1786
Langmuir 1994,10, 1786-1792
Electron Spin Resonance Study of the Effect of Urea on the Properties of AOT Reverse Micelles in Isooctane Fabio C. L. de Almeida, Hernan Chaimovich, and Shirley Schreier' Departamento de Bioquimica, Instituto de Quimica da Universidade de Stio Paulo, C.P. 20780, Stio Paul0 01498-970, Brazil Received January 3, 1994. I n Final Form: March 31, 1994@
The properties of supramolecular aggregates can be strongly influenced by the binding of small polar molecules. Here we describe the use of electron spin resonance to characterize the effect of urea on structural properties of reverse micelles. The electron spin resonance (ESR) spectra of water-soluble charged spin probes, 4-(trimethylammonio)-2,2,6,6-tetramethylpiperidine-l-oxyl iodide (trimethyl temphosphate (tempo phosphate, pamine, positively charged), and 2,2,6,6-tetramethylpiperidin-l-oxyl-4-y1 negatively charged), as well as the amphiphilic spin label 5-(4,4-dimethyloxazolidin-3-oxyl-2-yl)stearic acid (5-SASL)in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)reverse micelles in isooctane were examined with and without urea at increasing water contents (expressed as Wo, water:surfactant molar ratio). Micellar size, monitored by quasielastic light scattering (QELS), increased with increasing Wo and the increase was steeper in the presence of urea. The spectra of 5-SASL were also sensitive to changes in micellar size. As Wo increased, the spectra changed from those of a probe in the fast motional regime to that of a probe rotating anisotropically in a large particle. The data agree with the results of Haering et al. (J.Phys. Chem. 1988, 92, 3574). Rotational correlation times were calculated for spectra in the fast motion regime (low Wo) yielding particle radii in good agreement with those calculated by QELS. At higher Wo's, the spectra displayed inner and outer extrema, allowing the calculation of the order parameter, S. The values of S increased with increasing Wo, indicating a decrease in the amplitude of motion of the probe long molecular axis. Making use of the data of Haering et al., the probability of trans conformation along the hydrocarbon chain was also found to increase with Wo. Larger S values and slower motion about the long molecular axis were observed with urea. The isotropic hyperfine coupling constant, a,, increased with Wo, both with and without urea, indicating that the label is sensitive to the increasing hydration of the head group region. Spectra of the water-soluble probes were also sensitive to changes in Wo and to the addition of urea. At very low Wo (ca. 0.2)the probes yielded spectra indicative of orientation, both molecules rotating preferentially about the nitroxide x axis. As the water content increased, the probes displayed different spectral behavior: while tempo phosphate yielded lineshapes and a, values similar to those observed in bulk aqueous medium, the spectra of trimethyl tempamine indicated that the molecule was oriented, rotating preferentially about the nitroxide x axis at all Wo's examined, suggesting that the probe remained at the water-amphiphile interface region. Urea does not displace the probe from this region. Nevertheless, as suggested by the effects on line shapes and a,, the additive binds at the interface. In agreement with the results obtained for 5-SASL, the spectra of trimethyl tempamine were suggestive of slower probe motion in the presence of urea.
Introduction Reverse micelles and water in oil microemulsions, general denominations of ternary systems formed by water, detergent, and organic solvents, permit the investigation of solution properties of highly compartmentalized water.' In reverse micelles, water can be organized to solvate exclusively the detergent polar groups or form detergentsurrounded microdroplets.'S2 Among other parameters, water properties depend critically on the water:detergent molar ratio (Wo). The activity of water can be as low as 0.4at Wo < 1,tending asymptotically to 1as Wo increases above 12.3-9 * Author to whom correspondence should be addressed, FAX 5511-815 5579. e Abstract published in Advance ACS Abstracts, May 15, 1994. (1)(a) Eicke, H. F.; Rehak, J. Helv. Chim. Acta 1976, 59, 2883. (b) Luisi, P. L.; Giomini, M.; Pileni, M. P.;Robinson,B. H. Biochim. Eiophys. Acta 1988,947, 209. (c) Langevin, D. Acc. Chem. Res. 1988, 21, 255. (2) Kotlarghyk, M.; Huang, J. S.;Chen, S. H. J . Phys. Chem. 1985,89, 4382. (3) Wong, M.; Thomas, J. K.; Nowak, T. J. Am. Chem. SOC.1977,99, 4730. (4) Boicelli, C. A.; Giomini, M.; Giuliani, A. M. Appl. Spectrosc. 1984, 38,537. (5) Kon-No, K.; Kitahara, A. J . Colloid Interface Sci. 1971, 35, 409. (6) Politi, M. J.; Chaimovich, H. J. Phys. Chem. 1986, 90,282. (7) Keh, E.; Valeur, B. J . Colloid Interface Sci. 1981, 79, 465. (8) Zhang, J.; Bright, F. V. J. Phys. Chem. 1991, 95, 7900. (9) Bardez, E.; Monnier, E.; Valeur, B. J . Colloid Interface Sci. 1986, 112, 200.
