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Study on the aggregation number of mixed micelles in aqueous binary mixtures of the bile salts and nonionic surfactant
Study on the Aggregation Number of Mixed Micelles in Aqueous Binary Mixtures of the Bile Salts and Nonionic Surfactant M. UENO, *'1 Y. KIMOTO,* Y. IKEDA,* H. MOMOSE,* AND R. ZANAt *Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, 1-3, Kagurazaka, 162 Shinjuku-ku, Tokyo, Japan and ~fCNRS, Centre de Recherches sur les Macromolecules, 6, rue Boussingault, 67083 Strasbourg Cedex, France Received April 3, 1986; accepted July 21, 1986 The mixed micellar behavior of sodium cholate, NaC, and octa-oxyethylene glycol n-decyl ether, C10E8, has been studied from the viewpoints of the aggregation number and the polarity of the interior of the micelle. The polarity of the interior of the mixed micelle obtained from 11/13 and the lifetime of pyrene fluorescence shows that the solubilization site of pyrene varies with the molar fraction. This fact is considered to be caused by the differences in the chemical structure and the hydrophobic nature between NaC and Cl0Es. Concentration dependencies are seen for the aggregation number and 11/13. These dependencies indicate that the size and composition in the mixed micelles vary with the total concentration of surfactants, and that the initial micelles formed near the CMC contain higher molar fractions of C10E8 than do analytical molar fractions. Furthermore, theoretical calculations using regular solution theory and excess thermodynamic quantities have been performed with the values of CMCs. The calculated values of the molar fraction in the micelle were lower than those of the analytical molar fraction; however, there was a large difference between the values of the two methods. The experimental results agreed best with ones obtained from the excess thermodynamic quantities. © 1987Academic Press, Inc.
INTRODUCTION
The bile salts are biologically important molecules (1), and some of them are known to dissolve cholesterol gallstones (2). Furthermore, their mixed aggregates with biological lipids such as lecithin facilitate emulsification and absorption of lipids. The mechanism of bile salt association is different from that of ordinary surfactants, owing to their structure (3, 4). The bile salts have a steroidal nucleus as a hydrophobic part, and they also have a carboxyl group at the end of chain and hydrophilic parts. In general, micellization of the bile salts proceeds in the two stages (1). In the first stage, small micelles called primary micelles are formed. In the next stage, larger secondary micelles are formed by mutual assoTo whom correspondence should be addressed.
ciation of the primary micelles. Small et al. (5, 6) concluded that primary micelles are stabilized through hydrophobic interactions (back to back). On the other hand, Oakenfull and Fisher (7, 8) concluded that the driving force of primary micelle formation is essentially hydrogen bonding between hydroxyl groups of the bile salt molecules. Many studies on the mixed surfactant systems are focused on their critical micellar concentrations (CMC) (9-13) and surface properties (13, 14) and discuss these in terms ofintramicellar interactions. Two papers concerning the bile salts and ordinary surfactant mixtures (15, 16) have been published. However, these studies were performed with light scattering and NMR, and it seems difficult to confirm experimentally the concentration dependence for the aggregation number from the theoretical aspects of both methods. In recent
179 0021-9797/87 $3.00 Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
180
UENO ET AL.
years, fluorescence probing techniques have been extensively applied to micellar systems (17-21) in order to obtain information on the micelles. In particular, pyrene fluorescence can provide information on the polarity of the micelle interior (22, 23) and on the micelle aggregation number (24, 25). These techniques are completely insensitive to intermiceUar interactions, and information can be obtained at every concentration of surfactant and/or added electrolyte. In the present work, the mixed micellar behavior of sodium cholate (NaC) and octa-oxyethylene glycol n-decyl ether (Cl0E8) has been studied. In this paper, intramicellar interactions and molar fractions in the mixed micelles are compared on the basis of two treatments developed in recent years (30, 31). Also, the aggregation number and micelle polarity have been studied by pyrene fluorescence. EXPERIMENTS Materials
Sodium cholate, NaC (Sigma Chemicals), was purified several times by recrystallization and Soxhlet extraction was performed with acetone for 72 h (13). Octa-oxyethylene glycol n-decyl ether, C10E8 (Nikko Chemicals), was purified by gel chromatography (Wakogel C200, Wako Chemicals) with equivalently mixed solvents of acetone and n-hexane. The product gave a single spot on the TLC. Both surfactants were confirmed to be highly pure by the fact that there was no minimum around each of the CMCs in the surface tension vs concentration curves (26). Pyrene as a probe was purified by gel chromatography with cyclohexane. Dodecylpyridinium chloride, DPC1, as a quencher was used after several recrystallizations from ethylacetate-ethanol mixtures. Aqueous solutions of surfactants were prepared with aqueous phosphate buffer solution and were adjusted from pH 8.0 to 8.2. Water was deionized and distilled, and phosphates (Na2HPO4, NaH2PO4) were used as received. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
The total concentration of sodium ion of the buffer components was 8.41 × 10 -3 M. Methods
Surface tension was measured by the Wilhelmy plate method using a glass plate with a Shimadzu surface tensiometer ST-1. The I 1 / 13 ratio of the pyrene fluorescence emission spectrum was measured with a Hitachi fluorescence spectrophotometer 650-10s (excitation wavelength Xex= 335 nm), and the pyrene concentration was around 10-5 M. Time-dependent fluorescence decay curves were observed at low (around 10-5 M) and high pyrene concentrations, except for NaC and NaC: Ca0E8 = 9:1 systems, with an Ortec 7450 multichannel analyzer. The system of NaC:CloE8 = 9:1 was measured by a quenching method at a certain concentration of DPC1 quencher. All the decay curves were computer fitted to the equations using a nonlinear weighted leastsquares procedure, I = loexp[-t/ro]
[ 1]
I = Ioexp[--t/rE--
R(1 - exp(--kEt))]
[2]
I = Ioexp[--t/rq--
R(1 - exp(-kqt))],
[3]
where ro is the lifetime of pyrene fluorescence, rE and rq are pyrene fluorescence lifetimes with excimer formation and quenching, respectively. Rate constants of excimer formation, kE and of quenching by DPC1, kq, were obtained directly from Eqs. [2] and [3], respectively. Aggregation numbers, N, of C10E8 micelles and mixed micelles of C10E8 and NaC were calculated from the parameter R, using the equations N = R(CT -
CMC)/[pyrene]
[4]
N = R(CT-
CMC)/[DPC1],
[5]
where CT is total surfactant concentration. All the samples for fluorescence measurements were bubbled with argon gas for about 20-30 min for deoxygenation. Small-angle laser light-scattering measurements were performed only for the NaC sys-
A G G R E G A T I O N N U M B E R OF M I X E D MICELLES
tem because of the lesser solubility of pyrene into cholate micelles (27) and because of large interactions between cationic quenchers (25). A differential refractive index and scattered light intensity were measured with Chromatix KMX-16 and KMX-6, respectively. The wavelength of lasers at 632.8 nm was used as the light source in both measurements, and the scattering angle was set at 6 ° . Samples were filtered through microfilters (MiUipore) removing a trace of dust. The aggregation number for NaC was determined by Debye plots with extrapolation to the CMC. Throughout this work the temperature was 25 _+ 0.5°C.
