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Micelle formation and coacervation in mixtures of alkyltrimethylammonium bromides with di and trihydroxy bile salts
Micelle Formation and Coacervation in Mixtures of Alkyltrimethylammonium Bromides with Di and Trihydroxy Bile Salts B. W. B A R R Y AND G. M. T. G R A Y School of Pharmacy, Portsmouth Polytechnic, King Henry I Street, Portsmouth, U.K. Received December 16, 1974; accepted April 8, 1975 Coacervation of dihydroxy bile salts, sodium deoxycholate, and sodium chenodeoxycholate, in aqueous solutions of C12, C14, Cle homologous alkyltrimethylammonium bromides has been studied between 10 and 100°C. Coacervation occurred in mixtures containing cationic and anionic surfactant at close to eqnimolar amounts. Excess of either surfactant, or increasing temperature, tended to suppress coacervation. The areas of coacervation of sodium deoxycholate with the alkyltrimethylammonium bromides were larger than the corresponding areas of the sodium chenodeoxycholate mixtures. The interaction of tetradecyl-trimethylammonium bromide with the trihydroxy bile salt, sodium cholate, and the dihydroxy bile salt, sodium deoxycholate, in various mixtures, was studied by surface tension, conductivity, light scattering, and viscosity techniques at 25°C. The CMC values of the mixtures were much lower than those of the pure surfactants in water due to reduction of the unfavorable electrostatic interactions which tend to suppress micellization in the latter systems. The micelles in the sodium cholate-tetradecyl-trimethylammonium bromide mixtures were fairly small and approximately spherical, whereas those in the sodium deoxycholate-tetradecyl-trimethylammonium bromide mixtures grew large in solutions containing close to equimolar concentrations of the two surfactants. A mechanism for micelle growth in the latter system is proposed. INTRODUCTION I n a previous study (1) mixed micelle formation in a series of alkyltrimethylalnmoniuln cholates was investigated. The Inicelle consisted of a Inixture of long-chain cations and trihydroxy-substituted bile salt (steroidal) anions; it was small and approximately spherical and a model was proposed to account for the size and shape of the micelle. To investigate the effect of changing the composition on the size and shape of the micelle, the effect of altering the molar ratio of tetradecyl-trimethylamlnonium bromide to sodium cholate in the bulk solution was investigated b y surface tension conductivity, lightscattering, and viscosity techniques. I t was observed that approximately equimolar mixtures of the dihydroxy bile salts,
sodium deoxycholate and sodium chenodeoxycholate, with long-chain alkyltrimethylamInonium bromides separated into two liquid phases. Phase-separation diagrams for the mixtures of the dihydroxy bile salts with hexadecyl, tetradecyl, and dodecyl-trimethylammonium bromide were constructed. To examine further the differences between the dihydroxy and trihydroxy mixed systems, the effect of changing the molar ratio of tetradecyl-trimethylammoniuln bromide to sodium deoxycholate was also studied by Ineans of the techniques used for the trihydroxy systems. EXPERIMENTAL Materials Sodium cholate was described previously (1). Sodium chenodeoxycholate (sodium salt
327 Copyright O 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
328
B A R R Y A N D GRAY
of 3~, 7c~ dihydroxy-Sfl-cholanoic acid) was donated by Weddel Pharmaceuticals Ltd. (London). Sodium deoxycholate (sodium salt of 3a, 12~ dihydroxy-5fl-cholanoic acid) was obtained from Cambrian Chemicals Ltd. (Croydon, U.K.). It was recrystallized several times from methanol/acetone. The recrystallized deoxycholic acid and the chenodeoxycholic acid were analyzed by TLC (2-4). The deoxycholate contained less than 1% impurity, mainly chenodeoxycholate. Potentiometric titration showed that it contained 97.5-102.5% titratable groups. TLC showed that the chenodeoxycholate contained no detectable impurity. As the sample was used only in the phase-separation experiments, no further analysis was made. The molecular structure of each of the bile salts used is shown in Fig. 1. All bile salts were dried in vacuo at 100°C for 3-4 hr before use. The alkyltrimethylammonium bromides were as before (1, 5).
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Phase-Separation Diagrams These were constructed by titrating aqueous 200 raM1-1 alkyltrimethylammonium bromide solutions containing 25 raM1-1 of the appropriate bile salt against 25 raM1-1 aqueous solutions of either sodium deoxycholate or sodium chenodeoxycholate, maintained at various constant temperatures in a water bath. The concentration of bile salt was therefore unchanged throughout the titration. The volume of added solution at which persistent turbidity occurred was noted. The titration was then continued until the solution became clear and the volume added was noted again. Solutions were stirred with a magnetic stirrer and the temperature was controlled with a bead thermistor (calibrated against National Physical Laboratory thermometers) connected to a Wayne-Kerr Universal Bridge. To check results, the concentrations of reactants were maintained constant and the temperature was varied until the mixtures became turbid or cleared.
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Surface Tension Measurements
Surface tensions were measured at 25 -¢-0.1°C by the du Notiy method using a platinum ring. As reported previously (1), 10-20-min intervals were allowed between measurements on the same solution.
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FIG. 1. Molecular structures of the bile salts. (a) Sodium salt of 3a, 70~, 12¢z trihydroxy-Sfl-cholanoic acid (sodium cholate). (b) Sodium salt of 3a, 7~z dihydroxy5fl-cholanoic acid (sodium chenodeoxycholate). (c) Sodium salt of 3~x, 12~ dihydroxy-Sfl-cholauoic acid (sodium deoxycholate).
Conductivity Measurements
These were made at 25 -4- 0.02°C using the continuous-measuring technique reported previously (1). Turbidities, refractive index increments, and viscosities were determined as before (1). Stock solutions of bile salts, alkyltrimethylammonium bromides, and mixtures were prepared as w/v, and were diluted to the required concentration. The pH of the solutions was maintained between 8.5-9.5 with sodium hydroxide. After filtration of the solutions used in the light-scattering measurements and on completion of the viscosity determinations, the pH of a portion of each solution was remeasured. If the pH was less
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
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BILE SALT-CATIONIC M I X E D MICELLES
than 8.5, the results were discarded and the experiment was repeated. The water was distilled water passed through an Elgastat ion-exchange column and redistilled. It had a conductivity of 0.8-1.1 X 10-6 ohm -~ cm-1. RESULTS
The phase-separation diagrams for mixtures of dodecyl, tetradecyl, and hexadecyl-trimethylammonium bromides with sodium deoxycholate and sodium chenodeoxycholate are shown in Figs. 2a and 2b. Mixtures whose composition fell on or inside the boundaries of the curves were turbid. Light microscopy showed that the turbidity was caused by liquid droplets that were not visible in polarized light, indicating that they were isotropic. Such systems eventually separated into two distinct liquid phases. Phase separation was completely reversible. This behavior in colloidal systems is termed coacervation. Mixtures
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whose composition fell outside the boundaries of the curves were macroscopically homogeneous. It is evident that excess of either bile salt or alkyltrimethylammonium bromide suppressed coacervation; increasing the temperature also tended to suppress coacervation and each system exhibited a maximum temperature for coacervation, above which no coacervation occurred. Phase separation took place in solutions containing approximately equimolar concentrations of the cation and anion. Surface tension measurements on different molar mixtures of sodium cholate, or sodium deoxycholate, with tetradecyl-trimethylammonium bromide were plotted against the logarithm of concentration, expressed as total g m1-1 of bile salt and of tetradecyl-trimethylammonium bromide (Figs. 3 and 4). From these plots the CMC of each mixture was obtained assuming that the composition (i.e., ratio of anion to cation) of the micelles was equal to that of the bulk phase. The CMC values for the sodium cholate mixtures are
.
