Fluorescence quenching studies of the aggregation behavior of the mixed micelles of bile salts and cetyltrimethylammonium halides

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 aggregation behavior of cetyltrimethylammonium halide (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 intes- tinal 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. t University of Uppsala. t 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.;

(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,

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.

(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.;

(16) Shibata, 0.; Miyoshi, H.; Nagadome, S.; Sugihara, G. J. Colloid.

Plenum Press: New York, 1971, Vol. 1.

1987,91, 356.

Mogilevsky, L. J. Phys. Chem. 1990,94, 5078.

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 anion- ic,17J8 ~ a t i o n i c , l ~ * ~ ~ and 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.201~3 In 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-NaDC- water 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.

(18) VelBzquez, M. M.; Garcia-Mateos, F.; Lorente,F.; Valero,M.; Ror-

(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,

Colloid Interface Sci. 1990, 140, 93.

iguez, L. J. J. Mol. Liq. 1990, 45, 95.

A27 . (21) Ueno, M.; Kimoto, Y.; Ikeda, Y.; Momose, H.; Zana, R. J. Colloid

(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

(24) Vassiliades, A. E. In Cationic Surfactants; Jungermann, E., Ed.;

Interface Sci. 1987, 117, 179.

Interface Sci. 1985, 103, 375.

Marcel Dekker: New York, 1970; p 387.

0743-74631921 24O8-2396$03.00/0 0 1992 American Chemical Society

Aggregation Behavior of Mixed Micelles of Bile Salts

viscous and the other with little s u r f a ~ t a n t . ~ ~ * ~ ~ The coacervation observed in this system is believed to be caused by the growth of micelles to very large sizeseZ0 A similar coacervation region, 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 , ~ l * ~ ~ limited work has been reported on the application of fluorescence quenching to the study of bile salt micelles and bile salt mixed micelles.10J1p21p22 This 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. Cetyltri- methylammonium chloride (CTAC) was prepared by ion exchange from CTAB, on a Dowex 1-X8 resin. The product was freeze- dried and stored in a desiccator. The sodium salts of cholic acid (NaC, Sigma), deoxycholic acid (NaDC, Fluka; purity >99% 1, and chenodeoxycholic acid (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 intro- duced 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; American Chemical 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

Langmuir, Vol. 8, No. 10, 1992 2397

Sci. 1986, 110, 114.

Polym. Sci. 1988, 76, 68. (33) Almgren, M.; Alsins, J.; van Stam, J.; Mukhtar, E. Prog. Colloid

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 viscosity implying an insignificant change in aggregation behavior with pH.

Methods. Static fluorescence measurements were carried out on a SPEX Fluorolog 1680 combined with a SPEX Spectroscopy Laboratory Coordinator DMlB and performed at 25 OC.

Time-resolved fluorescence decay 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 tem- peratures, 25 and 40 "C.

The fluorescence quenching data were fitted to a generalization of the model for fluorescence deactivation proposed by Infelta et al.35,36 The equations used in the analysis of the surfactant- bile salt mixed micellar system are the following:3'

(1) B, = A, exp[-A,t + A3(exp(-A4t) - l)] where the expressions for the parameters Al-A, are given by

A, = Bo (la)

(1b) A, = k, + ( x ) , k ,

A3 = ( n ) ( l - tz),/(n))' ( I C )

(Id) Bo is the fluorescence intensity a t time t = 0, ko is the first-order decay constant, ko = 1/70, (z), is the average number of quenchers in micelles with 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

(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 ) << 1 is then expected and was generally found (Table I). Deviation occurred only when long polydispersed micelles were present and indicated that the model was inapplicable.

The natural lifetime of pyrene (TO) was determined in separate experiments without quencher. The solutions were not generally deoxygenated because of the risk for concentration changes due to frothing, resulting in difficulties to maintain identical con-

(z), /(n) = k J ( k , + kJ

(34) Almgren, M.; Alsins, J.; Mukhtar, E.; van Stam, J. J. Phys. Chem.

(35) Infelta, P. P.; Gratzel, M.; Thomas, J. K. J. Phys. Chem. 1974,78, 1988,92,4479.

190. (36) Tachiya, M. Chem. Phys. Lett. 1975, 33, 289. (37) Almgren, M.; Ldfroth, J.-E.; van Stam, J. J. Phys. Chem. 1986,

90,4431.

2398 Langmuir, Vol. 8, No. 10,1992 Vethamuthu et al.

400 1 1

1.2 1 t

A A

= NaDC A E NaC

-I 200 A I

Y = ,.I"

a

t 4 I

0.0 0.2 0.4 0.6 0.8 1 .o Mole fractlon of bile salt

Figure 1. Dependence of the III/I ratio of pyrene in the CTAC/ bile salt mixed micelles on the mole fraction of the bile salt, T = 25 OC.