18 1
390 1.2 370
.m 1.0
350 0.8 330
T0
I
I
0.5 X NaC
I
1.0
RESULTS A N D DISCUSSION
Effect of Composition on the Structure of the Mixed Micelles The ratio of the first and third vibronic peaks, I~/I3, in a monomeric pyrene fluorescence emission spectrum is known to be sensitive to the local polarity (22, 23) of the solubilization site of pyrene. Low values o f l l , I3 indicate that the environment of solubilized pyrene is apolar as in hydrocarbon solvent; for example, I1/I3 = 0.6 for cyclohexane and n-hexane. On the other hand, I1/I3 values in polar solvents are generally high. We obtained values for isopropanol, ethanol, polyoxyethylene (ethyleneoxide units are 400), and H20 of 1.05, 1.23, 1.75, and 1.83, respectively. Large values of I~/I3 above 1.8 suggest that
TABLE I The Values of l~/I~ and Lifetimes of Monomeric Pyrene Fluorescence with Various Molar Fractions (25 °C) Molar fraction Xr~,c
Id13
Lifetime ro (ns)
0.0 0.25 0.50 0.75 0.90 1.0
1.24 1.14 1.08 1,03 0.90 0.79
340 340 360 368 386 393
FIG. 1. Plots (O) of the 11/13 ratio vs molar fraction of NaC, XNac. Total surfactant concentrations were 0.1 M and concentrations of pyrene were around 10-5 M. Pyrene was excited at 335 nm. Plots (O) of the lifetime r0 vs molar fraction ofNaC, Xr4ac. Total surfactant concentrations were 0.1 M and concentrations of pyrene were around 10-5 M. Pyrene was excited at 335 n m (25°C).
most of pyrene is dispersed in aqueous phase. The opposite trend is found for the lifetime r0 (27), with large values in hydrocarbon solvents and low values in polar solvents. The values of 11/13 and r0 solutions of increasing molar fractions of NaC are listed in Table I. NaC has a low value of I1/I3 and a large value of r0. These values indicate that NaC micelles provide the solubilized pyrene with a microenvironment which is nearly as apolar as hydrocarbon solvents (27). The opposite trend was seen with the C10E8 micelle, and the I1/I3 value for CloE8 micelles was close to the value of polar solvents such as ethanol. Such large differences suggest that there is a clear difference between the solubilization sites ofpyrene in NaC and CloE8 micelles. For micelles of ordinary surfactants, the solubilization site of aromatic hydrocarbons such as pyrene is the micelle palisade layer owing to their slight surface activity (28). The solubilization site of pyrene in C10E8 miceUes also seems to be the palisade layer from the values of I1/I3 Journal of Colloidand InterfaceScience, Vol. 117,No. 1, May 1987
182
UENO ET AL. TABLE II
of the solubilization site of pyrene. It seems that the break points in both curves correspond The AggregationNumber and Rate Constant with to the transition point of the solubilization site Various Molar Fractions (25°C) ofpyrene from the palisade layer to the inside Aggregation of the micelle. Molar fraction number Rate constant XNaC N 10-7 k13 Aggregation numbers N and rate constants kE of pyrene excimer formation and kq of 0.0 69 1.31 quenching by DPC1 obtained from the analysis 0.25 55 1.39 of the pyrene fluorescence decay curves are 0.50 42 1.71 listed in Table II. There is clearly a trend in 0.75 26 2.05 0.90 16a 1.74a the decrease in N with XNaC. The small aggre1.0 6b -gation numbers in NaC-rich systems seem to a Those valueswere determined by a quenching method be caused by the nature of NaC. F r o m these results, the following concluwith DPC1quencher. The value of a rate constant means sions can be derived: the quenching rate constant kq. bFrom the results of small-anglelaser light scattering. The aggregation number for NaC was determined by (i) In the case of the micelles of pure NaC Debye plots with extrapolation to the CMC. or the mixed micelles at high molar fractions of NaC, solubilized pyrene is efficiently and T0. On the other hand, the low 11/13 and shielded from the aqueous phase (27) because large z0 values for NaC micelles suggest that of the rigid nature of the steroidic groups the solubilization site of pyrene is the micelle forming the micelle interior. interior. The same results have been reported (ii) In the range of XNaC from 0 to 0.75, the by Zana and Guveli (27). shape of the mixed micelles appears to be The values of I1/I3 are plotted as a function nearly spherical based on the value of N. The of the molar fraction of NaC, XNaC, in Fig. 1. solubilization site of pyrene is the palisade There is an obvious break point in the curve, layer of the mixed micelle. Above Xnac at 0.75, and after the break point, 11/13 values decrease NaC molecules seem to form an outer shell abruptly. Also the curve of T0 vs Xn~c, as with the hydroxyl groups facing the aqueous shown in Fig. 1, has a break point at the same phase, and the micellar inside becomes more molar fraction. An abrupt decrease in 11/13 and hydrophobic. Therefore, pyrene is considered an increase in z0 in the range of Xn~c from to be incorporated inside the micelles together 0.75 to 1.0 show the large increment o f a hy- with the alkyl chain of C10E8. These features drophobic nature in the micelle and the change are illustrated schematically in Fig. 2.