The internal water pool of reverse micelles incorporates a large variety of water-soluble additives, ranging from simple salts to high molecular weight biomolecules.' We have recently demonstrated that reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in hexane are formed in the presence of up to 10 M urea in the water Transitions pool at low aqueous volume fraction (4w).10 to bicontinous phases occur a t progressively lower #w as the urea concentration increases and these structural effects were rationalized in terms of interfacial urea interactions.10 The effects of urea on solution properties has been investigated for several decades and its denaturing properties have been used and examined mechanistically for a large variety of isolated molecules and macromolecular aggregates. Urea effects are attributed to either breaking of water structure'l and/or solute-urea association.12Urea does not affect the rate of proton transfer in bulk ~olutions'~ or in the inner pool of AOT reverse micelles,1° indicating (IO)Amara1,C.L. C.; Brino,O.; Chaimovich, H.;Politi, M. J. Langmuir 1992, 8, 2417.
(11)(a) Eisenberg, D.; Kauzmann, W. The Structure and Properties of Water;Oxford: London, 1969. (b) Bloemendal, M.; Somsen, G. J . Am. Chem. SOC.1985, 107, 3426. (12) (a)Tanaka, H.; Nakashini, K.;Touharam,H. J . Chem. Phys. 1985, 82, 5184. (13) Politi, M. J.; Chaimovich, H. J. Solution Chem. 1989, 18, 1055.
O~~3-7463~9~/24~O-1~86$04.50/ 0 0 1994 American Chemical Society
Effect of Urea on AOT Reverse Micelles urea insertion into proton-accepting, water-containing clusters. The effects of urea on association colloids, such as micelles in water, have also been investigated. Urea increases both the critical micelle concentration and the degree of counterion dissociation from aqueous ionic mi~e1les.l~ These phenomena can be rationalized in terms of monomer solubility increase and urea binding a t the water/micelle interface.14-19 The analysis of the electron spin resonance (ESR) spectra of spin probes is a powerful tool for investigating organizational and dynamic properties of supramolecular assemblies, particularly membranes, as well as other amphiphile aggregates.20P2l Spin labels have been used in reverse micelles to examine water activity, probe-interface interactions, changes in the dielectric constant at the interface, and size changes as a function of water content.22-33 In this work we have investigated the properties of reverse micelles using charged water-soluble spin probes 4-(trimethylammonio)-2,2,6,6-tetramethylpiperidine-loxy1 iodide (trimethyl tempamine) and 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl phosphate (tempo phosphate) and the amphiphilic fatty acid 5-(4,4-dimethyloxazolidin3-oxyl-2-y1)stearicacid (5-SASL) to examine the effect of urea upon sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in isooctane. Changes in the ESR spectra of 5-SASL with Wo allowed the estimation of micellar size and suggested that urea binding to the interface produces a decrease of the amplitude of motion and rate of rotation of the probe's long molecular axis. The effects on the ESR spectra of water-soluble probes were interpreted assuming urea binding to the micellar interface.
Materials and Methods Materials. 5-SASL was obtained from Sigma Chemical Co. (St. Louis, MO), trimethyl tempamine from Molecular Probes (Eugene, OR), and tempo phosphate from Syva (Palo Alto, CA). AOT (AldrichChemicalCo., St. Louis,MO) (99%) and isooctane (Merck Darmstad, Germany) (chromatography grade) were used without purification. Urea (Merck, Darmstad, Germany) was triply recrystallized from hot ethanol.1° The water used was bidistilled in all glass apparatus and all other reagents were analytical grade or better. (14) Muller, N. J. Phys. Chem. 1990, 94, 3856. (15) Abu-Hamdiyyah, M.; Kumari, K. J.Phys. Chem. 1991,95,5664. (16) Causi, S.; DeLisi, R.; Milioto, S.; Teroni, N. J.Phys. Chem. 1991, 95, 5664. (17) Baglioni, P.; Ferroni, E.; Kevan, L. J.Phys. Chem. 1990,94,4296.
