2396
Langmuir 1992,8, 2396-2404
Fluorescence Quenching Studies of the Aggregation Behavior of the Mixed Micelles of Bile Salts and Cetyltrimethylammonium Halides Martin Swanson Vethamuthu,t Mats Almgren,*lt Emad Mukhtar,? and Pratap Bahadurt Department of Physical Chemistry, University of Uppsala, Box 532, S - 751 21 Uppsala, Sweden, and Department of Chemistry, South Gujarat University, Surat 395007, India Received February 18, 1992. In Final Form: June 18, 1992
The fluorescence decay of pyrene quenched by dimethylbenzophenone(DMBP)has been used to study the aggregationbehavior of cetyltrimethylammoniumhalide (CTAX)in the presence of varied concentrations of two bile salts, sodium cholate (NaC) and sodium deoxycholate (NaDC). The study showed the different aggregation characteristics of mixed micelles. In both cases it was shown that the probe pyrene migrates from the palisade layer of the micelle to the interior of the mixed micelles with increase in bile salt concentration. The micelles were small for the CTAX/NaC system over the entire concentration range but tended to grow in size into rodlike micelles for the CTAX/NaDC system close to the equimolar concentrations, where phase separation into two micellar phases occurred. Dynamic light scattering and viscosity measurements also support this view. Introduction Bile salts are biologically important molecules that participate in many physiological processes, viz. in intestinal hydrolysis, in dispersion and digestion of lipids, cholesterol solubilization, and drug These substances possess hydrophobic and hydrophilic moieties and thus behave like surfactant^.^ The properties of bile salts micelles are markedly different from those of ordinary surfactants. These differences are due to the structure of the bile salts with a rigid nonplanar steroidal nucleus having one face polar due to hydroxyl groups and the other nonpolar. Micellization of bile salts in aqueous solutions is still debated and has been the subject of several review^^^^^^ and recent studies.*l6 The aggregation seems to proceed in two stages: At low concentration of di- and trihydroxy bile salts small primary micelles of aggregation number from 2 to 10 are formed through hydrophobic interactions. At higher concentration of dihydroxy bile salts,larger secondary micelles are formed possibly through hydrogen bonding between the hydroxyl groups.
* Author to whom correspondence should be addressed. University of Uppsala. South Gujarat University. (1) Hofmann, A. F.; Small, D. M. Annu. Rev. Med. 1967, 18, 333. (2) Small, D. M. Adu. Chem. Ser. 1968, No. 84, 31. (3) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971, Vol. 1. (4) Carey, M. C.; Small, D. M. Arch. Intern. Med. 1972,30, 506. (5) Fontell, K. Kolloid Z . 2.Polym. 1971,244,246,253; 1971,246,614, 700. (6) Kratovil, P. Adu. Colloid Interface Sci. 1986,26, 131. (7) Arias, M. I., Davidson, S. C., Eds. Physical Chemistry of Bile in Health and Disease. Hepatology; September-October 1984; Vol. 4, supplement. (8)Kratovil, J.; Hsu, W. P.; Jacobs, M.; Aminabhavi, T.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (9) Schurtenberger,P.; Mazer, N.; Kinzig, J. J. Phys. Chem. 1983,87, t t
308. (10) Hashimoto, S.; Thomas, J. K. J.Colloid Interface Sci. 1984,102, 152. (11) Zana, R.;Guveli, D. J. Phys. Chem. 1985, 89, 1687. (12) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987,91, 356. (13) Giglio, E.; Loreti, S.;Pavel, N. V. J. Phys. Chem. 1988,92, 2858. (14) Meyerhoffer, S. M.; McGown, L. B. Langmuir 1989,5, 187. (15) Zakrzewaska,J.; Markovic, V.; Vucelic, D.; Feigin, L.; Dembo, A.; Mogilevsky, L. J. Phys. Chem. 1990,94, 5078. (16) Shibata, 0.; Miyoshi, H.; Nagadome, S.; Sugihara, G. J. Colloid. Interface. Sci. 1991, 146, 594.