J
I
0
I
0.75 XNaC
1.0 ~ •
NaC C~oE8 Pyrene
FIG. 2. Schematicrepresentation of the micelles incorporated into pyrene. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
183
A G G R E G A T I O N N U M B E R OF MIXED MICELLES 70 1:0
-
•
Z
3
1
a.
1
1
•
1
9
50
30
10 C~oEs: NaC
0
I
20
I
40
I
I
60
[ I
100
80
CT(mM)
FIG. 3. Curves of the aggregation number N vs total surfactant concentration CT with various molar fractions; CIoEa:NaC = 1:0 (O), 3:1 (A), 1:1 (11), 1:3 (0), and 1:9 (v) (25°C).
Effect of Concentration on the Mixed Micelles In this section, concentration dependencies for the aggregation number and 11/I3 are described. However, a concentration dependence for the aggregation number of NaC could not be confirmed because of the low solubility of pyrene and the large interaction between NaC and the cationic quenchers such as DPC1. The low solubility of pyrene prevented the formation of enough excimers for the accurate determination of aggregation number. Also the quenching method could not be used because the large interaction between DPC1 and NaC gave a large value of the aggregation number N as compared to the real one (25), owing to the formation of partially insoluble complexes. Therefore, the aggregation number for NaC micelles was measured by small-angle laser light scattering, and the value of N was determined from the Debye plots with extrapolation to the CMC. It has been reported that the aggregation numbers of the bile salts are independent of the concentrations for the bile salts and added electrolyte (27). Therefore, in this experimen-
II:GoE~ 1.2
1.1
1.0
/
0.9-
081 o
I
O: NaC
s; CT(mM)
FIG. 4. Curves of the 11/13 ratio vs total surfactant concentration Cr with various molar fractions; C1oEs:NaC = 1:0 (e), 3:1 (A), 1:1 (11), 1:3 (0), 1:9 (v), and 0:1 (O)
(25°c). Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
184
UENO ET AL.
tal range, the value of N for NaC micelles is lization site and the microenvironment of the considered to be nearly constant. Further- initial micelles are considered t o be nearly the more, a transition point from a primary to a same as those ofC10E8 miceUe. Thus, the initial secondary micelle seems to be higher than 0.1 micelle in the mixed systems contains fewer M NaC concentration. NaC molecules, and as Ca- is increased, the The values of N at various molar fractions aggregation number N decreases and the miare plotted as a function of the total surfactant croenvironment of the micelle changes gradconcentration, Ct, in Fig. 3. It is seen that there uaUy. are clear trends in the decrease in N in the mixed micellar systems up to CT = 50 mM, Theoretical Analysis of the Composition in and that the values of N remain constant at the Mixed Micelles Cr > 50 mM. Dependencies of the 11/13values for all the mixed micellar systems are shown In Fig. 5, surface tensions, % with various in Fig. 4, and decreasing trends identical to molar fractions are plotted as a function of CT those for N are seen. These facts suggest that (in logarithmic scale), and there are obvious the size, the composition, and the aggregation break points corresponding to the CMCs. These CMCs are listed in Table III. The CMCs numbers vary with Cr. In Fig. 3 the aggregation numbers at Ca- for the mixed systems deviate negatively from lower than 50 m M are larger than the constant the ideal mixing curve (9, 10). The molar fracvalues above 50 m M of Cx. For XNaC = 0.25 tions in the mixed micelles can be estimated and 0.5, initial micelles formed at their CMCs by the two treatments published recently. seem to be organized by a high ratio of C10E8 Rubingh (30) has applied the regular solution molecules from the aggregation numbers at theory to the mixed micellar systems, and it low Cx. Near CMCs, I1/13 values of the mixed has been shown that the interaction palameter systems are close to the value of the C10E8 mi- can provide the molar fraction in the mixed celle, and this means that the pyrene solubi- micelles. Also Motomura et al. (31) have
!fo 60
40
T
i
0.1
i
I CT(rnM)
i
10
FIG. 5. Curves of the surface tension vs total suffactant concentration GT (in logarithmic scale) with various molar fractions. Same symbols as in Fig. 4 (25°C). Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
AGGREGATION NUMBER OF MIXED MICELLES TABLE III Critical Micellar Concentrations and Molar Fractions in the Mixed Micelles (25°C)
185
M o t o m u r a et al. is useful for the mixed micellar systems o f ordinary surfactants. REFERENCES
Molar fraction in the Molar fraction XN~c
0.0 0.25 0.50 0.75 0.90 1.0
Critical micellar concentrations CMC [raM]
1.02a 1.09 1.25 2.58 4.52 10.3
mixed micelles X~ b
X~ c
0.0 0.15 0.26 0.30 0.48 1.0
0.0 0.03 0.01 0.15 0.33 1.0
The CMC of CIoE8in this measurement agrees with the value in Refs. (29a) and (29b) within the experimental error. b Calculated by the regular solution theory (Ref. (30)). c Calculated by the excess thermodynamic quantities (Ref. (31)).
