controlled to 23 f 0.2 O C for all experiments, which included conductivity, fluorescence lifetime, and anisotropy measurements. The solutions were flowed during the laser experiments. 2. Optical Measurements. The fluorescence lifetimes were measured by sampled single photon counting using software discrimination. The frequency domain measurements were made using an apparatus very similar to one described previously,'* which employs the first four mode beats of a mode-locked ion laser for the modulation frequencies. The 476-nm line of the argon ion laser was used. The polarization was controlled to within 1 The one modification over the previously published instrument was that the polarization was controlled electronically by using a high-extinction (200:l) Pockels cell. The data were acquired by using ASYST to control a Keithley data acquisition and control bus. 3. Analysis. The simplex method was used for the nonlinear regression. x2 was calculated from where u was determined from a large pool of measurements. For a given SDS concentration, eight parameters were measured: the phase and amplitude anisotropies for four frequencies, which were 82, 164,246, and 328 MHz. The measurement of each parameter was repeated six times, giving 40 degrees of freedom, v. For all SDS concentrations, r(t) was characterized by regression of the eight phase and amplitude measurements to give four parameters: T ~712, ~ F, , and r(O), as in the general double-exponential decay. The fluorescence lifetimes were always determined from separate experiments at the magic angle polarization. Results and Discussion
The ratio of the amplitude for 0' polarization to that for 90° polarization, mo/m90, is
The position of protonation for acridine orange was confirmed with proton NMR. The pKa of the ground state is 10.5,19 which was verified by spectrophotometric titration. The pKa of the excited state is 11, which was determined by fluorimetric titration. Thus, for all fluorescence studies in neutral, aqueous solution, acridine orange remains in its protonated form. The protonated solute is referred to as acridine orange. 1. Behavior above the Cmc. The raw frequency domain data is illustrated graphically in Figure 3, which shows both the phase and amplitude data for several solutions obtained by using the four modulation frequencies. The decay parameters obtained from the simplex analysis of the data are summarized in Table I for
S($) and C(#) are the sine and cosine transforms of I(?,#). The
(17) Rosen, M. J. J . Colloid Interface Sci. 1981, 79, 587. (18) Wirth, M. J.; Chou, S.-H. Appl. Spectrosc. 1988, 42, 483. (19) Heterocyclic Compounds; V.9. Acridines, 2nd ed.; Acheson, R. M., Ed.: Interscience: New York, 1973.
$o
- $90 = tan-'
[
s90c0
- sOc90
Sa90
+c
~ c ~ ~ ](9)
The Journal of Physical Chemistry, Vol. 93, No. 22, 1989 7697
Rotational Diffusion of Acridine Orange TABLE I: Fluorescence Lifetime and Reorientation Data for Acridine Orange in Water and in Solutions above the Critical Micelle Concentrationa C(SDS) 71 72 F do) 7f X2 0 0.138 0.26 1.8 h 0.1 1.2
16.0
~~
0.009 0.010 0.020
1.67 1.68 u.68
0.280 0.272 0.277
0.349 0.348 0.34m
0.33 0.33 0.33
3.0 f 0.1 3.0 h 0.1 3.0 -+ 0.1
[I
.008-.020H .007H
3.1 2.7 3.7
‘C(SDS) indicates moles of SDS added per liter of solution. All time constants are in nanoseconds. The uncertainties in T,, 72,F,and r ( O ) , as determined by the sensitivity of the simplex fit, are as follows: each parameter changes 1% per 0.1 increase in xz.
acridine orange in SDS solutions above the cmc. The cmc at 23 OC was determined to be 8.3 mM by conductivity studies, in agreement with the literature.20 The decay parameters for acridine orange in pure water are also included in Table I, and it is single exponential, as is predicted from its structure and transition symmetry. For all micellar solutions above the cmc, the data fit well to a double-exponential decay. The parameters generally agree with the results of the previous transient dichroism experiment,’ where the decay parameters were reported to be r,, =