75
50[
50
25i
~s
20
I01 ~'5
C,rnMl-'
@
3'o \ x,20
2s
C,mMI-=
\~ 30
FIG. 2. Effect of concentration of alkyltrimethylammonium bromide, C, millimole/liter, on temperature of coacervafion of: (a) sodium deoxycholate-alkyltrimethylammonium bromide mixtures, and (b) sodium chenodeoxycholate-alkyltrimethylammonium bromide mixtures. Bile salt concentration in each case was 25 mmole/1. Alkyltrimethylammonium bromides were, m; hexadecyl-trimethylammonium bromide, e ; tetradecyl-trimethylammonium bromide; A , dodecyl-trimethylammonium bromide, in both cases. Coacervated mixtures were those which had a composition represented by points inside the phase-separation boundaries. Journal of Colloid and Interface Science, Vol. 52, No. 2, August 197S
330
BARRY AND GRAY
i
55
45[
40. ~
- 4"0
-5"0
-4'-0
'
Log(c,g ml -~) FIG. 3. Plots of surface tension, % dyne cm-~, versus logarithm of total concentration expressed in g m1-1, log C, for molar mixtures of sodium cholate and tetradecyl-trimethylammonium bromide, in water at 25°C. The plots have been separated for clarity. Mole ratio of sodium cholate to tetradecyltrimethylammonium bromide was: m, 4:1; Ak, 2:1; O, 1:1; O, 1:2; ~., 1:4; D, 1:7; v, 1:10.
given in Table I, those for sodium deoxycholate are listed in Table II. Conductivity measurements suggested CMC values of the same order as those determined from surface tension data. However, as the sodium and bromide ions have conductances far exceeding those of the surfactant ions,
they tended to obscure the exact position of the break in the specific conductivity versus concentration curve. The portion of the plots above the CMC was slightly curved, further obscuring the break. Conductivity CMC values are therefore not reported. Refractive index increments for the sodium
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4.=
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5
==
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=
35 -5.0
J -4'0
_-
-5-0 Log. (C,g ml-t)
-4"0
FIG. 4. Plots of surface tension, % dyne cm7 z, versus logarithm of total concentration expressed in g ml-z, log C, for molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide, in water at 25°C. The plots have been separated for clarity. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: , , 4:1; A , 3:1; v , 12:5; •, 2:1; O, 1:2; V, 5:12; /k, 1:3; [~, 1:4; X, I:10. Journal of Colloid and Interface Science, Vol. ,52, No. 2, A u g u s t 1975
BILE SALT-CATIONIC MIXED MICELLES TABLE I CMC, REFRACTIVE II~rDEX INCREMENTS, dn/dc, MICELLE MOLECULAR WEIGHTS, MMW, ASrD APPARENT INTRINSIC VISCOSITIES En]' o~ THE MICELLES IN MOLAR MIXTURES O1~ SODIUM: CHOLATE, NaG, WITH TETRADECYL-TRIMETHYLAM~IONIUM BROMIDE,
TTAB, IN WATER AT 25°C System
NaC/I-I20 mole ratio of NaC: TTAB 4:1 2:1 1:1 1:2 1:4 1:7 1:10 TTAB/H20
CMC (g m1-1 X 109
dn/dc (ml g-l)
MMW ( X 10-~)
[7]' (ml g-9
51.7~
--
approx 1a
3.9
1.39 1.18 1.00 0.91 1.13 1.38 1.60 11.1 b
0.186 0.182 0.176 0.169 0.164 0.158 0.167 --
8.6 12.3 14.6 16.1 13.9 16.8 16.4 27.3 b
3.7 3.2 3.2 4.0 5.0 -6.6 7.7
a Value taken from Ref. (14). b Value taken from Ref. (5). cholate mixtures are shown in T a b l e I ; those for the sodium deoxycholate mixtures are in T a b l e I I . Turbidities of the mixtures were treated according to D e b y e (6), assuming t h a t micellar and bulk compositions were the same. T h e t u r b i d i t y of the solvent was assumed equal to the t u r b i d i t y of a solution at the C M C and this was used as ro in the D e b y e plots. Where the plots were linear, they were treated b y the m e t h o d of least squares to obtain the intercept. Curved plots were extrapolated b y eye. D e b y e plots for the sodium cholatet e t r a d e c y l - t r i m e t h y l a m m o n i u m bromide mixtures are shown in Fig. 5; resultant micelle molecular weights are in T a b l e I. Corresponding plots for sodium deoxycholate systems are illustrated in Fig. 6; micelle molecular weights are in T a b l e I I . A p p a r e n t intrinsic viscosities were calculated from the equation: E ~ ] ' = {E(t -
to)/to]/(c
-
Co)]cC-~o)~O,
Eli
where t is the flow time of the solution; to is the flow time of the solvent; C is the total concentration of surfactant; and Co is the
331
concentration of surfactant a t the CMC. As shown previously (1), this equation gives values for the intrinsic viscosity t h a t are within experimental error of the values calculated using viscosities instead of flow times without determining the density of each solution. I n several of the systems containing tetrad e c y l - t r i m e t h y l a m m o n i u m bromide a t concentrations below the C M C of the system, flow times were high and variable. Close to, and just below, the C M C flow times decreased, reaching a m i n i m u m a t concentrations slightly greater than the CMC. W i t h further increase in concentration, flow times increased linearly. T y u z y o observed similar behavior in solutions of dodecylamine hydrochloride in water (7). F o r the present case, in solutions in which the mole ratio of sodium deoxycholate to tetrad e c y l - t r i m e t h y l a m m o n i u m bromide was greater than or equal to 1:2 and in solutions of the q u a t e r n a r y a m m o n i u m bromide in 0.15 M N a B r , this effect was suppressed. However, sodium cholate mixtures all exhibited anomaTABLE II CMC, REFRACTIVEI~EX INCREMENTS,dn/dc, MICELLE MOLECULAR WEIGHTS, MMW, ASr3
APPARENT INTRINSICVISCOSITIESEV]' Or THE MICELLES IN MOLAR MIXTURES OF
SODIU~ DEOX¥CHOLATE, NaDC, WlTI~ TETRADECYL-TRIMETHYLAMMONIUM
BROmDE, TTAB, IN WATER AT 25°C System
NaDC/H20 mole ratio of NaD C: TTAB 4:1 3:1 12:5 2:1 1:2 5:12 1:3 1:4 1:10
CMC (g ml-I x lo9
dn/dc (ml g-0
20.7~
--
0.69 0.60 0.53 0.52 0.54 0.56 0.57 0.67 0.73
0.187 0.183 0.183 0.181 0.166 0.167 0.165 0.161 0.157
MMW ( X 10-3)
[7]' (ml g-0
approx 2~ 6.1
14.8 17.6 65.5 110.0 50.0 37.0 16.3 17.7 19.6
4.9 5.1 -4.7 5.1 -4.8 5.1 --
Value taken from Ref. (14).