shown that the mixed miceUar composition o f surfactants can be derived from excess therm o d y n a m i c quantities. The results o f the m o lar fraction in the mixed micelles X ~ a n d X2~ calculated f r o m the treatment o f regular solution theory a n d excess t h e r m o d y n a m i c quantities, respectively, are also listed in Table III. It is seen that the values o f X ~ and X2M are low c o m p a r e d with their stoichiometric m o l a r fractions. Both analyses show that the initial micelles contain fewer N a C molecules. There is a large difference between X ~ and X ~ in the middle o f X_Nac, as shown in the results o f concentration dependencies o f N and 11/13. T h e initial micelles for the mixed systems were f o u n d to be organized by higher m o l a r fractions OfCloE8, as m e n t i o n e d above. O u r experimental results agree with the theoretical ones f r o m the treatment o f excess therm o d y n a m i c quantities. This treatment appears to be superior to the analysis with the regular solution theory. The availability for this mixed system including the extremely different chemical structures o f surfactants suggests that the m e t h o d o f analysis proposed by
1. Carey, M. C., in "The Liver: Biology and Pathobiology" (Arias, I., Popper, H., Schachter, D., and Shafritz, D. A., Eds.), Ch. 27, p. 429. Raven Press, New York, 1982. 2. Igimi, H., and Carey, M. C., J. Lipid Res. 22, 254 (1981). 3. Small, D. M., in "The Bile Acids" (Nair, P. P., and Kritchevsky, D., Eds.), Ch. 8, p. 249, Plenum, New York, 1971. 4. Small, D. M.,Adv. Chem. 84, 31 (1968). 5. Carey, M. C., and Small, D. M., Amer. J. Med. 49, 590 (1970). 6. Small, D. M., Pankett, S. A., and Chapman, D., Biochim. Biophys. Acta 176, 178 (1969). 7. Oakenfull, D. G., and Fisher, L. R., J. Phys. Chem. 81, 1838 (1977). 8. Fisher, L. R., and Oakenfull, D. G., Aust. J. Chem. 32, 31 (1979). 9. Clint, J. H., J. Chem. Soc. Faraday Trans. 1 76, 1327 (1975). 10. Lang, H., and Beck, H., Kolloid Z. Z. Polym. 251, 424 (1973). 11. (a) Rosen, M. J., and Hua, X. Y., J. Colloid Interface Sci. 86, 164 (1982); (b) Hua, X. Y., and Rosen, M. J., J. Colloid Interface ScL 90, 212 (1982). 12. (a) Moroi, Y., Nishilddo, N., and Matsuura, R., J. Colloid Interface Sci. 50, 344 (1975); (b) Moroi, Y., Nishikido, N., Saito, M., and Matsuura, R., J. Colloid Interface Sci. 52, 356 (1975). 13. Ueno, M., Obata, M., and Kimoto, Y., J. Japan. Med. Soc. Biol. Interface 15, 45 (1982). 14. Lucassen-Rynders,E. H., J. Colloid lnterface Sci. 85, 178 (1982). 15. Barry, B. W., and Gray, G. M. T., J. Colloidlnterface Sci. 52, 327 (1975). 16. Mesa, C. L., Khan, A., Fontell, K., and Lindman, B., J. Colloid lnterface Sci. 103, 373 (1980). 17. Turro, N. J., Gratzel, M., and Braun, A. M., Angew. Chem. Int. Ed. Engl. 19, 675 (1980). 18. Thomas, J. K., Chem. Rev. 80, 283 (1980). 19. (a) Tachiya, M., Chem. Phys. Lett. 33, 289 (1975); (b) J. Chem. Phys. 76, 340 (1982). 20. Singer, L. A., in "Solution Behavior of Surfactants" (Mittal, K. L., and Fendier, E., Eds.), Vol. 1, p. 73. Plenum, New York, 1982. 21. DeSchryver, F. C., Croonen, Y., Gelade, E., Van Der Auweraer, M., Dederen, J. C., Roalents, E., and Boens, N., in "Surfactants in Solution" (Mittal, IC L., and Lindman, B., Eds.), Vol. 1, p. 663. Plenum, New York, 1984. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
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UENO ET AL.
22. (a) Nakajima, A., Bull. Chem. Soc. Japan 44, 3272 (1971); (b) Spectrochim. Acta A 30, 860 (1974); (c) J. Mol. Spectrosc. 61,467 (1976); (d) J. Lumin. II, 429 (1976). 23. (a) Chen, M., Gratzel, M., and Thomas, J. K., J. Amer. Chem. Soc. 97, 2052 (1975); (b) Kalyanasundaram, K., and Thomas, J. K., J. Amer. Chem. Soc. 99, 2039 (1977). 24. (a) Lianos, P., and Zana, R., J. Colloid Interface Sci. 84, 100 (1981); (b) Lianos, P., Lang, J., and Zana, R., J. Colloid Interface Sci. 91, 276 (1983); (c) Llanos, P., Lang, J., Strum, J., and Zana, R., J. Phys. Chem. 88, 819 (1984). 25. Hasimoto, S., and Thomas, J. K., J. Colloidlnterface Sci. 102, 152 (1984). 26. Gibbs, J. W., in "The Collected Works ofJ. W. Gibbs"
Journalof Colloidand InterfaceScience,Vol. 117,No. 1, May 1987
27. 28. 29.