(18)Baglioni, P.;Rivara-Minten, E.; Dei,L.; Ferroni, E. J.Phys. Chem.
1990,94,8218. (19) Mukeriee. P.: Rav. A. J.Phvs. Chem. 1963.67. 190. (20) Berliner, L. J., Ed. Spin Libelling. Theory and Applications; Academic Press: New York, 1976. (21) Schreier, S.; Ernandes, J. R.; Cuccovia, I. M.; Chaimovich, H. J. Magn. Res. 1978, 30, 283. (22) Bratt, P. J.; Kevan, L. J.Phys. Chem. 1992,96,6894. (23) Haering, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988, 92, 3574. (24) Menger, F. M.; Saito, G.; Sanzero, G. V.; Dodd, J. R. J.Am. Chem. SOC. 1975, 97, 909. (25) Lim, Y. Y.; Fendler, J. H. J. Am. Chem. SOC. 1978,100, 7490. (26) Yoshioka, H. J. Colloid Interface Sci. 1981, 93, 214. (27) Yoshioka, H.; Kazama, S. J. Colloid Interface Sci. 1983,95,240. (28) Barelli, A.; Eicke, H. F. Langmuir 1986, 2, 780. (29) Kotake, Y.; Janzem, E. G. J. Phys. Chem. 1988,92, 6357. ~~
(30) Lossia, S. A,; Flore, S. G.; Nimmala, S.; Li, H.; Schlick, S. J.Phys. Chem. 1992, 96,6071. (31) Baalioni, P.; Nakamura, H.; Kevan, L. J. Phys. Chem. 1991,95,
3856. (32) Nakamura, H.; Baglioni, P.; Kevan, L.; Matau0,T.J. Phys. Chem. 1991,95, 1480. (33) Kang, Y. S.; McManus, H. J. D.; Kevan, L. J. Phys. Chem. 1992, 96,8647.
Langmuir, Vol. 10, No. 6, 1994 1787 Methods. Preparation of the Reverse Micellar Solutions. Adequate amounts of 0.05 M TrkHC1 buffer, pH 8.0, with or without 4.5 M urea, were added to a stock solution of 0.1 M AOT in isooctane. Stock chloroform solutions of 5-SASL were evaporated under vacuum (at least 0.5 h) and reverse micellar solutions were added subsequently. One microliter of methanol stock solutionof 0.0083M trimethyl tempamine or 2 pL of O.OO415 M tempo phosphate in water/methanol (1:l)were added to 0.2 mL of reverse micellar solutions to yield a final probe concentration of 4 X 1od M. ESR Spectra. Samples were placed in flat aqueous quartz cells (Wilmad) and the ESR spectra were obtained in a Bruker ER200 SRC spectrometer, at room temperature (22 f 2 "C). Quasielastic Light Scattering (QELS). The measurementa were made using a He-Ne laser (Hughes), a Thorn EM1 phototube, and a Brookhaven BI2030 autocorrelator. The samples were filtered using a 0.2-pm nylon filter. Measurements and Calculation of Spectral Parameters. Rotational correlation times ( T ~were ) calculated according to eq 1u
where AHo is the peak-to-peak width of the midfield line and h+l, b,and hl are the heights of the low, mid, and high field resonances, respectively. The order parameter, S, was calculated making use of eq 2 s
where 2Tl1and 2TL are the separations between the outer and inner extrema in the experimental spectra, respectively, and T,, TI,, and Tyyare the components of the hyperfine splitting tensor along the nitroxide principal axis (Tz, = 32.0 G, TI, = Tyy= 6.0 G35).S values were not corrected for polarity,%such corrections would not alter the values found by more than 3% . Isotropichyperfine splittings, an,were measured either directly from the spectra or making use of eq 3
for spectra of 5-SASL displaying inner and outer extrema.