0.0 0.2 0.4 0.6 0.8 1 .o Mole fractlon of blle salt

Figure 2. Dependence of the fluorescence lifetime of pyrene in the CTAB/bile salt mixed micelles on the mole fraction of the bile salt. T = 25 OC.

ditions for all samples.. It has been shown earlier38 that the effect of dissolved oxygen affects only the observed decay constant, ko, but not the use of eq 1. Deoxygenation was only carried out for samples where the exact influence of oxygen wastested. Similarly, the quenching by bromide ions in the system affect only ko.

In order to handle the effect of a short-lived fluorescing species in the bile salt surfactant which perturbed the initial part of the decay, a modified version of eq 1 was used, which contained terms representing the contribution of the perturbant39

400 """ i

-

E 200 _./ A

4 " 4

The different k{s and a{s were determined using quencher-free samples, employing a multiexponential fit program. The most long-lived component gave ko. In general one or two exponentials were needed to describe the initial perturbation for which the weight factors ai were calculated using

ai = A J ~ A , (5) where Ai is the amplitude for the different exponentials obtained from the fit. Using eq 4 in the evaluation allowed computer fittings much closer to zero time channel (ZTC) with retention of acceptable statistics and more reliable estimation of the parameters given in eq 1.

Dynamic light scattering measurements were performed using an apparatus and technique described before.a Laplace inversion of the correlation curves was performed using a constrained reg- ulization program to obtain the distribution of the decay times.

Viscosities of solutions were measured using a Ubbelhode capillary viscometer. The flow time always exceeded 150 s and no kinetic energy correction was necessary.

Rssults Fluorescence Spectra and Lifetime of Pyrene. The

ratio between the vibronic peaks III/I in the pyrene emission spectrum is often used as a measure of the polarity of the microenvironment around the p r ~ b e . ~ ~ ~ ~ ~ This ratio varies between 0.63 for a polar solvent (HzO) and 1.65 for nonpolar solvent (cyclohexane). The III/I ratio observed for pyrene in CTAC micelles as a function of the bile salt mole fraction is shown in Figure 1.

In the absence of bile salts, the III/I ratio is 0.82 which indicates that pyrene is located in the palisade layer of

1 .o 100

0.0 0.2 0.4 0.6 0.8

Mole fraction of bile salt

Figure 3. Dependence of the fluorescence lifetime of pyrene in the CTAC/bile salt mixed micelles on the mole fraction of the bile salt. The upper data points are the lifetimes in deaerated samples, T = 25 OC.

CTAC micelles.43 Successive addition of bile salt increases this ratio, showing that pyrene moves toward an increas- ingly nonpolar environment. Figure 1 also shows differences in the behavior of NaC and NaDC. The III/I ratio for NaC is similar to NaDC in the CTAC-rich region but lower in bile salt-rich region. Close to equimolar ratio of CTAC and NaDC coacervation occurred and the III/I ratio could not be determined.

Above a mole fraction of 0.7 of bile salta the III/I ratio attains a near constant value slowly reaching that of pure bile salt micelles. High III/I ratio has also been observed in the bile salts-rich region for bile salt/nonionic surfactant micelles.21

The measured lifetime in CTAB micelles at different mole fraction of bile salts is given in Figure 2. The lifetime is found to increase with the addition of bile salta. The increase is slightly larger for the NaC system below the equimolar concentration and for NaDC at higher bile salt/ CTAB mole fraction. For both systems the lifetime is shorter at higher temperature. A similar behavior is observed in the CTAC/bile salt system shown in Figure 3, where the quenching effect of Br- ions is eliminated by replacing it with C1-ions. This can be seen from the longer lifetime of pyrene in the pure CTAC micelles. It can be noted that pyrene in CTAC/bile and CTAB/bile micelles have similar lifetimes at high concentration of bile salts,

~~ ~

(38) van Stam, J.; Almgren, M.; Lindblad, C. h o g . Colloid Polym. Sci. 1991.84, 15. (39) Almqen, M.; Haneeon, P.; Makhtar, E.; van Stam, J. Langmuir,

in Drew. (40) Nicolai, T.; Brown, W.; Johnsen, R. M.; Sthpanek, P. Macro-

(41) Naknjima, A. Bull Chem. SOC. Jpn. 1971,44, 3272. (42) Kalyanaeundaram,K.;Thomaa, J. K. J . Am. Chem. SOC. 1977,99,

molecules 1990,23, 1165.

7. (43) Almgren, M.; Medhage, B.; Mukhtar, E. J. Photochem. Photo-

biol. A: Chem. 1991, 59, 332.