30.
31.
(Longmans, eds.), Vol. 1, p. 219. Green and Co., New York, 1931. Zana, R., and Guveli, D., J. Phys. Chem. 89, 1687 (1985). (a) Cardinal, J. R., and Mukerjee, P., J. Phys. Chem. 82, 1614 (1978); (b) Mukerjee, P,, and Cardinal, J. R., J. Phys. Chem. 82, 1620 (1978). (a) Ueno, M., Takasawa, Y., Miyasige, H., Tabata, Y., and Meguro, K., Colloid Polym. Sci. 259, 761 (1981); (b) Ueno, M., Takasawa, Y., Nakahashi, N., Tabata, Y., and Meguro, K., J. Colloid Interface Sci. 83, 50 (1981). Rubingh, D. N., in "Solution Chemistry of Surfactams" (Mittal, K. L., Ed.), Vol. 1, Plenum, New York, 1980. Motomura, K., Yamanaka, M., and Aratono, M., Colloid & Polym. Sci. 262, 948 (1984).
INTRODUCTION
The bile salts are biologically important molecules (1), and some of them are known to dissolve cholesterol gallstones (2). Furthermore, their mixed aggregates with biological lipids such as lecithin facilitate emulsification and absorption of lipids. The mechanism of bile salt association is different from that of ordinary surfactants, owing to their structure (3, 4). The bile salts have a steroidal nucleus as a hydrophobic part, and they also have a carboxyl group at the end of chain and hydrophilic parts. In general, micellization of the bile salts proceeds in the two stages (1). In the first stage, small micelles called primary micelles are formed. In the next stage, larger secondary micelles are formed by mutual assoTo whom correspondence should be addressed.
ciation of the primary micelles. Small et al. (5, 6) concluded that primary micelles are stabilized through hydrophobic interactions (back to back). On the other hand, Oakenfull and Fisher (7, 8) concluded that the driving force of primary micelle formation is essentially hydrogen bonding between hydroxyl groups of the bile salt molecules. Many studies on the mixed surfactant systems are focused on their critical micellar concentrations (CMC) (9-13) and surface properties (13, 14) and discuss these in terms ofintramicellar interactions. Two papers concerning the bile salts and ordinary surfactant mixtures (15, 16) have been published. However, these studies were performed with light scattering and NMR, and it seems difficult to confirm experimentally the concentration dependence for the aggregation number from the theoretical aspects of both methods. In recent
179 0021-9797/87 $3.00 Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
180
UENO ET AL.
years, fluorescence probing techniques have been extensively applied to micellar systems (17-21) in order to obtain information on the micelles. In particular, pyrene fluorescence can provide information on the polarity of the micelle interior (22, 23) and on the micelle aggregation number (24, 25). These techniques are completely insensitive to intermiceUar interactions, and information can be obtained at every concentration of surfactant and/or added electrolyte. In the present work, the mixed micellar behavior of sodium cholate (NaC) and octa-oxyethylene glycol n-decyl ether (Cl0E8) has been studied. In this paper, intramicellar interactions and molar fractions in the mixed micelles are compared on the basis of two treatments developed in recent years (30, 31). Also, the aggregation number and micelle polarity have been studied by pyrene fluorescence. EXPERIMENTS Materials
Sodium cholate, NaC (Sigma Chemicals), was purified several times by recrystallization and Soxhlet extraction was performed with acetone for 72 h (13). Octa-oxyethylene glycol n-decyl ether, C10E8 (Nikko Chemicals), was purified by gel chromatography (Wakogel C200, Wako Chemicals) with equivalently mixed solvents of acetone and n-hexane. The product gave a single spot on the TLC. Both surfactants were confirmed to be highly pure by the fact that there was no minimum around each of the CMCs in the surface tension vs concentration curves (26). Pyrene as a probe was purified by gel chromatography with cyclohexane. Dodecylpyridinium chloride, DPC1, as a quencher was used after several recrystallizations from ethylacetate-ethanol mixtures. Aqueous solutions of surfactants were prepared with aqueous phosphate buffer solution and were adjusted from pH 8.0 to 8.2. Water was deionized and distilled, and phosphates (Na2HPO4, NaH2PO4) were used as received. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
The total concentration of sodium ion of the buffer components was 8.41 × 10 -3 M. Methods
Surface tension was measured by the Wilhelmy plate method using a glass plate with a Shimadzu surface tensiometer ST-1. The I 1 / 13 ratio of the pyrene fluorescence emission spectrum was measured with a Hitachi fluorescence spectrophotometer 650-10s (excitation wavelength Xex= 335 nm), and the pyrene concentration was around 10-5 M. Time-dependent fluorescence decay curves were observed at low (around 10-5 M) and high pyrene concentrations, except for NaC and NaC: Ca0E8 = 9:1 systems, with an Ortec 7450 multichannel analyzer. The system of NaC:CloE8 = 9:1 was measured by a quenching method at a certain concentration of DPC1 quencher. All the decay curves were computer fitted to the equations using a nonlinear weighted leastsquares procedure, I = loexp[-t/ro]
[ 1]
I = Ioexp[--t/rE--
R(1 - exp(--kEt))]
[2]
I = Ioexp[--t/rq--
R(1 - exp(-kqt))],
[3]
where ro is the lifetime of pyrene fluorescence, rE and rq are pyrene fluorescence lifetimes with excimer formation and quenching, respectively. Rate constants of excimer formation, kE and of quenching by DPC1, kq, were obtained directly from Eqs. [2] and [3], respectively. Aggregation numbers, N, of C10E8 micelles and mixed micelles of C10E8 and NaC were calculated from the parameter R, using the equations N = R(CT -
CMC)/[pyrene]
[4]
N = R(CT-
CMC)/[DPC1],
[5]
where CT is total surfactant concentration. All the samples for fluorescence measurements were bubbled with argon gas for about 20-30 min for deoxygenation. Small-angle laser light-scattering measurements were performed only for the NaC sys-
A G G R E G A T I O N N U M B E R OF M I X E D MICELLES
tem because of the lesser solubility of pyrene into cholate micelles (27) and because of large interactions between cationic quenchers (25). A differential refractive index and scattered light intensity were measured with Chromatix KMX-16 and KMX-6, respectively. The wavelength of lasers at 632.8 nm was used as the light source in both measurements, and the scattering angle was set at 6 ° . Samples were filtered through microfilters (MiUipore) removing a trace of dust. The aggregation number for NaC was determined by Debye plots with extrapolation to the CMC. Throughout this work the temperature was 25 _+ 0.5°C.