Results Apparent hydrodynamic diameters (&) of particles in solution can be obtained from diffusion coefficienta measured by QELS and from rotational correlation times calculated from ESR spectra. Usually both calculations make use of the Stokes-Einstein relationship. In the present work, the particles were assumed to be spherical. Table 1 lists the &'S obtained from QELS and ESR measurementa, as well as the corresponding rotational correlation times ( r C )of AOT reverse micelles with and without urea. The values of Dh increased sharply with Wo (Table 1)and addition of urea to the water pool led to a steeper dependence (Table 1).lbJoThe maximal Wo achieved for the urea-containing reverse micelles without observing phase separation was 10. A simple geometrical calculation shows that ureacontaining AOT micelles cannot be spherical a t high Wo. Considering Dh = 33.2 nm for Wo = 8.8 (Table 1) and taking the area per head group as 0.45 nm2,1 a spherical micelle would require an internal water pool volume of ca. (34) Snipes, W.; Keith, A. D. Res. Deu. 1970, 22, 26. (35) Schreier, S.; Polnaszek, C. F.; Smith, I. C. P. Biochim.Biophys. Acta 1978,515, 375.
de Almeida et al.
1788 Langmuir, Vol. 10, No. 6, 1994 Table 1. Rotational Correlation Times ( T J a n d Hydrodynamic Diameters (&) of 0.1 M AOT Reverse Micelles i n Isooctane in t h e Absence o r i n t h e Presence of 4.5 M Urea 4.5 M urea
buffer
Wo
7,.(XIOB, wad-')
0.2 1.9 2.2 4.4 5.5 8.8 26 39
1.47 2.14
I
0 6 1
Dh (nm)
T~
(XI@, wad-')
Dh (nm)
~~~
2.80 3.2a 2.42
3.3" 7.96
6.3b
04;
0
33.2b 14.56 16.g6
0 Obtained making use of use of the Stokes-Einstein equation, from the T~ values calculated from the pseudoisotropic ESR spectra of 5-SASL. Obtained from the diffusion coefficient (QELS) and the Stokes-Einsten equation.
C3Tlwater
1 2
2
-: (io=iO
AOT/4
-
5
" r i-
V
urea
?
is,ss
Figure 1. ESR spectra of 5-SASL in 0.1 M AOT reverse micelles as a function of Wo without or with 4.5 M urea.
135pL/mL of the total volume. Since the sample contains 20 pL/mL, clearly the aggregate cannot be spherical. Therefore, Dh values for urea-containing micelles can only be taken as apparent, especially for Wo = 8.8 (Table 1). Similar considerations apply for Wo 4.4 in the presence of urea. The ESR spectra of 5-SASL in AOT reverse micelles were affected by changes in Wo, indicating that the probe was incorporated in the micelle and was sensitive to changes in aggregate structure (Figure 1). 5-SASL is expected to be located in the AOT monolayer with an orientation of the long molecular axis similar to that of the surfactant~.~3 The motionally averaged spectra of 5-SASL a t low Wo (Figure 1)were similar to those displayed by the probe in aqueous micelles.21 These spectra have been interpreted as being due to the freedom of molecular motion (segmental and of the long molecular axis) in the hydrocarbon region plus the fast rotation of the entire micelle, which is small enough to present short correlation times in the experimental time s ~ a l e . Lindblom ~ ~ ? ~ ~ and Wennestrom demonstrated that diffusion of the label on the micellar surface also contributes to the observed line shape.36 Assuming that the spectra were pseudoisotropic, rotational correla-
w2 Figure 2. Order parameter ( S ) ,calculated using eq 3, from 5-SASL spectra as a function Wo, in AOT reversed micelles and with ( 0 )4.5 M urea. formed without (0)
tion times ( 7 c )were calculated for Wo 0.2 and 1.9 without urea and for Wo 2.2 in the presence of the additive, according to eq 1(Methods). Assuming that the particle is a rigid sphere, the corresponding diameters were obtained using the Stokes-Einstein equation (Table 1). In spite of the oversimplifications involved in these calculations, the values of Dh calculated from ESR spectra are in reasonable agreement with those obtained by linear extrapolation16 of QELS-derived Dh values obtained at higher Wo. The increase in Wo led to larger particles, as indicated by the QELS measurements (Table 1). These particles are large enough to appear as immobile in the ESR time scale. As a result, the spectra of 5-SASL displayed inner and outer extrema and consist of the summation of spectra of probes rotating anisotropically about their long molecular axes at allorientations with respect to the magnetic field. The effect of increase in the reverse micellar size on the spectra of fatty acid-derived spin labels has been previously reported by Haering et The order parameter ( S )was calculated from the spectra of 5-SASL displaying inner and outer extrema according to eq 2 (Methods). S increased with Wo reaching a value of 0.55 without urea at Wo > 15 (Figure 2). Similar S values were measured in the presence of urea at Wo = 8 (Figure 2). S includes the contribution of the amplitude of motion of the long molecular axis (So) plus the probability of gauche conformations (Pg) along the hydrocarbon chain.37 Infrared and Raman measurements of the same system have yielded contradictory results indicating decrease,38 or increase,39of the probability of gauche conformations along the hydrocarbon chain, with increasing Wo. An equation relating the measured S values to So and the occurrence of intrachain trans-gauche isomerization has been given by Hubbell and M ~ C o n n e l l ~ ~ log s = n log P, + log s, + c
(4)
Pt + P, = 1
(5)
where n is the position of the oxazolidine ring in the fatty (36) Lindblom, G.; WennerstrBm, H. Biophys. Chem. 1977, 6, 167. (37) Hubbell, W. L.; McConnell, H. M. J . Am. Chem. SOC.1971, 93, 314. (38)Marin, G. A.; Magid, L. J. J. Phys. Chem. 1981, 85, 3938. (39) Maitra, A.; Jain, T. K. Colloids Surf. 1987, 22, 19.