Table I. Aggregation Numbers with Respect to CTA+ (&), Quenching Rate Constants (kq), (r),/(n),,, and xz of the Mixed

Langmuir, Vol. 8, No. 10, 1992 2399

Micelles of CTAB at Varied Bile Salt Mole Fractions at 25 and 40 OC

Nw, lo-' k,, s-l ( x ) d ( n ) , X 2

bile salt mole fraction 25 O C 40 O C 25 "C 40 O C 25 O C 40 O C 25 "C 40 "C

A

A

2 1

NaC 0 0.17 0.29 0.37 0.44 0.50 0.62 0.67

108 85 66 43 34 26 9 8

86 1.1 73 0.79 54 0.68 41 0.97 28 1.80 25 2.12

1.55 1.21

NaDC 0.17 106 83 0.70 0.29 112 78 0.36 0.62 15 0.67 0.67 12 0.58

Table 11. Aggregation Numbers with Respect to CTA+ (&), Quenching Rate Constants (kq), (x),/(n)q, and x2 of

the Mixed Micelles of CTAC at Varied Bile Salt Mole Fractions at 25 "C

bile salt Nag 10-7k,, s-l ( ~ ) d ( n ) ~ X 2

NaC 0 0.17 0.29 0.37 0.44 0.50 0.55 0.67

82 68 54 35 26 18 10 7

1.50 1.00 0.90 1.24 1.57 1.65 1.36 1.28

0.01 0.99 0.009 1.04 0.007 1.17 0.01 0.97 0.006 1.07 0.01 1.03 0.03 1.03 0.01 1.10

NaDC 0.17 68 1.11 0.02 1.07 0.28 60 0.87 0.02 1.06 0.62 12 0.88 0.01 1.02 0.67 10 0.84 0.006 1.07

showing that the halide ions are expelled from the micelle surface a t these concentrations. A similar dependence of the fluorescence lifetime of monomeric pyrene on surfactant concentration in mixed micelles has been observed before1al4 and attributed to the dependence of the pyrene lifetime on the microenvironment. This behavior can again be explained by the migration of pyrene into the micelle interior. However, such a dependence of the lifetime can also result from a shielding of pyrene from the dissolved oxygen in the solution.39 In order to determine the effect of oxygen quenching, the lifetimes of pyrene in deaerated samples of CTAC/ bile salts micelles are also given in Figure 3. A similar dependence of the bile salt concentration is observed but the lifetimes were longer as expected. This point will be further examined in the discussion section.

Time-Resolved Fluorescence Quenching. The results obtained from fitting the fluorescence quenching decay curves to eq 4 for the CTAB/bile salt and CTAC/ bile salt mixed micelles are given in Tables I and 11, respectively. In addition to the x2 values given in the table, the goodness of the fit was also checked by the weighted residuals and the autocorrelation function.

The aggregation number Nagg with respect to CTA+ and the quenching rate constant k, for the CTAB and CTAC bile salt mixed micelles given in Tables I and I1 are also plotted as a function of the mole fraction of the bile salts in Figures 4 and 5, respectively. Examining first the results for the CTA+/NaC micelles, we observe that both for CTAB and CTAC (Figure 4), the aggregation number shows a

(44) Zachariasae, K. A.; Kozankiewicz, B.; Kuhnle, W. In Surfactants in Solutions; Mittal, K. L., Ed.; Plenum: New York, 1084, p 565.

2.13 1.29 1.8 1.95 2.51 3.02

1.28 1.35

0.02 0.03 0.03 0.03 0.007 0.004 0.04 0.02

0.02 0.08 0.007 0.006

0.007 0.02 0.013 0.07 0.02 0.03

0.03 0.02

1.10 1.13 1.09 1.09 1.10 1.11 1.08 1.30

1.09 1.22 1.03 1.05

1.07 1.07 1.11 1.006 1.14 1.08

1.16 1.002

e 0)

P

I NaDC A = NaC

A

A

A

0.0 0.2 0.4 0.6 0.8


W1

I = NaDC A NaC

4

A

A

gradual decrease with increase in the mole fraction of the NaC. A similar trend was also observed a t 40 "C (see Table I).

The values for the quenching rate constant k, for the CTA+/NaC system are shown in Figure 5. Usually k, decreases with increasing size of the aggregate. Figure 5 however shows an initial decrease in k, followed by a gradual increase with increasing bile salt concentration up to equimolar ratio. Further increase in the NaC concentration leads to a decrease in k,, while the aggregation number over this range shows a continuous decrease. This behavior can be explained by an increase in the local viscosity for pyrene as the concentration of the bile salt

2400 Langmuir, Vol. 8, No. 10, 1992

0

Vethamuthu et al.

W I

2.2 I I

= 1.1 *.