In recent years much attention has also been paid to the aggregation and phase behavior of mixed systems containing bile salt and other surfactants. This stems from the fact that most of the biological functions of bile salts are based on their ability to associate with molecules such as cholesterol and lecithin to form mixed micellar aggregate structure^.^ Therefore, mixtures of bile salts with anionic,17J8~ a t i o n i c , and l ~ *nonionicz1Pz2 ~~ surfactants have been investigated and the micellar characteristics examined for varying mole ratios using different methods. The phase behavior of cationic surfactant-bile salt mixtures in water has also been determined.201~3In contrast to binary aqueous systems of ordinary surfactants, bile salts form no liquid crystalline phases with water. However in mixtures with other amphiphiles, Mesa et al.23 found that the bile salts are often accepted in large proportions in the existing liquid crystalline phases, and sometimes new phases are formed. In the CTAB-NaDCwater system the hexagonal phases can thus accept up to 30% of bile salt, and in the center of the phase diagram a cubic phase is formed. No lamellar phase is present, however, and most prominent is the isotropic micelle phase, L1. In similarity to many other anionidcationic systems, a narrow two-phase area, the coacervation region, is found in the L1 region at low surfactant concentrations close to equimolar ratio of the two surfactants. Coacervation here refers to the separation of the system into two liquid phases.24 Initially on mixing the Surfactants, turbidity is caused by the coacervate, but on standing over a period of time a distinct separation into two isotropic liquids occurs, one is rich in surfactant and therefore usually (17) Shilnikov, G. V.; Sarvazyan, A. P.; Zakrzewska, J.; Vucelic, D. J. Colloid Interface Sci. 1990, 140, 93. (18) VelBzquez,M. M.;Garcia-Mateos,F.;Lorente,F.;Valero,M.;Roriguez, L. J. J. Mol. Liq. 1990, 45, 95. (19) Jana, P. K.; Moulik, S. P. J. Phys. Chem. 1991, 95, 9525. (20) Barry, B. W.; Gray, G. M. T. J. Colloid Interface Sci. 1975,52, A27 .
(21) Ueno, M.; Kimoto, Y.; Ikeda, Y.; Momose, H.; Zana, R.J. Colloid Interface Sci. 1987, 117, 179. (22) Asano, H.; Aki, K.; Ueno, M. Colloid Polym. Sci. 1989,267,935. (23) Mesa, C. La.; Khan, A.; Fontell, K.; Lindman, B. J. Colloid Interface Sci. 1985, 103, 375. (24) Vassiliades, A. E. In Cationic Surfactants;Jungermann, E., Ed.; Marcel Dekker: New York, 1970; p 387.
0743-7463192124O8-2396$03.00/0 0 1992 American Chemical Society
Langmuir, Vol. 8, No. 10, 1992 2397
Aggregation Behavior of Mixed Micelles of Bile Salts
viscous and the other with little s u r f a ~ t a n t . The ~ ~ *coac~~ ervation observed in this system is believed to be caused by the growth of micelles to very large sizeseZ0A similar coacervationregion, somewhat less prominent, is also found with chenodeoxycholate (NaCDC), but not with the trihydroxy bile salt, NaC. The remarkable difference exhibited by the bile salts in CTAX solutions prompted us to further investigate the mixed micelles formed in these systems with the fluorescence quenching technique. Although it has proved to be a very useful technique in characterizing micellar system^^'-^ and mixed m i ~ e l l e s , limited ~ l * ~ ~work has been reported on the application of fluorescence quenching to the study of bile salt micelles and bile salt mixed micelles.10J1p21p22This is also interesting from the point of view of the transformation of CTAX with the addition of organic or other strongly adsorbed counterions, with highly specific effects on the growth of the micelles into long rods or threadlike micelles.33
Experimental Section Materiale. The surfactant cetyltrimethylammonium bromide (CTAB) purchased from Serva was used as supplied. Cetyltrimethylammoniumchloride (CTAC)was prepared by ion exchange from CTAB, on a Dowex 1-X8 resin. The product was freezedried and stored in a desiccator. The sodium salts of cholic acid (NaC, Sigma), deoxycholic acid (NaDC, Fluka; purity >99% 1, and chenodeoxycholicacid (NaCDC,Sigma; especially pure) were used without further purification. The problem of impurities in pure bile salt micelle research is well documented.6 The bile salts showed trace amount8 of a fluorescing species that perturbed the initial decay pattern of monomeric pyrene emission. This perturbation was impossible to eliminate even when small amount of the salts were further purified using several techniques; however, this problem was successfully handled in the data evaluation model as described later. Although the sample purity is important in characterizing the micelles of pure bile salts, we believe that this effect is reduced in the case of mixed micelles. Pyrene (Aldrich) was recrystallized twice from ethanol, and dimethylbenzophenone (DMBP, Aldrich), with