Effect of Urea on AOT Reverse Micelles
Langmuir, Vol. 10, No. 6,1994 1789
16.0 I
I
AOT/woter
I
water
AOT/4.5
M urea
4 5 M urea
1401
0
1
10
20
30
40
50
I
60
wo Figure 3. Isotropic hyperfine splitting (a3 for the ESR spectra of 5-SASL as a function of Wo in AOT reverse micelles without (0) or with ( 0 )4.5 M urea. For pseudoisotropic spectra, a. was
calculated from the separation between the low- and high-field resonances, at the baseline; for spectra displaying inner and outer extrema, a. was calculated using eq 4. The dashed and dotted lines indicate the values of a. without and with urea in bulk solution, respectively.
acid chain, Pt is the probability of trans conformation, and C is a correction factor. A simplified form of eq 4 has been presented by Knowles et aL40 log S = n log Pt + log So
(6)
We have applied eq 6 to the data of Hearing et obtained in AOT reverse micelles without added urea, for stearic acid labeled at n = 5,6, and 7. We find So (0.87, 0.91,0.95) and Pt (0.85,0.89,0.89) for Wo 15,30, and 50, respectively. Thus, while Soincreases with Wo, Pt tends to a constant value of 0.89. The increase in Soreflects the decrease in amplitude of motion of the probe’s long molecular axis, because of the change in acyl chain packing due to the increase in aggregate size. These calculations also suggest that the acyl chain becomes more extended (Pt increases) with increasing Wo. In the presence of 4.5 M urea, similar qualitative effects were observed a t a lower Wo (Figures 1and 2). Broader lines, resulting from a slower rotation about the probe’s long molecular axis, are displayed in the spectra obtained in the presence of urea. Since the probe’s head group is anchored at the interface, this could be a result of urea intercalation between the head groups, leading to a slower rotation. Isotropic hyperfine splittings (an), measured from the spectra of 5-SASL are given in Figure 3. The values of an increased with Wo, both with and without urea, never reaching those measured in bulk solutions (Figure 3). The extent of water penetration into the hydrocarbon region of amphiphilic assemblies remains controversial.41 Our results can be taken as an indication of the increasing hydration of the interface region and/or an increase in local polarity due to urea adsorption. This hypothesis was further substantiated by the results obtained with trimethyl tempamine (see below). The behavior of molecules in the aqueous compartment of AOT reverse micelles was examined using cationic (40)Knowlee, P. F.; Marsh, D.; Rattle, H. W. E. Magnetic Resonance of Biomolecules;J. Wiley and Sons: New York, 1976. (41) (a) Tanford. The HydrophobicEffect; Wiley & Sons: New York, 1980. (b)Ieraelachvili, J. N. Intermolecularand Surface forces, 2nd ed.; Academic Press: New York, 1992.