0

2.2

= 1.1 -e

[AI

A A

A

A

=NeDC A =NaC

0.0 0.2 0.4 0.6 0.8

Mole fractlon 01 bile salt

[ B l

I

A

4

A

A A

I NaDC A i NaC

0.0 0.2 0.4 0.6 O B


Figure 5. First-order quenching rate constant k, for (a) CTAB and (b) CTAC in NaDC and NaC mixed micelles, T = 25 “C.

increases, a behavior compatible with the indication from III/I ratio that pyrene migrates to the interior of the micelle.

A different behavior was observed for the mixed micelles of CTAB and CTAC with the bile salt NaDC. The Nwg and k, are shown in Figures 4 and 5, respectively. This behavior can be divided into three separate regions: (i) small micelles formed a t high and low bile salt concentration; (ii) large aggregates formed in two regions, between 0.3 and 0.46 mmol fractions and also between 0.56 and 0.6; (iii) liquid phase separation (coacervation) occurred close to equimolar ratio. The nature of the aggregates in the three regions will now be discussed in detail. To further clarify the discussion, the decay curves recorded in these regions are given in Figure 6. The natural lifetime of pyrene has been shown to depend on the concentration of the bile salt (Figures 2 and 3). This will therefore complicate the direct comparison of the quenching behavior in the different regions. In Figure 6, therefore, we have eliminated the effect of the variation in the natural lifetime by multiplying the decay curve by exp(kot,) where ko is the decay constant measured a t the given CTAC/ NaDC concentration without quencher. For comparison the corresponding curves for the CTAC/NaC system are also shown in Figure 7. The curves are all of the type expected for small micelles as described by eqs 1 and 4 where at long times the fluorescence decays a t rate similar to the unquenched pyrene.

(i) The fluorescence quenching resulta in this region could satisfactorily be fitted to the model given in eqs 1 and 4. At low concentration of NaDC (Figure 6A,B) CTA+- rich micelles are formed with Nwg close to that of CTAB and decrease slowly in size with increasing NaDC concentration. At high bile salt concentration (Figure 611,

I A / c l

Figure 6. Fluorescence quenching curves of CTAC/NaDC mixed micelles freed from the influence of the natural decay by multiplication of the measured intensities with the appropriate factor of exp(k0t). All the curves are for samples with pyrene concentration of 2.5 X 10” M and DMBP concentration of 0.3 mM. The concentration of CTAC was 25 mM and the mole fraction of NaDC was (a) 0.0, (b) 0.17, (c) 0.29, (d) 0.37, (e) 0.44, tf) 0.46, (g) 0.55, (h) 0.58, and (i) 0.67.

4.2

- 4

a

I# 3.8

3

w

d a - =. LL

0

3.6

0 200 400 600 800 1000 Tlme (ne)

Figure 7. Fluorescence quenching curves of CTAC/NaC mixed micelles produced in a similar way to those in Figure 6. The mole fraction of NaC was (a) unquenched, (b) 0.67, (c) 0.50, (d) 0.44, (e) 0.37, (f) 0.29, (9) 0.17, and (h) 0.0.

small bile salt-rich micelles are formed, decreasing grad- ually in size to that observed for pure NaDC. Assuming all NaDC being in micelles, the aggregation number with respect to NaDC is obtained as 24 when the mole fraction of NaDC is 0.62 and 0.67.

(ii) The fluorescence quenching decay curves in this region (Figure 6C-F) could not be fitted satisfactorily to the model given in eqs 1 and 4, and therefore no reliable estimate of Nagg could be made. In this concentration range, a transition from small spherical micelles to large aggregates occurs. The reverse transition from large aggregates to small bile-rich micelles seems to occur in Figure 6G,H. The decay curves Figure 6C and Figure 6H appear biphasic probably due to a polydisperse distribution of micellar aggregates. The decay curves in Figure 6D-G were fitted to the model for quenching in infinite rod micelles,33,34 and good agreement was obtained. Further experimenta are being carried out on the behavior of the


E NaDC A = NaC

400

Langmuir, Vol. 8, No. 10, 1992 2401

A m N8C ( T . d C ) + = N8DC ( T ~ l o ° C ~ 0 s N8DC (T.40°C)

2.5 * NsDC (T.6o0C1

c /

t ------J i b, I . . A , ?

0

0.3 0 .4 0.5 Mole traction ot bile salt

Figure 8. Hydrodynamic radius Rh for mixed micelles of CTAB/ bile salt as a function of mole fraction of bile salt with CTAB concentration fixed at 25 mM.

system in this region and will be reported in a forthcoming communication.

At a bile salt concentration of 0.29mole fraction of NaDC (Figure 6C) the decay curves obtained seem to be influ- enced by the concentration of the quencher. At a quencher concentration of 0.3 mM (curve I) the decay curve has a form typical of long micelles and could not be fitted to eq 1. When the quencher concentration was reduced to 0.15 mM, however, a decay curve (curve 11) typical of small micelles was observed which could be fitted to eq 1. At this concentration the system seems to be on the border of the transition from small to large aggregate and could therefore be perturbed by the presence of the quencher. Small micelles were also observed for the same composition when the temperature was raised to 40 OC. The transition from small to large aggregate therefore is also temperature dependent.