purity >99 7% ,was used as supplied. All solutions were prepared in distilled water. In the preparation of samples for fluorescence measurements a stock solution of pyrene and the quencher DMBP in ethanol was used. To introduce the probe into the sample, an appropriate amount of the stock solution was placed in a volumetric flask and the solvent evaporated by passing a gentle stream of nitrogen over it. The pyrene was then solubilized in the surfactant solution to give the desired concentration. DMBP was similarly introduced into a part of the surfactant solution with probe to give a single concentration of probe, quencher, and surfactant. This new stock solution was diluted further by the solution without quencher to give other desired concentrations of quencher. The concentration of pyrene was 2.5 pM in all the solutions with varying quencher concentrations. The pyrene concentration was low enough for excimer formation to be avoided. All solutions (25) Scamehorn, J. F., Ed. Phenomena in Mixed Surfactant System; ACS Symp. Ser. 311; AmericanChemical Society: Washington, DC, 1986. (26) Stellner, K. L.; Amante, J. C.; Scamehorn, J. F.; Harwell, J. H. J. Colloid Sci. 1988, 123, 186. (27) Tachiya, M. InKinetics ofNonhomogeneow Processes;Freemen, G. R., Ed.;John Wiley and Sons: New York, 1987; pp 576-650. (28) h a , R. In Surfactant Solutions. NewMethodsofInuestigation;
Surfactant Science Series; h a , R., Ed.; Marcel Dekker: New York and Basel, 1987; p 214. (29) Auweraer, M. van der; De Schryver, F. C. In Inverse Micelles, Studies in Physical and Theoretical Chemistry; Pileni, M. P., Ed.; Elsevier: Amsterdam, 1990; Vol. 65, p 77. (30) Almgren, M. In Kinetics and Catalysis in Microheterogenous Systems; Griitzel, M., Kalyanasundaram, K., Ed.; Marcel Dekker, Inc.: New York, 1991; pp 63-108. (31) Lang, J. J. Phys. Chem. 1990, 94, 3734. (32) Malliaris, A.; Binana-Limbele, W.; Zana, R. J. Colloid Interface Sci. 1986, 110, 114. (33) Almgren, M.; Alsins, J.; van Stam, J.; Mukhtar, E. Prog. Colloid Polym. Sci. 1988, 76, 68.
were thoroughly stirred mechanically for more than 4 h until the probe and quencher were completely solubilized and then allowed to stand sufficiently to ensure equilibrium conditions. The variation in the natural pH of the various compositions was measured and found to change within about one pH unit. For the NaDC/CTAB system the pH varied between 6.6 and 7.8 pH units in the mole fraction of bile salt between 0.17 and 0.67. No effort was made to adjust the pH of the mixture using buffer solutions since this small pH variation should have a negligible effect on the aggregation behavior of the mixed micelle system. This has also been confirmed by intrinsic viscosity measurements by comparing neutral samples with samples prepared in 10% phosphate buffer solutions maintained at pH 8.0. Similar viscosity curves were obtained indicating no change in intrinsic viscosityimplying an insignificantchange in aggregation behavior with pH. Methods. Static fluorescencemeasurements were carried out on a SPEX Fluorolog 1680combined with a SPEX Spectroscopy Laboratory Coordinator DMlB and performed at 25 OC. Time-resolved fluorescencedecay data were collected with the single photon counting technique, as described earlier." The setup uses a mode-locked Nd-YAG laser to synchronously pump a cavity-dumped dye laser for the excitation, using DCM as dye, and a KDP crystal for frequency doubling. The excitation wavelength was 323 nm and the pyrene monomer emission was measured at 395 nm. Measurements were performed at two temperatures, 25 and 40 "C. The fluorescencequenching data were fitted to a generalization of the model for fluorescence deactivation proposed by Infelta et al.35,36The equations used in the analysis of the surfactantbile salt mixed micellar system are the following:3'
B, = A, exp[-A,t
+ A3(exp(-A4t)- l)]
(1)
where the expressions for the parameters Al-A, are given by
A, = Bo
(la)
A, = k, + ( x ) , k ,
(1b)
A3 = ( n ) ( l - tz),/(n))'
(IC)
(Id)
Bo is the fluorescence intensity at time t = 0, ko is the first-order decay constant, ko = 1/70, (z), is the average number of quenchers in micelleswith a surviving excited probe during the stationary part of the fluorescence decay, and (a)is the average number of quenchers in a micelle. The aggregation number N,, is obtained from where S, and Q, are the concentrations of the aggregated surfactant and quenchers in the micellar phase. Equation 1 has the same form as the Infelta-Tachiya36*% equation but the exprewions for the parameters differ. In the Infelta-Tachiya case
(z),/(n) = k J ( k , + k J
(3)
and eq 1 reduces to the parameters in the Infelta case. In the system under study neither probe nor quencher is expected to migrate appreciably during the time window of measurement (about 2 ps). A value of ( x ) J ( n )