Journal of Colloid and Interface Science, VoL 52, No. 2, August 1975
332
BARRY AND GRAY 15
10
2:5
~o
7:s
x
o~
is I
10
|
•
•
Sodium cholate, however, did not have this effect. In addition, large concentrations of inorganic salt will tend to shield the silicate ions from the cationic ions, thus preventing adsorption of the latter onto the glass. For systems in which anomalous flow times were observed, the value of to was taken as the minimum in the curve of flow time versus concentration. For convenience, Co was taken as the concentration at which the minimum flow time occurred. Flow times of solutions, and to, used in computation of apparent intrinsic viscosities were reproducible within 4-0.05 sec. Plots of [ ( t - to)/to]/(C- Co) versus C - Co are shown in Figs. 7 and 8, assuming that the composition of the micelles and bulk solution was identical. The plots were
•
20
s~
2:s
g.o
72s
(C-Co) x 10~, gml-'
FIG. 5. Debye plots of molar mixtures of sodium
15
cholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium cholate to tetradecyl-trimethylammonium bromide was: II, 4:1 ; A, 2:1; Q, 1:1; O, 1:2; A, 1:4; V, 1:7; E3, 1:10.
1
lous flow times at concentrations below the CMC. Solutions of sodium cholate, or sodium deoxycholate, alone did not exhibit this effect. Davies and Rideal (8) have shown that glass is nonwetted by low concentrations of cationic detergents, due to the adsorption of the detergent onto the negatively charged silicate ions of the glass. Thus, a hydrophobic layer m a y be formed on the glass of the viscometer capillary, obstructing the flow of aqueous solutions. This effect is not observed with anionic detergents. At higher concentrations of the cationic detergent the surface tension is lowered and a hydrophilic double layer may be built up until a concentration is reached at which complete wetting of the glass results. In solutions with an excess of sodium deoxycholate it seems that interaction of the bile salt with the cationic prevents interaction of the latter with the silicate ions of the glass.
x
215
520
7-5
(C-Col n 10 3, gmV'
FIG. 6. Debye plots of molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: I , 4:1; A , 3:1; V , 12:5; O, 2:1; O, 1:2; V , 5:12; A, 1:3; [], 1:4; X, 1:10.
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
BILE SALT-CATIONIC MIXED MICELLES
333
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st_
6.5
T O)
o
o, 5.5 []
[]
~
o
0
[]
o "7
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o
w
A
•
w
2'.0
~
4'.0
¢,
a
6'.o
s'.o
( c - c o ) x l O ~, g m1-1
FIG. 7. Plots of apparent reduced viscosity, F(t-to)/to-l/(C-Co), ml g-l, versus micellar concentration, ( C - C o ) , g ml -z, for molar mixtures of sodium cholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium cholate to tetradecyl-trimethylammonium bromide was : • , 4:1 ; • , 2:1 ; • , 1 : 1 ; A , 1 : 2; [], 1 : 4; y , 1 : 10; O , sodium cholate alone; V, tetradecyl-trimethyla m m o n i u m bromide alone.
treated by the least-squares method to obtain apparent intrinsic viscosities at infinite dilution of the micelles. The resultant values of apparent intrinsic viscosity are shown in Tables I and II. DISCUSSION
As aggregation numbers and solubilities of dihydroxy bile salts are extremely variable below pH 7-8 (9), all solutions were adjusted to pH 8.5-9.5. Even at this pH, however, mixtures of dihydroxy bile salts with the alkyltrimethylammonium bromides coacervated at close to equimolar mixing ratios as shown in
Fig. 1, whereas mixtures of the trihydroxy compound, sodium cholate with the cationic surfactants did not. The phase-separation diagrams show that the areas of coacervation of the 3a, 7a dihydroxy-compound, sodium chenodeoxycholate, with the three homologous cationic compounds are smaller than the corresponding areas of coacervation of the 3a, 12a dihydroxy-compound, sodium deoxycholate. This is consistent with the observation that sodium chenodeoxycholate has a higher CMC than sodium deoxycholate (10). Carey and Small attributed this to the 7a-hydroxyl group being a better hydrophilic group than
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
334
BARRY AND GRAY
v ~
v
V.VV
V
v
~
V
6"0
5"0
4"0
?
2:0
4:0
6!0
8~0
6:0
8~0
6.0
u
5"0 #,
0
2:0
~
±
A
A A
4!0 (c-cs),10]gml -L
F~G. 8. Plots of apparent reduced viscosity, [-(t -- t o ) / t o ~ / ( C -- Co), ml g-l, versus micellar concentration, (C -- Co), g m1-1, for molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: II, 4:1; A , 3:1; e , 2:1; O, 1:2; /x, 1:3; [~, 1:4; V , sodium deoxycholate alone.
the 12a-hydroxyl group (11). The phaseseparation studies described above indicate, therefore, that the position, as well as the number, of hydroxyl groups on the steroid nucleus is instrumental in establishing the solubility of the mixed micelles in water. It was hoped that light-scattering and viscosity studies of the sodium cholate-cationic and sodium deoxycholate-cationic mixed micelles would give an insight into the mechanisms of interaction in the two systems. To estimate the CMC values and to calculate the micelle molecular weights and intrinsic viscosities, it was assumed that the composition of the micelle was identical to the composition of the bulk phase. A number of workers have attempted to measure the exact composi-
tion of mixed micelles (12, 13). However, for the present systems, it was considered reasonable to assume identical composition of micellar and bulk phases. First, sodium deoxycholate and the sample of tetradecyltrimethylammonium bromide used have CMC values in water of 3-5 mmole 1-1 (14) and 3.4 mmole 1-1 (5), respectively, i.e., the tendency of these compounds to micellize is almost identical. The CMC of sodium cholate in water is of the order of 10-12 mmole 1-1 (14), which does not differ greatly from*that of the cationic. Second, an excess of ions*of one kind in the micelle or bulk phase would tend to attract ions of opposite charge into the phase, thus leveling the tendency of both surface active ions to micellize. This behavior would be especially true at close to 1 : 1 mixing ratios. Third, as comparative results were the main aim of these experiments, it was considered that small deviations from the assumption of identical composition of micellar and bulk phases could be tolerated. The CMC values thus found for the mixtures (Tables I and II) are much lower than the CMC values of the pure compounds in water. This is attributed to a significant decrease in the unfavorable electrostatic interactions which tend to suppress micellization in homoionic surfactant systems. In Fig. 9, the CMC values are plotted versus mole percent of tetradecyl-trimethylammonium bromide. For the sodium cholate-quaternary ammonium bromide mixtures, the CMC curve is approximately symmetrical about a mixing ratio of cationic to bile salt of slightly greater than one. For the sodium deoxycholatequaternary ammonium bromide mixtures the CMC curve is almost symmetrical about the 1:1 molar mixing ratio. This lends further support to the validity of the assumption that the tendency of both the anionic and cationic surfactive ions to form micelles is similar, and hence, that the bulk and micellar phases have similar compositions. The CMC values in sodium deoxycholate mixtures are approximately one-half those of the corresponding sodium cholate mixtures.
Journal of Colloid and Inlerface Science. Vol. 52, No. 2, AugusC1975
335
BILE SALT-CATIONIC MIXED MICELLES 2"0
/ /
\ 1.5
Tm E *~
1"0
\
x
o
/
0"5
2~
go
71
loo
Mole Percent TTAB
FIG. 9. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on critical micelle concentrations, CMC, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. O, sodium cholate-tetradecyl-trimethylammonium bromide mixtures, I t , sodium deoxycholate-tetradecyl-trimethylammoniumbromide mixtures.