(iii) The phase separation observed in this region is a common feature of anionicfcationic mixtures. When this mixture of 0.5 mole fraction NaDC was allowed to settle for few days, two distinct isotropic phases could be separated, an upper low-viscosity phase and a lower high- viscosity phase with a volume ratio of about 1 to 10.

Fluorescence quenching studies were also performed on the CTAB/sodium chenodeoxycholate (NaCDC) system. This dihydroxy bile salt behaves similar to sodium deoxycholate where the equimolar solution with cationic surfactant also forms coacervates, but in this case the phase separation is shifted from about 80 OC for NaDC to below 30 OC.zo At 30 OC isotropic solutions were obtained for the equimolar concentration but the fluorescence quenching decay curves could not be fitted to the model of eqs 1 and 4. The decay curve has a form typical of rodlike micelles as shown in Figure 6F.

Dynamic Light Scattering. The hydrodynamic radii, Rh, of mixed micelles of CTAB/NaDC and NaC salt systems were obtained from diffusion coefficients (D) using the Stokes-Einstein equation

D = kT/6~qR, (6) where 7 is the viscosity and k T the energy term. Figure 8 shows the dependence of hydrodynamic radius upon the mole fraction of added bile salts at 25 "C. The CTAB/ NaC micelles were small for all NaC concentrations; the Rh values were in the range 12-20 A. Even at equimolar concentration these micelles did not show any signifi- cant increase in size. Small spherical micelles for alkyl- trimethylammonium cholates with aggregation numbers

$ 2

1 5

1

0 0.25 0.5 Mole lraction 01 blle aalt

Figure 9. Relative viscosity of CTAB/bile salt at different tem- peratures as a function of the bile salt mole fraction. CTAB concentration was 25 mM.

19-32 were observed by Barry and Gray.45 For the CTAB/ NaDC system, the hydrodynamic radius increased mono- tonically with increasing NaDC concentration. A distinct growth of micelles was thus noticed for the CTAB/NaDC system, particularly at NaDC concentrations close to the CTAB concentration and at low temperature. The hydrodynamic radius in such cases increased to a few hundred angstroms.

Viscosity. Figure 9 shows the variations in relative viscosity of 25 mM CTAB solution with varied bile salt mole fraction. In the concentration/composition range studied, the solutions display a Newtonian flow. For both CTAB/NaC and CTAB/NaDC systems, a small initial decrease in viscosity was observed up to about 0.29 mole fraction of bile salt. This decrease may be ascribed to the incorporation of bile salt molecules in the CTAB micelles, changing the micelle size or concentration. For CTABf NaC, the relative viscosity did not show any change at high bile salt concentration, whereas the CTAB/NaDC system behaved quite differently. The viscosity of the solution showed a dramatic increase with the NaDC concentration. This increase was more pronounced at lower temperature. The different behavior shown by two bile salts supports the fluorescence quenching and dynamic light scattering results. More concentrated solutions of CTAB (100 mM) with NaDC (up to 100 mM) were highly viscous and gel-like.

Discussion The fluorescence quenching studies were performed with

solutions containing a constant concentration of CTAC or CTAB and varying amounts of the bile salt. Addition of NaC reduces the aggregation number with respect to CTA+ monotonously, and also the total aggregation number and the size of the micelles is decreased. Addition of NaDC gives first a small reduction of the number of CTA+ surfactants in the aggregate, the total aggregation number increases slightly, but already with 0.29 mole fraction of NaDC the decay pattern, Figure 6c, changes in a way that makes the model of eq 1 inapplicable. At 0.37 mole fraction of NaDC, the decay, Figure 6c, is similar to that recorded in CTA-ClOS- indicating that long rodlike micelles are p r e ~ e n t . ~ ~ ? ~ From the results in the study of the latter system it can be concluded that the aggregation numbers are much larger than 1OOO. The change in the quenching curve with further addition of bile salt, Figure 6E-G, can be understood from a reduction of the number of quench-

(45) Barry, B. W.; Gray, G. M. T. J. Colloid Interface Sci. 1976,52, 314.

2402 Langmuir, Vol. 8, No. 10, 1992

ers per length unit of the rods when more bile salt is inserted into the micelles and possibly also a reduction of the diffusion coefficients of the reactants. The decay pattern in the case with 0.29 mole fraction NaDC still appears biphasic, however, and is probably to be regarded as a superposition of decays in small micelles and very large ones, which suggests that there is a very broad polydis- persity in this region.

The decay pattern for the micelles at bile salt concentrations just above the coacervation region are similar to those just before; i.e. long rodlike micelles seem to be formed also here. At high bile salt concentrations small bile-rich micelles are found. The results from dynamic light scattering, Figure 8, and viscosity measurements, Figure 9, for bile concentrations lower than equimolar corroborate this interpretation.