18 1
390 1.2 370
.m 1.0
350 0.8 330
T0
I
I
0.5 X NaC
I
1.0
RESULTS A N D DISCUSSION
Effect of Composition on the Structure of the Mixed Micelles The ratio of the first and third vibronic peaks, I~/I3, in a monomeric pyrene fluorescence emission spectrum is known to be sensitive to the local polarity (22, 23) of the solubilization site of pyrene. Low values o f l l , I3 indicate that the environment of solubilized pyrene is apolar as in hydrocarbon solvent; for example, I1/I3 = 0.6 for cyclohexane and n-hexane. On the other hand, I1/I3 values in polar solvents are generally high. We obtained values for isopropanol, ethanol, polyoxyethylene (ethyleneoxide units are 400), and H20 of 1.05, 1.23, 1.75, and 1.83, respectively. Large values of I~/I3 above 1.8 suggest that
TABLE I The Values of l~/I~ and Lifetimes of Monomeric Pyrene Fluorescence with Various Molar Fractions (25 °C) Molar fraction Xr~,c
Id13
Lifetime ro (ns)
0.0 0.25 0.50 0.75 0.90 1.0
1.24 1.14 1.08 1,03 0.90 0.79
340 340 360 368 386 393
FIG. 1. Plots (O) of the 11/13 ratio vs molar fraction of NaC, XNac. Total surfactant concentrations were 0.1 M and concentrations of pyrene were around 10-5 M. Pyrene was excited at 335 nm. Plots (O) of the lifetime r0 vs molar fraction ofNaC, Xr4ac. Total surfactant concentrations were 0.1 M and concentrations of pyrene were around 10-5 M. Pyrene was excited at 335 n m (25°C).
most of pyrene is dispersed in aqueous phase. The opposite trend is found for the lifetime r0 (27), with large values in hydrocarbon solvents and low values in polar solvents. The values of 11/13 and r0 solutions of increasing molar fractions of NaC are listed in Table I. NaC has a low value of I1/I3 and a large value of r0. These values indicate that NaC micelles provide the solubilized pyrene with a microenvironment which is nearly as apolar as hydrocarbon solvents (27). The opposite trend was seen with the C10E8 micelle, and the I1/I3 value for CloE8 micelles was close to the value of polar solvents such as ethanol. Such large differences suggest that there is a clear difference between the solubilization sites ofpyrene in NaC and CloE8 micelles. For micelles of ordinary surfactants, the solubilization site of aromatic hydrocarbons such as pyrene is the micelle palisade layer owing to their slight surface activity (28). The solubilization site of pyrene in C10E8 miceUes also seems to be the palisade layer from the values of I1/I3 Journal of Colloidand InterfaceScience, Vol. 117,No. 1, May 1987
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UENO ET AL. TABLE II
of the solubilization site of pyrene. It seems that the break points in both curves correspond The AggregationNumber and Rate Constant with to the transition point of the solubilization site Various Molar Fractions (25°C) ofpyrene from the palisade layer to the inside Aggregation of the micelle. Molar fraction number Rate constant XNaC N 10-7 k13 Aggregation numbers N and rate constants kE of pyrene excimer formation and kq of 0.0 69 1.31 quenching by DPC1 obtained from the analysis 0.25 55 1.39 of the pyrene fluorescence decay curves are 0.50 42 1.71 listed in Table II. There is clearly a trend in 0.75 26 2.05 0.90 16a 1.74a the decrease in N with XNaC. The small aggre1.0 6b -gation numbers in NaC-rich systems seem to a Those valueswere determined by a quenching method be caused by the nature of NaC. F r o m these results, the following concluwith DPC1quencher. The value of a rate constant means sions can be derived: the quenching rate constant kq. bFrom the results of small-anglelaser light scattering. The aggregation number for NaC was determined by (i) In the case of the micelles of pure NaC Debye plots with extrapolation to the CMC. or the mixed micelles at high molar fractions of NaC, solubilized pyrene is efficiently and T0. On the other hand, the low 11/13 and shielded from the aqueous phase (27) because large z0 values for NaC micelles suggest that of the rigid nature of the steroidic groups the solubilization site of pyrene is the micelle forming the micelle interior. interior. The same results have been reported (ii) In the range of XNaC from 0 to 0.75, the by Zana and Guveli (27). shape of the mixed micelles appears to be The values of I1/I3 are plotted as a function nearly spherical based on the value of N. The of the molar fraction of NaC, XNaC, in Fig. 1. solubilization site of pyrene is the palisade There is an obvious break point in the curve, layer of the mixed micelle. Above Xnac at 0.75, and after the break point, 11/13 values decrease NaC molecules seem to form an outer shell abruptly. Also the curve of T0 vs Xn~c, as with the hydroxyl groups facing the aqueous shown in Fig. 1, has a break point at the same phase, and the micellar inside becomes more molar fraction. An abrupt decrease in 11/13 and hydrophobic. Therefore, pyrene is considered an increase in z0 in the range of Xn~c from to be incorporated inside the micelles together 0.75 to 1.0 show the large increment o f a hy- with the alkyl chain of C10E8. These features drophobic nature in the micelle and the change are illustrated schematically in Fig. 2.