Figure 4. ESR spectra of trimethyl tempamine in AOT reverse micelles as a function of Wo without or with 4.5 M urea. The bottom spectra were obtained in bulk aqueous solution. (trimethyl tempamine) and anionic (tempo phosphate) spin probes. These probes are essentially insoluble in isooctane,yielding exchange narrowed spectra when added to the solvent. These spectra (not shown) are characteristic of undissolved, aggregated probe.42 When incorporated in AOT reverse micelles, both probes yielded spectra (Figures4 and 5)corresponding to statistically distributed probe.21 At low Wo, both probes gave rise to spectra that displayed low field resonances narrower than the midfield resonances (Figures 4 and 5). This feature is typical of nitroxides rotating preferentially about the x axis, that coincides with the direction of the N 0 bond.4 Thus, both molecules,in spite of their opposite charge, are located in the head group region a t low Wo. Similar results have been described for another positively charged probe, as well as for a zwitterionic nitroxide in AOT reverse micelles.23 The spectra of trimethyl tempamineremained indicative of rotation about the xaxisat all Wo values, with or without urea (Figure 4). We calculated the ratio of the low field (h+l)to the midfield (ho) resonances as a mean to evaluate this preferential rotation (Table 2). Without urea, at Wo = 4.1, h+l/ho was considerably higher than in aqueous solution. The ratio further increased up to Wo = 13 and remained constant thereafter. With urea, the value of h+Jho was even larger with respect to bulk solution, remained constant from Wo 2.2 to 8.8, and was of the same order of magnitude (1.29 f 0.005)as that obtained without urea at Wo = 9.5 (Table 2). The values of h+l/ho indicate anisotropic rotation. Rough estimates of the order parameter S according to
-
(42) Wertz, J. E.; Bolton, J. R. Electron Spin Resonance. Elementary Theory and Practical Applications; McGraw Hill. New York, 1972. (43)Griffith,O. H.; Joet,P.InSpinLabellingTheoryandApplications; Berliner, L. W., Ed.; Academic Press: New York, 1976; pp 453-523.
de Almeida et al.
1790 Langmuir, Vol. 10, No. 6, 1994 AOT/4 5 M urea
AOT/water
16.8
--
16.5
-
wo 0
2
4
6
8
1
0
- -- - - -- - *_ - - - - - - - - - 1 16.8 water 4 . 5 M urea
7
16.5
16.2 15.9 15.6
15.e
A 15.3 0
10
20
30
40
50
9
-
I
15.3
,
60
wo
Figure 6. Isotropic hyperfine splitting (a,) in the spectra of trimethyl tempamine in AOT reverse micelles as a function of Wo. a, was estimated from the separation between the low field and midfield (filledsymbols) or the midfield and high field (open symbols) resonances: A, without urea; b, without ( 0 , 0)and with (m, 0)4.5 M urea. Note that the scale in the abscissa has been expanded in B. The dashed lines represent the values of a, in bulk solution without (A) and with (B) 4.5 M urea. wo 0
2
4
6
8
10
20 Gauss
Figure 5. ESR spectra of tempo phosphate in AOT reverse micelles as a function of Wo without or with 4.5 M urea. The two bottom spectra were obtained in bulk aqueous solution. Table 2. Effect of the Water/Detergent Molar Ratio (Wo) on the b+l/boand bolh-1 Ratios Measured from ESR Spectra of Water-Soluble Spin Probes Incorporated in AOT Reverse Micelles in the Absence and in the Presence of Urea wo
trimethyl tempamine
tempo phosphate
h+ilho
h+ilhn
holh-i
1.23 0.998
2.82 1.36
0.991
1.22
0.98 0.98 0.98
1.16 1.13 1.12
1.13 1.03 0.983 0.98
2.07 1.60 1.36 1.30
1.00 0.96
1.10 1.19
holh-i
Buffer 0.2 4.1 6.8 7 9.5 13 27 48
1.16 1.27 1.28
2.01 2.31 2.27
55
1.31 1.33 1.35 1.36 1.35
2.31 2.30 2.42 2.28 2.27
2.2 4.4 6.6 8.8
1.29 1.30 1.29 1.30
4.5 M Urea 2.44 2.45 2.42 2.64
water 4.5 M urea
1.00 0.99
Bulk Aqueous Solution 1.06 1.06
Polnaszek (private communication) yielded values of S < 0.2. Hence, we measured the ratios of the midfield (ho)