Most remarkable in the results is the striking difference of the effects of the trihydroxy bile salt, NaC, and the dihydroxy salts, NaCDC and NaDC. Similar differences have been noted in the interaction of the bile salts with lecithins; these systems have been studied e x t e n s i ~ e l y . ~ ~ From 2H NMR studies using selectively deuterated bile salts, Saito et al.so concluded that deoxycholate and chenodeoxycholate were incorporated perpendicular with respect to the surface of a lecithin bilayer membrane, whereas Ulmius et al.51 have suggested that cholate molecules most probably are placed flat on the surface, this conclusion drawn from the observed reduction of the deuterium order parameter for deuterated segments of specifically deuterated lecithins on adsorption of cholate on the membrane. Results obtained by Schubert et al.52 on the binding of bile salts to egg lecithin vesicles are in line with these proposals.

The reason for these differences in the behavior is found in the structure of the bile salts. These molecules do not have a polar head and a hydrophobic tail like most common amphiphiles. They are instead presenting a polar face, which gest its hydrophilic character from the hydroxy groups and the carboxylate group, and a nonpolar face made up of the hydrocarbon segments of the steroid backbone. As was pointed out by Mazer et al.153 a large fraction of the polar face remains exposed hydrocarbon elements, between the polar groups. They estimated the water-exposed hydrocarbon area as close to 70 A2 for cholate and as large as 160 A2 for the dihydroxy cholates, slightly larger for deoxycholate than for chenodeoxycholate. This is in the same order as the hydrophobicity quantified by ~hromatography.5~ The flat position on a interface with all polar groups in contact with water is much more favorable for cholate than for the dihydroxy salts. It has further been suggested that the bile salts form dimers when inserted in hydrophobic environments so that the loss of hydration of the hydroxy groups could be compensated for by formation of hydrogen bonds. For geometrical reasons, hydrogen bonds could be formed only by one of the pairs of hydroxy groups in dimers of DC or CDC (but possibly between all three in cholate). A

(46) Carey, M. C. Hepatology 1984,4, 1385. (47) Mazer, N. A.; Benedek, G. B.; Carey, M. C. Biochemistry 1980,

(48) Malloy, R. C.; Binford, J. S. J. Phya. Chem. 1990,94, 337. (49) Schubert, R.; Schmidt, K. H. Biochemistry 1988, 27, 8787. (50) Saito, H.; Sugimoto, Y.; Tabeta, R.; Suzuki, S.; Kodama, M.;

(51) Ulmius, J.; Lindblom, G.; Wenneretrdm, H.; Johanseon, L.-B.;

(62) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry

(53) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B.

(54) Bloch, C. A.; Watkins, J. B. J. Lipid Res. 1978, 19, 510.

19, 601.

Toyoshima, S.; Nagata, Ch. J. Biochem. (Tokyo) 1983, 94, 1877.

Fontell, K.; SBderman, 0.; Arvidson, G. Biochemistry 1982,21, 1553.

1986,25, 6263.

Biochemistry 1979, 18, 3064.

Vethamuthu et al.

distribution between flat positioning at the surface and inserted dimers may be possible for the bile salts, with the flat mode preferred by cholate and the inserted one by DC and CDC.

Turning now to the CTAX-bile salt mixed micelles it is readily understood that the insertion of a bile molecule at the interface with the polar face exposed to water would add a large area and tend to decrease the size of the micelle as observed with NaC. Insertion of bile molecules between the cationic surfactants, with only the negatively charged carboxylate in the head group region, would decrease the average area per surfactant molecule, due to the mutual attraction between the opposite charges, and give a comparable or somewhat larger contribution to the core volume than that from the surfactant. The critical length would be mainly determined by the long surfactant chain, but in the cylindrical, and in particular the lamellar geometries, the short extension of the bile salt body would decrease it. Taken together, the effect must be an increase of the value of the packing parameter of Israelachvili et al.,55 U/aolcrit, where u is the volume of the hydrophobic tail, a. the effective surface area of the amphiphile, and lcrit the critical length. For spherical micelles the value should be below l/3, and for cylindrical shapes, or oblate and prolate ellipsoids, the value is between and l/2; above the latter value lamellar shapes or bilayer vesicles are allowed. The CTAC and CTAB micelles are well- known to easily undergo a transformation to rodlike micelles. Such a transformation is thus the effect to be expected from an insertion of amphiphiles that increase the packing parameter.