Micelle molecular weights, for a twocomponent system, are usually obtained using the equation given by Debye (6) : H(C
-- Co)/(r
- - to)
= 1/~
+ 2 B ( c - co),
[-23
where H is a constant; C and r are the concentration and turbidity of the bulk phase; Co and ro are the values at the CMC; M is the molecular weight; and B is the second virial coefficient, which describes the extent of interaction between the scattering particles. In these cases, a plot of I t ( C - C o ) / ( r - to) versus C - - Co yields a straight line with slope 2B and intercept (infinite dilution of the micelles) equal to the reciprocal of micelle molecular weight. However, where the third, and higher, virial coefficients are important, i.e., when interaction between the micelles is large, or where other nonideal behavior is present, the plots may be curved. Plots of this nature were observed by Anacker (15) in studies of the light scattered by micelles formed from surfactants of double long-chain salts. This is also the case in mixtures of sodium deoxycholate with tetradecyl-trimethylammonium bromide close to the phase-sepa-
ration point (Fig. 6), where interaction is expected to be large. The calculation of micelle molecular weights for these systems, therefore becomes more uncertain. However, with the emphasis on comparison rather than on absolute values, a plot of micelle molecular weights versus mole percent of tetradecyl-trimethylammonium bromide, as shown in Fig. 10, demonstrates a major difference between the sodium cholate and the sodium deoxycholate mixed systems. While the micelles in the former systems are always relatively small, the micelles in the deoxycholate mixed systems close to the phaseseparation point are large and grow exponentially on approaching the phase-separation point. The apparent intrinsic viscosities of the mixed micelles support the conclusions drawn from the light scattering experiments. A plot of apparent intrinsic viscosities versus mole percent of tetradecyl-trimethylammonium bromide is shown in Fig. 11. The sodium cholatetetradecyl-trimethylammonium bromide plot shows a minimum value at close to an equimolar mixture of anionic and cationic detergents. With excess of either detergent the
Journal of Colloid and Interface Science.
Vol. 52, No. 2, August 1975
336
BARRY AND GRAY
,,o[ loo~ \
76
\ \
.50
o x
2~
o
2'5
~o
7~
loo
Mole Percent TTAB
Fro. 10. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on micelle molecular weights, MMW, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. 0 , sodium cholate-tetradecyl-trimethylammonium bromide mixtures, B, sodium deoxycholate-tetradecyl-trimethylammonium bromide mixtures.
intrinsic viscosities of the micelles increase. This effect is attributed to an increased micellar charge as excess of either of the micellized ions increases. An electroviscous effect, caused by interaction between the double layers of the charged micelles, results (16). As has been shown previously (1), this effect is absent in equimolar solutions of sodium cholate and tetradecyl-trimethylam-
monium bromide in water; the value of 3.2 ml g-1 for this system suggests that the micelles are approximately spherical with a small degree of hydration. The data for the sodium deoxycholatetetradecyl-trimethylammonium bromide mixed micelles suggest that the micelles, although not spherical, are not very asymmetric. The light-scattering and viscosity studies,
9.0
7~
5"(
o
2~
5b
7~
loo
Mole Percent TTAB
FIG. 11. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on apparent intrinsic viscosities of the micelles [~]', ml g-l, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. O, sodium cholate-tetradecyl-trimethylammonium bromide mixtures, B, sodium deoxycholate-tetradecyl-trimethylammonium bromide mixtures. Journal of Colloid and Interface Science, Vol. 52, No. 2, August. 1975
BILE SALT-CATIONIC MIXED MICELLES
337
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Fio. 12. (a) Proposed shape of a]kyltrimethylammoniumcholate micelle. (b), (c), (d) Possible mechanisms of micellegrowth in alkyltrimethylammoniumbromide-dihydroxybile salt mixtures. therefore, show that phase separation in the sodium deoxycholate-tetradecyl-trimethylammonium bromide mixed systems is caused by growth of the mixed micelles to a large size, with a resultant separation of the Inicelles into a colloid-rich phase, the coacervate (17, 18). Phase separation in the sodium chenodeoxycholate-tetradecyl-trimethylammonium bromide systems is probably the result of a similar mechanism. The light scattering and viscosity studies also support the phase-separation studies in demonstrating the importance of the hydroxyl groups in determining the shape and size of the mixed micelles. The authors have previously proposed what they believe is the most probable form of the alkyltrimethylammonium cholate micelle (1). For convenience, this is reproduced in Fig. 12a. This micelle remains relatively small to minimize the unfavorable coulombic interactions of the quaternary ammonium groups at the surface of the micelle. Although essentially electro-neutral, it is maintained in aqueous solution by hydrogen bonding between the hydroxyl groups on the hydrophilic face of the steroid nucleus and the surrounding water molecules. The importance of the
steroidal hydroxyl groups, as demonstrated by the studies reported above, support the suggested model of the micelle. If these groups were in the interior of the micelle they would have no effect on water solubility and probably little effect on micelle size. Assuming, then, that the fundamental micelle is as shown in Fig. 12a, growth of the dihydroxy bile salt-tetradecyl-trimethylammonium bromide mixed micelles may be attributed to three possible mechanisms: (i) The micelle may expand, resulting in the bimolecular leaflet becoming very large (short cylindrical shape), as shown in Fig. 12b; (if) the small fundamental micelles may interact with each other through the opposite charges on the micelle surface, forming chain-like aggregates (rod shape), as depicted in Fig. 12c; or (iii) aggregation of the small micelles may occur because of hydrophobic interactions between the portions of the water-facing side of the steroid nucleus not covered by hydroxyl groups. In solutions that contain approximately equimolar concentrations of dihydroxy bile salt and alkyltrimethylammonium bromide, and therefore where the micelle is effectively electro-neutral, a three-dimensional
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 197S
338
BARRY AND GRAY
aggregate similar to that shown in Fig. 12d may be formed. Mechanism (i) is unlikely because a large number of quaternary ammonium groups would be concentrated in the center of the surface of the micelle, causing a high electrical free energy at this point. Such a micelle would also be very asymmetric and the viscosity studies show that this is not the case. Mechanism (ii) would also lead to asymmetric aggregates. Of equal importance is that neither mechanism (i) nor (ii) explains the differences observed between trihydroxy and dihydroxy systems. The fundamental mixed micelle shown in Fig. 12a is approximately spherical (1) and aggregation by mechanism (iii) would form a nearly spherical agglomerate. Furthermore, aggregation by mechanism (iii) may account for the dependence of micelle size on the number and position of the hydroxyl groups on the steroid nucleus. The sodium cholate molecule contains three hydroxyl groups which are regularly-spaced over the water-facing hydrophilic side of the steroid nucleus, thus preventing aggregation of the micelles by mechanism (iii). Examination of molecular models shows that the hydroxyl groups of the 3a, 12a dihydroxy compound (sodium deoxycholate) are situated along the same side of the hydrophilic face of the steroid nucleus in such a manner as to allow hydrophobic bonding between adjacent micelles. However, the hydroxyl groups of the 3a, 7a dihydroxy compound (sodium chenodeoxycholate) are situated on opposite sides of the hydrophilic face of the steroid nucleus, thus decreasing the possibility of hydrophobic bonding between adjacent micelles, although not completely preventing it. In this way, the differences in solubilities and aggregation numbers between alkyltrimethylammonium bromide-bile salt mixed systems, in which the number and position of the hydroxyl groups on the steroid nucleus are different, may be simply explained. Small (9) attributed the differences in aggregation numbers of dihydroxy and trihydroxy bile salt micelles in aqueous salt solutions to dehydration of the weaker nonionic moeity of the dihydroxy bile salts by