and high field (h-1) resonances and analyzed them as indicators of probe mobility (Table 2). Without urea the holh-1 ratios followed a pattern similar to that of the h+J ho ratios: a much higher value (2.01) than that in water was obtained for Wo = 0.2 and a higher and constant value (2.31 f 0.05) from Wo = 4.1 on. In the presence of the additive the holh-1 ratio was constant for all Wo's (2.28.8)) much higher than that in bulk aqueous solution and somewhat higher (2.48 f 0.1) than that obtained in the absence of urea at Wo >9.5 (Table 2). In agreement with the variation in h+llhoand holh-1 ratios in the absence of urea, the values of a n for trimethyl tempamine increased in going from Wo 0.2 to 27 and
6'
16.5 16.2
1
1 U
0
A
'
10
'
20
"30 ' 40 I 50
i
i
60
1
B
16.5
16.2
'
I
,
wo
Figure 7. Isotropic hyperfine splitting (an)in the spectra of tempo phosphate in AOT reverse micelles as a function of Wo without (A) or with 4.5 M urea (B). a, was estimated from the
separation between the low field and midfield resonances (filled symbols)and midfield and high field (open symbols) resonances. The dashed lines represent the values of an in bulk solution without (A) and with (B) urea. remained constant with increasing water. A similar behavior was observed in the presence of urea. The values of an were always smaller than those in aqueous solution (Figure 6). Our results suggest that trimethyl tempamine rotates preferentially about the nitroxide x axis a t all Wo values and its motion is considerably slower than that in aqueous solution. The preferential rotation about the x axis is maintained in the presence of urea indicating that the additive does not displace the probe from the micellar interface. The rate of motion seems to be further decreased by urea, in agreement with the results found for 5-SASL. The behavior of tempo phosphate in reverse AOT micelles was markedly different from that of trimethyl tempamine with increasing Wo. Without urea, at Wo 0.2, tempo phosphate also yielded spectra indicative of rotation about the nitroxide x axis and of slow motion (Figure 5 ) . As the water content increased, the spectra of tempo phosphate approached those of the probe in aqueous solution (Figure 5 ) . Both ratios h+llhoand holh-1 decreased steeply with increasing Wo, reaching the values found in aqueous solution for Wo 4.1 and 13, respectively (Table 2). In agreement with these results, a, also increased steeply with Wo to reach the values in aqueous solution (Figure 7). In the presence of urea, the variations of the line height ratios followed a pattern similar to that found without the
Effect of Urea on AOT Reverse Micelles
Langmuir, Vol. 10, No. 6,1994 1791
Table 3. Rotational Correlation Times ( r c )for Tempo Phosphate in AOT Reverse Micelles T, (xlO-*Owad-*) buffer 4.5 M urea
wo
0.2 2.2 4.1 4.4 6.6 7.0 8.8 13 27 48 aqueous solution
8.39 4.25 1.79 2.71 1.87
1.11 1.56 0.884 0.740 0.680 0.590
1.02
additive (Table 2). However, the probe displayed spectral features characteristic of slower motion a t higher Wo’s. The values of a, were indicative of an environment with the same polarity as that of bulk water (Figure 7). The isotropic nature of the spectra of tempo phosphate in reverse micelles above Wo 2.2 allowed the calculation of T~ (eq 1,Methods). The effects of urea on the T~ values of the probes in reverse micelles have to be compared with the effect of the additive in bulk solution. For trimethyl tempamine the value of T~ measured in bulk solution (3.5 X 10-l1 wad-’) is unaffected by the addition of 4.5 M urea. In contrast, 4.5 M urea almost doubled the value of T~ for tempo phosphate in bulk solution (Table 3), suggesting complex formation between urea and tempo phosphate. In reverse micelles the values of T~ for tempo phosphate approached that in water with increasing Wo, strongly suggesting that the probe is distributed mainly in the aqueous pool above Wo = 4.1 (Table 3). In the presence of urea the trend was similar (Table 3).