Pyrene Lifetime and III/I Ratio. The 11111 ratio of pyrene in CTAC is 0.82, suggesting a more polar environment for pyrene than in SDS (III/I = 0.92). Based on this and other evidence, it has been suggested that pyrene is preferentially solubilized in the head group region of CTAC and other quaternary ammonium surfactant mi- c e l l e ~ . ~ ~ Addition of bile salta increases the value of the ratio toward the value representative of pyrene in pure bile salt micelles. Simultaneously, the lifetime of the fluorescence increases with the bile salt addition. Both effects indicate that pyrene is better shielded from the aqueous environment in the bile-containing micelles. A similar trend was observed in nonionic surfactant (C&8)/ bile salt mixed micelles21+22 and in the study of pyrene solubilization by sodium taurocholate.1° In the former system a breakpoint was observed at a certain composition, indicating a transition to a new type of bile-rich micelle with particularly good shielding of pyrene from the aqueous environment. No such break is seen in the lifetime or III/I data for the CTAX/bile salt systems; on the contrary the values of both parameters change smoothly with the bile salt concentration. Even the values on either side of the two-phase region in CTAX/NaDC seem to belong to the same smooth curve. The coacervation is thus not accompanied by a micellar rearrangement.

From the III/I data, and the lifetimes in degassed solution (Figures 1 and 3); NaDC is seen to give better shielding than NaC, in particular a t a mole fraction above 0.4. The behavior at high concentrations is probably due to the larger size of the NaDC mixed micelles.

The lifetime data allow in principle a more quantitative interpretation. A t least three effects are operative in

(55) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. SOC.,

(56) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. SOC. 1980, Faraday Trans. 2 1976, 72, 1525.

102,3188.


Table I11 (a) Fluorescence Lifetimes of Pyrene in CTAB/NaC Mixed

Micelles, mr (Aerated), and CTAC/NaC Mixed Micelles, T&

(Aerated) and TO (Deaerated), the Calculated Fraction of Pyrene in Contact with Water (P), the Fraction of Bound Bromide Ions (&), and the Oxygen Shielding Factor cf, as a Function of Bile

Salt Mole Fraction NaCMolefraction mr,ns z&,ns 70,ns P BBr f

0.00 0.17 0.29 0.37 0.44 0.50 0.62 0.67 1.00

114 142 181 213 229 241 251 286

156 181 205 226 240 244 254 288 332

308 325 350 369 385 391 394 402 424

0.30 0.75 1.00 0.25 0.58 0.77 0.19 0.32 0.64 0.14 0.18 0.54 0.11 0.17 0.49 0.10 0.05 0.48 0.09 0.04 0.44 0.08 0.03 0.31 0.04 0.21

(b) Fluorescence Lifetimes of Pyrene in CTAB/NaDC Mixed Micelles, 7Br (Aerated), and CTAC/NaDC Mixed Micelles, 7,,jr

(Aerated) and TO (Deaerated), the Calculated Fraction of Pyrene in Contact with Water (P), the Fraction of Bound Bromide Ions (Ber), and the Oxygen Shielding Factor cf, as a Function of Bile

Salt Mole Fraction NaDCmolefraction TBr, ns 7&,ns 70, ne P @Br f

0.00 114 156 308 0.30 0.75 1.00 0.17 138 181 327 0.24 0.68 0.78 0.29 160 196 357 0.17 0.65 0.73 0.37 193 217 384 0.12 0.51 0.63 0.44 216 235 403 0.08 0.45 0.55 0.50 0.62 281 284 424 0.05 0.10 0.36 0.67 305 307 427 0.04 0.05 0.29 1.00 377 456 0.00 0.15

reducing the lifetime of pyrene fluorescence from a value of about 450 ns in a dilute, oxygen-free hydrocarbon solution.67 First, water has in itself an effect; the lifetime indeaerated water was measured to be 175 ns. The second factor to consider is the bromide ion quenching in the CTAB system. Finally oxygen quenches at a diffusion- controlled rate, reducing the value in air-saturated water to 125 ne; the value in an air-saturated hydrocarbon is much smaller due to the higher oxygen solubility. These three factors will now be considered in more details.

The Effect of Water. If it is assumed that the change of the lifetime, TO, when bile salts are added to CTAC micelles in deaerated solutions is due solely to that the contact between pyrene and water is reduced, the fraction, P, of pyrene in contact with water can be estimated by aesuming that the observed decay rate is the weighted average of the decay rate in water taken to be 1 / ~ , ~ = 5.7 X 106 s-l and that in the interior of the micelle, assumed equal to that in a hydrocarbon solvent (1/7hc = 2.2 X lo6 8-1)

(7) The resulta from the measured values are reported for the NaC and NaDC micelles in parta a and b of Table 111, respectively. In this calculation it has been assumed that the pyrene molecules exchange rapidly between the interior and the surface, otherwise the decays without added quenchera should have been nonexponential. On comparison of the calculated values for NaC and NaDC, it is obaerved that slightly lower values are obtained for NaDC. The P values are also plotted in Figure 10.