inorganic salts, resulting in hydrogen bonding between the hydroxyl groups of adjacent dihydroxy bile salt micelles. For the mixed surfactant systems described above, this would lead to an aggregate of micelles identical to that formed by mechanism (iii) above. However, in our systems, no inorganic salt, other than the counter-ions of the surfactants, was added and salting-out of the hydroxyl groups in this case is unlikely. It is possible, however, that hydrogen bonding between hydroxyl groups on adjacent micelles in the interior of alkyltrimethylammonium-dihydroxy bile salt agglomerates occurs as a result of aggregation of the basic micelles. The aggregate shown in Fig. 12d would therefore be much more stable than that illustrated in Fig. 12c because of the additional hydrophobic and hydrogen-bonding modes. If we consider the possibility that the mixed micelle is of a more usual long-chain paraffin salt shape, with the bile salt molecules interdigitated between the paraffin chains, we see that this model could not explain the differences in behavior between dihydroxy and trihydroxy-mixed systems at concentrations close to the phase-separation point. Furthermore, systems in which long-chain paraffin salt-type micelles are present, and which are close to phase-separation boundaries, tend to become viscoelastic due to formation of asymmetric micelles (19, 20). No viscoelasticity was observed in the present systems. The following conclusions may therefore be drawn for the sodium cholate-tetradecyltrimethylammonium bromide mixed micelles in water. In mixtures in which there is no large excess of the quaternary ammonium bromide the mixed micelles are similar to that proposed for the alkyltrimethylammonium cholates, shown in Fig. 12a. Aggregation of these micelles does not occur. Elementary calculations, based on the micelle molecular weights and assuming 6-8 A for the crosssectional diameter of the sodium cholate molecule (14) and 40 A2 for the area occupied by each quaternary ammonium group (21) at the micelle surface, show that the maximum ratio of cationic to bile salt that will allow a
Journal of Colloid and Interface Science, Vol. 52, No. 2, A u g u s t 1975
BILE SALT-CATIONIC MIXED MICELLES complete circumference of bile salt molecules is about 2:1. At higher cationic to bile salt ratios, however, it is not evident from the present data whether there is a mixture of micelles, i.e., mixed micelles as shown in Fig. 12a, with the remainder of the tetradecyltrimethylammonium bromide forming normal long-chain paraffin salt micelles, or whether the mixed micelle assumes this latter form, with the bile salt molecules interposed between the hydrocarbon chains, perhaps with hydrogen-bonding between pairs of bile salt molecules, as suggested by Dervichian for the bile salt-lecithin mixed micelle in organic solvents (22). The conclusions derived from the data for the sodium deoxycholate-tetradecyl-trimethyla m m o n i u m bromide mixed systems are similar to those obtained for the system just described, except that, in the former case, as the micelles approach the situation where there is an equal number of positive and negative ions in the micelle, i.e., the fundamental micelles are approaching electro-neutrality and are therefore less likely to repel each other through electrostatic interactions, aggregation of these micelles occurs primarily through hydrophobic bonding, and then through hydrogen and electrostatic bonding between adjacent micelles. This finally causes coacervation. REFERENCES 1. BARRY, B. W., Aim GRAY, G. M. T., J. Colloid Interface Sci., 52, 314 (1975).
339
2. HOFMANN, A. F., in "New Biochemical Separations" (L. ]. Morris and A. T. James, Eds.), p. 261. Van Nostrand, London, 1964. 3. SUNDARAM,G. S., AND SODttI, H. S., J. Chromatogr.
61, 370 (1971). 4. SUNDARAM,G. S., SINGH, H., AXn) SODHI, H. S., Clin. Chim. Acta 34, 425 (1971). 5. BARRY, B. W., AND RUSSELL, G. F. ]., J. Colloid Interface Sci. 40, 174 (1972). 6. DEBX% P., Ann. N. Y. Acad. Sci. 51, 575 (1949). 7. TY~zYo, K., Kolloid-Z. 175, 40 (1960).
8. DAVIES, J. T., AXD RII)EAL, E. K., "Interfacial Phenomena," p. 435. Academic Press, New York, 1963. 9. S~ALL,D. M., Advan. Chem. Ser. 84, 31 (1968). 10. HOFMANN,A. F., Biochem. Y. 89, 57 (1963). 11. CAI~Y, M. C., Aim SMALL, D. M., Arch. Intern. Med. 130, 506 (1972). 12. MYSELS,K. J., ANDOTTER,R. J., J. Colloid Sci. 16, 462 (1961). 13. SHANKLAND, W., J. Colloid Interface Sci. 34, 9 (1970). 14. SMALL,D. 1VL,in "The Bile Acids" (P. P. Nair and D. Kritchevsky, Eds.), Vol. 1, p. 249. Plenum Press, New York, 1971. 15. ANACKER,E. W., J. Colloid Sci. 8, 402 (1953). 16. BOOTH,F., Proc. Roy. Soc., Set. A. 203, 533 (1950). 17. COHEN,I., ANDVASSILIADES,T., J. Phys. Chem. 65,
1774 (1961). 18. BARRY,B. W., AND GRAY, G. M. T., J. Pharm. Sci.
63, 548 (1974). 19. COHEN,I., ANDVASSlLIADES,T., J. Phys. Chem. 65, 1781 (1961). 20. BARKER, C. A., SAUL, D., TIDDY, G. J. T., WHEELER, B. A., Aim WILLIS, E., J. Chem. Soc., Faraday Trans. I 70, 154 (1974). 21. WEINER, N. D., Aim ZOORAFI,G., J. Pharm. Sci.
54, 436 (1965). 22. DERVICHIAN,D. G., Advan. Chem. Ser. 84, 78 (1968).
Journal of Colloid and Interface Science. Vol. 52, No. 2, August 1975
sodium deoxycholate and sodium chenodeoxycholate, with long-chain alkyltrimethylamInonium bromides separated into two liquid phases. Phase-separation diagrams for the mixtures of the dihydroxy bile salts with hexadecyl, tetradecyl, and dodecyl-trimethylammonium bromide were constructed. To examine further the differences between the dihydroxy and trihydroxy mixed systems, the effect of changing the molar ratio of tetradecyl-trimethylammoniuln bromide to sodium deoxycholate was also studied by Ineans of the techniques used for the trihydroxy systems. EXPERIMENTAL Materials Sodium cholate was described previously (1). Sodium chenodeoxycholate (sodium salt
327 Copyright O 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
328
B A R R Y A N D GRAY
of 3~, 7c~ dihydroxy-Sfl-cholanoic acid) was donated by Weddel Pharmaceuticals Ltd. (London). Sodium deoxycholate (sodium salt of 3a, 12~ dihydroxy-5fl-cholanoic acid) was obtained from Cambrian Chemicals Ltd. (Croydon, U.K.). It was recrystallized several times from methanol/acetone. The recrystallized deoxycholic acid and the chenodeoxycholic acid were analyzed by TLC (2-4). The deoxycholate contained less than 1% impurity, mainly chenodeoxycholate. Potentiometric titration showed that it contained 97.5-102.5% titratable groups. TLC showed that the chenodeoxycholate contained no detectable impurity. As the sample was used only in the phase-separation experiments, no further analysis was made. The molecular structure of each of the bile salts used is shown in Fig. 1. All bile salts were dried in vacuo at 100°C for 3-4 hr before use. The alkyltrimethylammonium bromides were as before (1, 5).
®
Phase-Separation Diagrams These were constructed by titrating aqueous 200 raM1-1 alkyltrimethylammonium bromide solutions containing 25 raM1-1 of the appropriate bile salt against 25 raM1-1 aqueous solutions of either sodium deoxycholate or sodium chenodeoxycholate, maintained at various constant temperatures in a water bath. The concentration of bile salt was therefore unchanged throughout the titration. The volume of added solution at which persistent turbidity occurred was noted. The titration was then continued until the solution became clear and the volume added was noted again. Solutions were stirred with a magnetic stirrer and the temperature was controlled with a bead thermistor (calibrated against National Physical Laboratory thermometers) connected to a Wayne-Kerr Universal Bridge. To check results, the concentrations of reactants were maintained constant and the temperature was varied until the mixtures became turbid or cleared.