Discussion The combined use of amphiphilic and water-soluble spin labels to probe both the hydrocarbon and the aqueous microenvironments of reversed micelles can provide considerable information about these supramolecular aggregates. The size of reverse micelles increases upon addition of water due to the formation of a detergent-surrounded inner aqueous pool where the properties of water are increasingly similar to those of the bulk so1vent.l The Wo-dependent size increase has been determined using a variety of experimental methods. Our light-scattering data are in good agreement with published values.’ The spectra of micelle-incorporated fatty acid spin probes have been related to micellar size in AOT micelles in is0octane.~3 5-SASL is bound to the amphiphile monolayer surrounding the internal water droplet and its spectra are sensitive to the change in size of the reverse micelle (Figure 1). The probe’s rotational correlation times are comparable to the micelle’s rotational correlation time (Table 1). The value of S increases with Wo reaching a plateau (Figure 2). This effect can be explained by the high monolayer curvature of the small AOT micellar size. The increase in Wo and, consequently, micellar size leads to a decrease in curvature. This effect is observed in both the presence and absence of urea. The variation of a, for 5-SASL as a function of Wo suggests that the monolayer-bound probe reports a polarity increase as the water pool increases. This effect can be ascribed to several factors that cannot be separated with the present data: (i) water molecules can penetrate the
monolayer;4 (ii) the alkyl chain of 5-SASL bends, increasing the probability of the nitroxide moiety to reach the interface; (iii)increasing polarity a t the interface results in a polarity dependent interface-nitroxide interaction.* The addition of urea to the aqueous pool does not alter significantly the effect of polarity increase with micellar size. Trimethyl tempamine is located in the head g r o u p water interface a t all Wo’s, as indicated by the behavior of both ratios h+llho and hol h-1 (Table 3) as a function of water content. The increase in a, as a function of Wo (Figure 6) does not indicate probe partitioning into the micellar water pool. Rather, the increase in this polarity parameter seems to imply an increase of water penetration a t the interface. This interpretation is confirmed by the increase of a, found with the amphiphilic probe 5-SASL (Figure 3). The values of a, calculated for trimethyl tempamine never reach that in aqueous solution, in contrast with tempo phosphate. At low Wo, in spite of the charge difference both probes display immobilized spectra, indicating interfacial binding. As Wo increases, negative charge develops a t the interface, a discrete water pool is formed, and tempo phosphate is coulombically excluded from the interface while the positively-charged trimethyl tempamine remains interfacially bound a t all Wo’s. The spectral behavior of tempo phosphate (e.g., variation of h+l/ho, of hdho and of a,, Table 3 and Figure 6, respectively) clearly indicates that the aqueous phase of AOT reversed micelles has the properties of bulk water a t wo>13, a result that is in very good agreement with data obtained by a variety of other meth0ds.l Urea may intercalate between the headgroups in aqueous micelles.l”8 Our data strongly suggest that in reverse micelles urea also shows a preference for intercalation between the aggregate head groups. The addition of urea decreases the rate of motion of 5-SASL about the long molecular axis. Urea also decreases the rate of motion of trimethyl tempamine along the x-axis. The values of a, of both 5-SASL and trimethyl tempamine fail to reach those in water or 4.5 M urea. The ESR spectra of trimethyl tempamine suggest that, with or without urea, the probe remains a t the headgroup region. The additive therefore does not displace trimethyl tempamine from the interface but rather decreases the rate of probe motion. For the negatively charged tempo phosphate spin probe the value of a, with urea reaches that in bulk solution of comparable composition. Other spectral parameters (e.g. T ~h+llho , and holh-I), however, indicate that urea produces a decrease in the motional freedom of the probe, even a t high Wo. The negatively charged probe therefore must reside in the aqueous pool of the reverse micelle, yet the detailed properties of the urea-containing aqueous pool are not those of bulk solution a t the same concentration. The transition from a free particle regime to a bicontinous phase is obtained at a lower aqueous volume fraction with urea containing reverse micelles.1° The decrease of the rate of probe motion, as estimated here, may therefore reflect a change in particle geometry or a difference in local viscosity of the urea-containing aqueous pool. (44) Griffith, 0. H.; Dehlinger, P. J.; Van, S . P.J . Membr. B i d . 1974, 15, 159. (45) Seelig, J.; Limacher, H.; Bader, P. J. Am. Chem. SOC.1972, 94,
6364.
1792 Langmuir, Vol. 10, No. 6,1994
In conclusion, we have used ESR to confirm that the growth of reverse micelles can be detected using an amphiphilic spin probe. The analysis of the spectra of 5-SASL demonstrated that the acyl chain becomes more extended, and the long molecular axis displays a lower amplitude of motion as Wo increases. In addition the nitroxide reports a more polar environment. Urea in the aqueous pool induces probe immobilization at lower Wo’s. The spectra of the water-soluble spin probes also showed that urea induces a motional restriction at both the interface and the water pool. These data, taken together,
de Almeida et al.
strongly suggest that urea binds to the interface of reverse micelles of AOT in isooctane.
Acknowledgment. This work was partially supported by grants from the following Brazilian agencies: FundacBo de Amparo h Pesquisa (FAPESP, Projeto Temdtico) and Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico (CNPq, and Projetos Integrados). We acknowledge predoctoral fellowship from CNPq (FLCA) and research fellowships from CNPq (H.C. and S.S.) and CAPES (S.S.).