Bromide Ion Effect. The effect of the bromide ions can be inferred from a comparison of the lifetimes in the CTAC and CTAB/bile salt systems. The increased lifetime

(57) Birh, J. B. Photophysics of Aromatic Molecules; John Wiley: London, 1970.

1/70 = P(1/Taq) + (1 -fl(1/7hc)

1 . 2

0.8 - 5

Q - - 0,

0 . 4

0

1.2

0.8 s

m - 0.4

0

Langmuir, Vol. 8, No. 10, 1992 2403 I - - . . I ' . - . I ' " ' I ' . . -

[ A I

0 0 2 0 4 0 6 0 8 1

Mole fraction of NaC

0 0.2 0.4 0.6 0.8 1

Mole fraction of NaDC

Figure 10. Calculated parameters P, B, and f from eqs 7,8, and 9 aa a function of the mole fraction of the bile salts (a) NaC; (b) NaDC.

in the CTAB/bile system (Figure 2) with increasing bile salt concentration is due to both the retreat of pyrene from the surface and the replacement of bromide as coun- terion by the bile salt anion. Assuming that the fraction of cationic micelle charges neutralized by 'bound" bromide ions is dBr, with the value for the CTAB micelle taken as 0.75,5a we have

1/7Br = 1/7hr + PkdB, (8) where indices Br and air refer to the lifetimes in CTAB and CTAC mixed micelles, respectively, and k is a rate coefficient, the value of which is 10.5 X 108 s-l and determined from the measurements without bile ealta. This assumption is an approximation; the rate coefficient could very well be dependent on the micelle structure. The values of P are taken from the calculations of the water effect. The dBr values calculated using eq 8 are given in Table I11 and Figure 10.

The Oxygen Effect. This presents another problem. It cannot a priori be assumed that pyrene in the interior of the micelles is perfectly shielded from oxygen. Normal micelles, such as SDS micelles, seem to give no protection whatsoever. The oxygen bimolecular quenching constant is approximately the same in SDS micelles as in water." To quantify the shielding effect, a shielding factor, f, is calculated from

1/7hr = 1/70 fk,q[o,laq (9) where 7hr is the measured lifetime in the air-saturated solution, TO the lifetime in the deaerated eolution, k, the

(58) Thalberg, K.; van Stam, J.; Lindblad, C.; Almqen, M.; Lindman, B. J. Phys. Chem. 1991,95, 8975.

2404 Langmuir, Vol. 3, No. 10, 1992

second-order quenching constant in water, and [ 0 2 I a q the concentration of oxygen in the air-saturated solution (taken as 2.6 X lo4 M at 25 "C and 1 atm pressure). From a calculated k,, of 1.22 X 10-lo M-' s-l assuming a shielding factor of 1 in pure CTAC micelles the results are given in Figure 10 and Table 111.

It is clear from Figure 10 and Table I11 that both bile salts give a similar and strong shielding effect toward quenching by oxygen. The fraction of pyrene exposed to water is also similar in both systems. NaDC gives better protection in the bile-rich micelles, probably due to the formation of larger micelles. The estimation of the fraction of Br- at the micelle surface is the least reliable of the quantities not only due to the small difference between measured lifetimes but also due to the factor P. It seems clear however that the bromide ions have better access to pyrene at the surface of the rodlike CTA+/NaDC micelles than at the small CTA+/NaC micelles. The 0~~ values cannot be regarded as estimates of bromide ion binding, however, since it appears very unlikely that mixed micelles containing, for instance, a mole fraction of 0.44 DC-, i.e. a mole ratio of 0.78, could still keep the surface concentration of Br- at 60% of that in pure CTAB micelles. This inconsistency probably means that the change of the water

Vethamuthu et al.

exposure, given by the factor P, is overestimated, or in other words that the increase of the pyrene fluorescence lifetime with the bile salt concentration is not solely due to the decreased water contact.

Conclusion The fluorescence quenching technique has been shown

to provide information regarding the aggregation behavior of the CTAX/bile salt mixed micelles. The transition from small monodispersed micelles to large aggregate could be observed as the mole fraction of the bile salt increases. This behavior was found to depend on the nature of the bile salt and temperature. Further work is underway in studying the large aggregate formed using the appropriate models for rodlike micelles and in accurately determining the phase behavior in the coacervation region.

Acknowledgment. This work has been supported by the Swedish Natural Science Research Council and The Knut and Alice Wallenberg Foundation.

Registry No. CTAB, 57-09-0; CTAC, 14002-56-3; NaC, 361- 09-1; NaDC, 302-95-4; NaCDC, 2646-38-0; DMBP, 54323-31-8; pyrene, 129-00-0.

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Fluorescence quenching studies of the aggregation behavior of the mixed micelles of bile salts and cetyltrimethylammonium halides