J
I
0
I
0.75 XNaC
1.0 ~ •
NaC C~oE8 Pyrene
FIG. 2. Schematicrepresentation of the micelles incorporated into pyrene. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
183
A G G R E G A T I O N N U M B E R OF MIXED MICELLES 70 1:0
-
•
Z
3
1
a.
1
1
•
1
9
50
30
10 C~oEs: NaC
0
I
20
I
40
I
I
60
[ I
100
80
CT(mM)
FIG. 3. Curves of the aggregation number N vs total surfactant concentration CT with various molar fractions; CIoEa:NaC = 1:0 (O), 3:1 (A), 1:1 (11), 1:3 (0), and 1:9 (v) (25°C).
Effect of Concentration on the Mixed Micelles In this section, concentration dependencies for the aggregation number and 11/I3 are described. However, a concentration dependence for the aggregation number of NaC could not be confirmed because of the low solubility of pyrene and the large interaction between NaC and the cationic quenchers such as DPC1. The low solubility of pyrene prevented the formation of enough excimers for the accurate determination of aggregation number. Also the quenching method could not be used because the large interaction between DPC1 and NaC gave a large value of the aggregation number N as compared to the real one (25), owing to the formation of partially insoluble complexes. Therefore, the aggregation number for NaC micelles was measured by small-angle laser light scattering, and the value of N was determined from the Debye plots with extrapolation to the CMC. It has been reported that the aggregation numbers of the bile salts are independent of the concentrations for the bile salts and added electrolyte (27). Therefore, in this experimen-
II:GoE~ 1.2
1.1
1.0
/
0.9-
081 o
I
O: NaC
s; CT(mM)
FIG. 4. Curves of the 11/13 ratio vs total surfactant concentration Cr with various molar fractions; C1oEs:NaC = 1:0 (e), 3:1 (A), 1:1 (11), 1:3 (0), 1:9 (v), and 0:1 (O)
(25°c). Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
184
UENO ET AL.
tal range, the value of N for NaC micelles is lization site and the microenvironment of the considered to be nearly constant. Further- initial micelles are considered t o be nearly the more, a transition point from a primary to a same as those ofC10E8 miceUe. Thus, the initial secondary micelle seems to be higher than 0.1 micelle in the mixed systems contains fewer M NaC concentration. NaC molecules, and as Ca- is increased, the The values of N at various molar fractions aggregation number N decreases and the miare plotted as a function of the total surfactant croenvironment of the micelle changes gradconcentration, Ct, in Fig. 3. It is seen that there uaUy. are clear trends in the decrease in N in the mixed micellar systems up to CT = 50 mM, Theoretical Analysis of the Composition in and that the values of N remain constant at the Mixed Micelles Cr > 50 mM. Dependencies of the 11/13values for all the mixed micellar systems are shown In Fig. 5, surface tensions, % with various in Fig. 4, and decreasing trends identical to molar fractions are plotted as a function of CT those for N are seen. These facts suggest that (in logarithmic scale), and there are obvious the size, the composition, and the aggregation break points corresponding to the CMCs. These CMCs are listed in Table III. The CMCs numbers vary with Cr. In Fig. 3 the aggregation numbers at Ca- for the mixed systems deviate negatively from lower than 50 m M are larger than the constant the ideal mixing curve (9, 10). The molar fracvalues above 50 m M of Cx. For XNaC = 0.25 tions in the mixed micelles can be estimated and 0.5, initial micelles formed at their CMCs by the two treatments published recently. seem to be organized by a high ratio of C10E8 Rubingh (30) has applied the regular solution molecules from the aggregation numbers at theory to the mixed micellar systems, and it low Cx. Near CMCs, I1/13 values of the mixed has been shown that the interaction palameter systems are close to the value of the C10E8 mi- can provide the molar fraction in the mixed celle, and this means that the pyrene solubi- micelles. Also Motomura et al. (31) have
!fo 60
40
T
i
0.1
i
I CT(rnM)
i
10
FIG. 5. Curves of the surface tension vs total suffactant concentration GT (in logarithmic scale) with various molar fractions. Same symbols as in Fig. 4 (25°C). Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
AGGREGATION NUMBER OF MIXED MICELLES TABLE III Critical Micellar Concentrations and Molar Fractions in the Mixed Micelles (25°C)
185
M o t o m u r a et al. is useful for the mixed micellar systems o f ordinary surfactants. REFERENCES
Molar fraction in the Molar fraction XN~c
0.0 0.25 0.50 0.75 0.90 1.0
Critical micellar concentrations CMC [raM]
1.02a 1.09 1.25 2.58 4.52 10.3
mixed micelles X~ b
X~ c
0.0 0.15 0.26 0.30 0.48 1.0
0.0 0.03 0.01 0.15 0.33 1.0
The CMC of CIoE8in this measurement agrees with the value in Refs. (29a) and (29b) within the experimental error. b Calculated by the regular solution theory (Ref. (30)). c Calculated by the excess thermodynamic quantities (Ref. (31)).