HO~COONa
Surface Tension Measurements
Surface tensions were measured at 25 -¢-0.1°C by the du Notiy method using a platinum ring. As reported previously (1), 10-20-min intervals were allowed between measurements on the same solution.
®
©
HO~COONa H~COONa
FIG. 1. Molecular structures of the bile salts. (a) Sodium salt of 3a, 70~, 12¢z trihydroxy-Sfl-cholanoic acid (sodium cholate). (b) Sodium salt of 3a, 7~z dihydroxy5fl-cholanoic acid (sodium chenodeoxycholate). (c) Sodium salt of 3~x, 12~ dihydroxy-Sfl-cholauoic acid (sodium deoxycholate).
Conductivity Measurements
These were made at 25 -4- 0.02°C using the continuous-measuring technique reported previously (1). Turbidities, refractive index increments, and viscosities were determined as before (1). Stock solutions of bile salts, alkyltrimethylammonium bromides, and mixtures were prepared as w/v, and were diluted to the required concentration. The pH of the solutions was maintained between 8.5-9.5 with sodium hydroxide. After filtration of the solutions used in the light-scattering measurements and on completion of the viscosity determinations, the pH of a portion of each solution was remeasured. If the pH was less
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
329
BILE SALT-CATIONIC M I X E D MICELLES
than 8.5, the results were discarded and the experiment was repeated. The water was distilled water passed through an Elgastat ion-exchange column and redistilled. It had a conductivity of 0.8-1.1 X 10-6 ohm -~ cm-1. RESULTS
The phase-separation diagrams for mixtures of dodecyl, tetradecyl, and hexadecyl-trimethylammonium bromides with sodium deoxycholate and sodium chenodeoxycholate are shown in Figs. 2a and 2b. Mixtures whose composition fell on or inside the boundaries of the curves were turbid. Light microscopy showed that the turbidity was caused by liquid droplets that were not visible in polarized light, indicating that they were isotropic. Such systems eventually separated into two distinct liquid phases. Phase separation was completely reversible. This behavior in colloidal systems is termed coacervation. Mixtures
1001
100
®
751
i
whose composition fell outside the boundaries of the curves were macroscopically homogeneous. It is evident that excess of either bile salt or alkyltrimethylammonium bromide suppressed coacervation; increasing the temperature also tended to suppress coacervation and each system exhibited a maximum temperature for coacervation, above which no coacervation occurred. Phase separation took place in solutions containing approximately equimolar concentrations of the cation and anion. Surface tension measurements on different molar mixtures of sodium cholate, or sodium deoxycholate, with tetradecyl-trimethylammonium bromide were plotted against the logarithm of concentration, expressed as total g m1-1 of bile salt and of tetradecyl-trimethylammonium bromide (Figs. 3 and 4). From these plots the CMC of each mixture was obtained assuming that the composition (i.e., ratio of anion to cation) of the micelles was equal to that of the bulk phase. The CMC values for the sodium cholate mixtures are
.
75
50[
50
25i
~s
20
I01 ~'5
C,rnMl-'
@
3'o \ x,20
2s
C,mMI-=
\~ 30
FIG. 2. Effect of concentration of alkyltrimethylammonium bromide, C, millimole/liter, on temperature of coacervafion of: (a) sodium deoxycholate-alkyltrimethylammonium bromide mixtures, and (b) sodium chenodeoxycholate-alkyltrimethylammonium bromide mixtures. Bile salt concentration in each case was 25 mmole/1. Alkyltrimethylammonium bromides were, m; hexadecyl-trimethylammonium bromide, e ; tetradecyl-trimethylammonium bromide; A , dodecyl-trimethylammonium bromide, in both cases. Coacervated mixtures were those which had a composition represented by points inside the phase-separation boundaries. Journal of Colloid and Interface Science, Vol. 52, No. 2, August 197S
330
BARRY AND GRAY
i
55
45[
40. ~
- 4"0
-5"0
-4'-0
'
Log(c,g ml -~) FIG. 3. Plots of surface tension, % dyne cm-~, versus logarithm of total concentration expressed in g m1-1, log C, for molar mixtures of sodium cholate and tetradecyl-trimethylammonium bromide, in water at 25°C. The plots have been separated for clarity. Mole ratio of sodium cholate to tetradecyltrimethylammonium bromide was: m, 4:1; Ak, 2:1; O, 1:1; O, 1:2; ~., 1:4; D, 1:7; v, 1:10.
given in Table I, those for sodium deoxycholate are listed in Table II. Conductivity measurements suggested CMC values of the same order as those determined from surface tension data. However, as the sodium and bromide ions have conductances far exceeding those of the surfactant ions,
they tended to obscure the exact position of the break in the specific conductivity versus concentration curve. The portion of the plots above the CMC was slightly curved, further obscuring the break. Conductivity CMC values are therefore not reported. Refractive index increments for the sodium
5(
50
'v
45
4.=
4O
4(
5
==
>.:
=
35 -5.0
J -4'0
_-
-5-0 Log. (C,g ml-t)
-4"0
FIG. 4. Plots of surface tension, % dyne cm7 z, versus logarithm of total concentration expressed in g ml-z, log C, for molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide, in water at 25°C. The plots have been separated for clarity. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: , , 4:1; A , 3:1; v , 12:5; •, 2:1; O, 1:2; V, 5:12; /k, 1:3; [~, 1:4; X, I:10. Journal of Colloid and Interface Science, Vol. ,52, No. 2, A u g u s t 1975
BILE SALT-CATIONIC MIXED MICELLES TABLE I CMC, REFRACTIVE II~rDEX INCREMENTS, dn/dc, MICELLE MOLECULAR WEIGHTS, MMW, ASrD APPARENT INTRINSIC VISCOSITIES En]' o~ THE MICELLES IN MOLAR MIXTURES O1~ SODIUM: CHOLATE, NaG, WITH TETRADECYL-TRIMETHYLAM~IONIUM BROMIDE,
TTAB, IN WATER AT 25°C System
NaC/I-I20 mole ratio of NaC: TTAB 4:1 2:1 1:1 1:2 1:4 1:7 1:10 TTAB/H20
CMC (g m1-1 X 109
dn/dc (ml g-l)
MMW ( X 10-~)
[7]' (ml g-9
51.7~
--
approx 1a
3.9
1.39 1.18 1.00 0.91 1.13 1.38 1.60 11.1 b
0.186 0.182 0.176 0.169 0.164 0.158 0.167 --
8.6 12.3 14.6 16.1 13.9 16.8 16.4 27.3 b
3.7 3.2 3.2 4.0 5.0 -6.6 7.7