shown that the mixed miceUar composition o f surfactants can be derived from excess therm o d y n a m i c quantities. The results o f the m o lar fraction in the mixed micelles X ~ a n d X2~ calculated f r o m the treatment o f regular solution theory a n d excess t h e r m o d y n a m i c quantities, respectively, are also listed in Table III. It is seen that the values o f X ~ and X2M are low c o m p a r e d with their stoichiometric m o l a r fractions. Both analyses show that the initial micelles contain fewer N a C molecules. There is a large difference between X ~ and X ~ in the middle o f X_Nac, as shown in the results o f concentration dependencies o f N and 11/13. T h e initial micelles for the mixed systems were f o u n d to be organized by higher m o l a r fractions OfCloE8, as m e n t i o n e d above. O u r experimental results agree with the theoretical ones f r o m the treatment o f excess therm o d y n a m i c quantities. This treatment appears to be superior to the analysis with the regular solution theory. The availability for this mixed system including the extremely different chemical structures o f surfactants suggests that the m e t h o d o f analysis proposed by
1. Carey, M. C., in "The Liver: Biology and Pathobiology" (Arias, I., Popper, H., Schachter, D., and Shafritz, D. A., Eds.), Ch. 27, p. 429. Raven Press, New York, 1982. 2. Igimi, H., and Carey, M. C., J. Lipid Res. 22, 254 (1981). 3. Small, D. M., in "The Bile Acids" (Nair, P. P., and Kritchevsky, D., Eds.), Ch. 8, p. 249, Plenum, New York, 1971. 4. Small, D. M.,Adv. Chem. 84, 31 (1968). 5. Carey, M. C., and Small, D. M., Amer. J. Med. 49, 590 (1970). 6. Small, D. M., Pankett, S. A., and Chapman, D., Biochim. Biophys. Acta 176, 178 (1969). 7. Oakenfull, D. G., and Fisher, L. R., J. Phys. Chem. 81, 1838 (1977). 8. Fisher, L. R., and Oakenfull, D. G., Aust. J. Chem. 32, 31 (1979). 9. Clint, J. H., J. Chem. Soc. Faraday Trans. 1 76, 1327 (1975). 10. Lang, H., and Beck, H., Kolloid Z. Z. Polym. 251, 424 (1973). 11. (a) Rosen, M. J., and Hua, X. Y., J. Colloid Interface Sci. 86, 164 (1982); (b) Hua, X. Y., and Rosen, M. J., J. Colloid Interface ScL 90, 212 (1982). 12. (a) Moroi, Y., Nishilddo, N., and Matsuura, R., J. Colloid Interface Sci. 50, 344 (1975); (b) Moroi, Y., Nishikido, N., Saito, M., and Matsuura, R., J. Colloid Interface Sci. 52, 356 (1975). 13. Ueno, M., Obata, M., and Kimoto, Y., J. Japan. Med. Soc. Biol. Interface 15, 45 (1982). 14. Lucassen-Rynders,E. H., J. Colloid lnterface Sci. 85, 178 (1982). 15. Barry, B. W., and Gray, G. M. T., J. Colloidlnterface Sci. 52, 327 (1975). 16. Mesa, C. L., Khan, A., Fontell, K., and Lindman, B., J. Colloid lnterface Sci. 103, 373 (1980). 17. Turro, N. J., Gratzel, M., and Braun, A. M., Angew. Chem. Int. Ed. Engl. 19, 675 (1980). 18. Thomas, J. K., Chem. Rev. 80, 283 (1980). 19. (a) Tachiya, M., Chem. Phys. Lett. 33, 289 (1975); (b) J. Chem. Phys. 76, 340 (1982). 20. Singer, L. A., in "Solution Behavior of Surfactants" (Mittal, K. L., and Fendier, E., Eds.), Vol. 1, p. 73. Plenum, New York, 1982. 21. DeSchryver, F. C., Croonen, Y., Gelade, E., Van Der Auweraer, M., Dederen, J. C., Roalents, E., and Boens, N., in "Surfactants in Solution" (Mittal, IC L., and Lindman, B., Eds.), Vol. 1, p. 663. Plenum, New York, 1984. Journal of Colloid and Interface Science, Vol. 117, No. 1, May 1987
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22. (a) Nakajima, A., Bull. Chem. Soc. Japan 44, 3272 (1971); (b) Spectrochim. Acta A 30, 860 (1974); (c) J. Mol. Spectrosc. 61,467 (1976); (d) J. Lumin. II, 429 (1976). 23. (a) Chen, M., Gratzel, M., and Thomas, J. K., J. Amer. Chem. Soc. 97, 2052 (1975); (b) Kalyanasundaram, K., and Thomas, J. K., J. Amer. Chem. Soc. 99, 2039 (1977). 24. (a) Lianos, P., and Zana, R., J. Colloid Interface Sci. 84, 100 (1981); (b) Lianos, P., Lang, J., and Zana, R., J. Colloid Interface Sci. 91, 276 (1983); (c) Llanos, P., Lang, J., Strum, J., and Zana, R., J. Phys. Chem. 88, 819 (1984). 25. Hasimoto, S., and Thomas, J. K., J. Colloidlnterface Sci. 102, 152 (1984). 26. Gibbs, J. W., in "The Collected Works ofJ. W. Gibbs"
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