a Value taken from Ref. (14). b Value taken from Ref. (5). cholate mixtures are shown in T a b l e I ; those for the sodium deoxycholate mixtures are in T a b l e I I . Turbidities of the mixtures were treated according to D e b y e (6), assuming t h a t micellar and bulk compositions were the same. T h e t u r b i d i t y of the solvent was assumed equal to the t u r b i d i t y of a solution at the C M C and this was used as ro in the D e b y e plots. Where the plots were linear, they were treated b y the m e t h o d of least squares to obtain the intercept. Curved plots were extrapolated b y eye. D e b y e plots for the sodium cholatet e t r a d e c y l - t r i m e t h y l a m m o n i u m bromide mixtures are shown in Fig. 5; resultant micelle molecular weights are in T a b l e I. Corresponding plots for sodium deoxycholate systems are illustrated in Fig. 6; micelle molecular weights are in T a b l e I I . A p p a r e n t intrinsic viscosities were calculated from the equation: E ~ ] ' = {E(t -
to)/to]/(c
-
Co)]cC-~o)~O,
Eli
where t is the flow time of the solution; to is the flow time of the solvent; C is the total concentration of surfactant; and Co is the
331
concentration of surfactant a t the CMC. As shown previously (1), this equation gives values for the intrinsic viscosity t h a t are within experimental error of the values calculated using viscosities instead of flow times without determining the density of each solution. I n several of the systems containing tetrad e c y l - t r i m e t h y l a m m o n i u m bromide a t concentrations below the C M C of the system, flow times were high and variable. Close to, and just below, the C M C flow times decreased, reaching a m i n i m u m a t concentrations slightly greater than the CMC. W i t h further increase in concentration, flow times increased linearly. T y u z y o observed similar behavior in solutions of dodecylamine hydrochloride in water (7). F o r the present case, in solutions in which the mole ratio of sodium deoxycholate to tetrad e c y l - t r i m e t h y l a m m o n i u m bromide was greater than or equal to 1:2 and in solutions of the q u a t e r n a r y a m m o n i u m bromide in 0.15 M N a B r , this effect was suppressed. However, sodium cholate mixtures all exhibited anomaTABLE II CMC, REFRACTIVEI~EX INCREMENTS,dn/dc, MICELLE MOLECULAR WEIGHTS, MMW, ASr3
APPARENT INTRINSICVISCOSITIESEV]' Or THE MICELLES IN MOLAR MIXTURES OF
SODIU~ DEOX¥CHOLATE, NaDC, WlTI~ TETRADECYL-TRIMETHYLAMMONIUM
BROmDE, TTAB, IN WATER AT 25°C System
NaDC/H20 mole ratio of NaD C: TTAB 4:1 3:1 12:5 2:1 1:2 5:12 1:3 1:4 1:10
CMC (g ml-I x lo9
dn/dc (ml g-0
20.7~
--
0.69 0.60 0.53 0.52 0.54 0.56 0.57 0.67 0.73
0.187 0.183 0.183 0.181 0.166 0.167 0.165 0.161 0.157
MMW ( X 10-3)
[7]' (ml g-0
approx 2~ 6.1
14.8 17.6 65.5 110.0 50.0 37.0 16.3 17.7 19.6
4.9 5.1 -4.7 5.1 -4.8 5.1 --
Value taken from Ref. (14).
Journal of Colloid and Interface Science, VoL 52, No. 2, August 1975
332
BARRY AND GRAY 15
10
2:5
~o
7:s
x
o~
is I
10
|
•
•
Sodium cholate, however, did not have this effect. In addition, large concentrations of inorganic salt will tend to shield the silicate ions from the cationic ions, thus preventing adsorption of the latter onto the glass. For systems in which anomalous flow times were observed, the value of to was taken as the minimum in the curve of flow time versus concentration. For convenience, Co was taken as the concentration at which the minimum flow time occurred. Flow times of solutions, and to, used in computation of apparent intrinsic viscosities were reproducible within 4-0.05 sec. Plots of [ ( t - to)/to]/(C- Co) versus C - Co are shown in Figs. 7 and 8, assuming that the composition of the micelles and bulk solution was identical. The plots were
•
20
s~
2:s
g.o
72s
(C-Co) x 10~, gml-'
FIG. 5. Debye plots of molar mixtures of sodium
15
cholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium cholate to tetradecyl-trimethylammonium bromide was: II, 4:1 ; A, 2:1; Q, 1:1; O, 1:2; A, 1:4; V, 1:7; E3, 1:10.
1
lous flow times at concentrations below the CMC. Solutions of sodium cholate, or sodium deoxycholate, alone did not exhibit this effect. Davies and Rideal (8) have shown that glass is nonwetted by low concentrations of cationic detergents, due to the adsorption of the detergent onto the negatively charged silicate ions of the glass. Thus, a hydrophobic layer m a y be formed on the glass of the viscometer capillary, obstructing the flow of aqueous solutions. This effect is not observed with anionic detergents. At higher concentrations of the cationic detergent the surface tension is lowered and a hydrophilic double layer may be built up until a concentration is reached at which complete wetting of the glass results. In solutions with an excess of sodium deoxycholate it seems that interaction of the bile salt with the cationic prevents interaction of the latter with the silicate ions of the glass.
x
215
520
7-5
(C-Col n 10 3, gmV'
FIG. 6. Debye plots of molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: I , 4:1; A , 3:1; V , 12:5; O, 2:1; O, 1:2; V , 5:12; A, 1:3; [], 1:4; X, 1:10.
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
BILE SALT-CATIONIC MIXED MICELLES
333
7".*
st_
6.5
T O)
o
o, 5.5 []
[]
~
o
0
[]
o "7
'~'4.s
•
•
_i
3-5 ":*
o
w
A
•
w
2'.0
~
4'.0
¢,
a
6'.o
s'.o
( c - c o ) x l O ~, g m1-1
FIG. 7. Plots of apparent reduced viscosity, F(t-to)/to-l/(C-Co), ml g-l, versus micellar concentration, ( C - C o ) , g ml -z, for molar mixtures of sodium cholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium cholate to tetradecyl-trimethylammonium bromide was : • , 4:1 ; • , 2:1 ; • , 1 : 1 ; A , 1 : 2; [], 1 : 4; y , 1 : 10; O , sodium cholate alone; V, tetradecyl-trimethyla m m o n i u m bromide alone.
treated by the least-squares method to obtain apparent intrinsic viscosities at infinite dilution of the micelles. The resultant values of apparent intrinsic viscosity are shown in Tables I and II. DISCUSSION
As aggregation numbers and solubilities of dihydroxy bile salts are extremely variable below pH 7-8 (9), all solutions were adjusted to pH 8.5-9.5. Even at this pH, however, mixtures of dihydroxy bile salts with the alkyltrimethylammonium bromides coacervated at close to equimolar mixing ratios as shown in
Fig. 1, whereas mixtures of the trihydroxy compound, sodium cholate with the cationic surfactants did not. The phase-separation diagrams show that the areas of coacervation of the 3a, 7a dihydroxy-compound, sodium chenodeoxycholate, with the three homologous cationic compounds are smaller than the corresponding areas of coacervation of the 3a, 12a dihydroxy-compound, sodium deoxycholate. This is consistent with the observation that sodium chenodeoxycholate has a higher CMC than sodium deoxycholate (10). Carey and Small attributed this to the 7a-hydroxyl group being a better hydrophilic group than
Journal of Colloid and Interface Science, Vol. 52, No. 2, August 1975
334
BARRY AND GRAY
v ~
v
V.VV
V
v
~
V
6"0
5"0
4"0
?
2:0
4:0
6!0
8~0
6:0
8~0
6.0
u
5"0 #,
0
2:0
~
±
A
A A
4!0 (c-cs),10]gml -L
F~G. 8. Plots of apparent reduced viscosity, [-(t -- t o ) / t o ~ / ( C -- Co), ml g-l, versus micellar concentration, (C -- Co), g m1-1, for molar mixtures of sodium deoxycholate and tetradecyl-trimethylammonium bromide in water at 25°C. Mole ratio of sodium deoxycholate to tetradecyl-trimethylammonium bromide was: II, 4:1; A , 3:1; e , 2:1; O, 1:2; /x, 1:3; [~, 1:4; V , sodium deoxycholate alone.
the 12a-hydroxyl group (11). The phaseseparation studies described above indicate, therefore, that the position, as well as the number, of hydroxyl groups on the steroid nucleus is instrumental in establishing the solubility of the mixed micelles in water. It was hoped that light-scattering and viscosity studies of the sodium cholate-cationic and sodium deoxycholate-cationic mixed micelles would give an insight into the mechanisms of interaction in the two systems. To estimate the CMC values and to calculate the micelle molecular weights and intrinsic viscosities, it was assumed that the composition of the micelle was identical to the composition of the bulk phase. A number of workers have attempted to measure the exact composi-
tion of mixed micelles (12, 13). However, for the present systems, it was considered reasonable to assume identical composition of micellar and bulk phases. First, sodium deoxycholate and the sample of tetradecyltrimethylammonium bromide used have CMC values in water of 3-5 mmole 1-1 (14) and 3.4 mmole 1-1 (5), respectively, i.e., the tendency of these compounds to micellize is almost identical. The CMC of sodium cholate in water is of the order of 10-12 mmole 1-1 (14), which does not differ greatly from*that of the cationic. Second, an excess of ions*of one kind in the micelle or bulk phase would tend to attract ions of opposite charge into the phase, thus leveling the tendency of both surface active ions to micellize. This behavior would be especially true at close to 1 : 1 mixing ratios. Third, as comparative results were the main aim of these experiments, it was considered that small deviations from the assumption of identical composition of micellar and bulk phases could be tolerated. The CMC values thus found for the mixtures (Tables I and II) are much lower than the CMC values of the pure compounds in water. This is attributed to a significant decrease in the unfavorable electrostatic interactions which tend to suppress micellization in homoionic surfactant systems. In Fig. 9, the CMC values are plotted versus mole percent of tetradecyl-trimethylammonium bromide. For the sodium cholate-quaternary ammonium bromide mixtures, the CMC curve is approximately symmetrical about a mixing ratio of cationic to bile salt of slightly greater than one. For the sodium deoxycholatequaternary ammonium bromide mixtures the CMC curve is almost symmetrical about the 1:1 molar mixing ratio. This lends further support to the validity of the assumption that the tendency of both the anionic and cationic surfactive ions to form micelles is similar, and hence, that the bulk and micellar phases have similar compositions. The CMC values in sodium deoxycholate mixtures are approximately one-half those of the corresponding sodium cholate mixtures.
Journal of Colloid and Inlerface Science. Vol. 52, No. 2, AugusC1975
335
BILE SALT-CATIONIC MIXED MICELLES 2"0
/ /
\ 1.5
Tm E *~
1"0
\
x
o
/
0"5
2~
go
71
loo
Mole Percent TTAB
FIG. 9. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on critical micelle concentrations, CMC, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. O, sodium cholate-tetradecyl-trimethylammonium bromide mixtures, I t , sodium deoxycholate-tetradecyl-trimethylammoniumbromide mixtures.
Micelle molecular weights, for a twocomponent system, are usually obtained using the equation given by Debye (6) : H(C
-- Co)/(r
- - to)
= 1/~
+ 2 B ( c - co),
[-23
where H is a constant; C and r are the concentration and turbidity of the bulk phase; Co and ro are the values at the CMC; M is the molecular weight; and B is the second virial coefficient, which describes the extent of interaction between the scattering particles. In these cases, a plot of I t ( C - C o ) / ( r - to) versus C - - Co yields a straight line with slope 2B and intercept (infinite dilution of the micelles) equal to the reciprocal of micelle molecular weight. However, where the third, and higher, virial coefficients are important, i.e., when interaction between the micelles is large, or where other nonideal behavior is present, the plots may be curved. Plots of this nature were observed by Anacker (15) in studies of the light scattered by micelles formed from surfactants of double long-chain salts. This is also the case in mixtures of sodium deoxycholate with tetradecyl-trimethylammonium bromide close to the phase-sepa-
ration point (Fig. 6), where interaction is expected to be large. The calculation of micelle molecular weights for these systems, therefore becomes more uncertain. However, with the emphasis on comparison rather than on absolute values, a plot of micelle molecular weights versus mole percent of tetradecyl-trimethylammonium bromide, as shown in Fig. 10, demonstrates a major difference between the sodium cholate and the sodium deoxycholate mixed systems. While the micelles in the former systems are always relatively small, the micelles in the deoxycholate mixed systems close to the phaseseparation point are large and grow exponentially on approaching the phase-separation point. The apparent intrinsic viscosities of the mixed micelles support the conclusions drawn from the light scattering experiments. A plot of apparent intrinsic viscosities versus mole percent of tetradecyl-trimethylammonium bromide is shown in Fig. 11. The sodium cholatetetradecyl-trimethylammonium bromide plot shows a minimum value at close to an equimolar mixture of anionic and cationic detergents. With excess of either detergent the
Journal of Colloid and Interface Science.
Vol. 52, No. 2, August 1975
336
BARRY AND GRAY
,,o[ loo~ \
76
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.50
o x
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Mole Percent TTAB
Fro. 10. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on micelle molecular weights, MMW, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. 0 , sodium cholate-tetradecyl-trimethylammonium bromide mixtures, B, sodium deoxycholate-tetradecyl-trimethylammonium bromide mixtures.
intrinsic viscosities of the micelles increase. This effect is attributed to an increased micellar charge as excess of either of the micellized ions increases. An electroviscous effect, caused by interaction between the double layers of the charged micelles, results (16). As has been shown previously (1), this effect is absent in equimolar solutions of sodium cholate and tetradecyl-trimethylam-
monium bromide in water; the value of 3.2 ml g-1 for this system suggests that the micelles are approximately spherical with a small degree of hydration. The data for the sodium deoxycholatetetradecyl-trimethylammonium bromide mixed micelles suggest that the micelles, although not spherical, are not very asymmetric. The light-scattering and viscosity studies,
9.0
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Mole Percent TTAB
FIG. 11. Effect of changing the mole percent of tetradecyl-trimethylammonium bromide, TTAB, on apparent intrinsic viscosities of the micelles [~]', ml g-l, in mixtures of bile salts and tetradecyl-trimethylammonium bromide in water at 25°C. O, sodium cholate-tetradecyl-trimethylammonium bromide mixtures, B, sodium deoxycholate-tetradecyl-trimethylammonium bromide mixtures. Journal of Colloid and Interface Science, Vol. 52, No. 2, August. 1975
BILE SALT-CATIONIC MIXED MICELLES
337
®
® ®
Fio. 12. (a) Proposed shape of a]kyltrimethylammoniumcholate micelle. (b), (c), (d) Possible mechanisms of micellegrowth in alkyltrimethylammoniumbromide-dihydroxybile salt mixtures. therefore, show that phase separation in the sodium deoxycholate-tetradecyl-trimethylammonium bromide mixed systems is caused by growth of the mixed micelles to a large size, with a resultant separation of the Inicelles into a colloid-rich phase, the coacervate (17, 18). Phase separation in the sodium chenodeoxycholate-tetradecyl-trimethylammonium bromide systems is probably the result of a similar mechanism. The light scattering and viscosity studies also support the phase-separation studies in demonstrating the importance of the hydroxyl groups in determining the shape and size of the mixed micelles. The authors have previously proposed what they believe is the most probable form of the alkyltrimethylammonium cholate micelle (1). For convenience, this is reproduced in Fig. 12a. This micelle remains relatively small to minimize the unfavorable coulombic interactions of the quaternary ammonium groups at the surface of the micelle. Although essentially electro-neutral, it is maintained in aqueous solution by hydrogen bonding between the hydroxyl groups on the hydrophilic face of the steroid nucleus and the surrounding water molecules. The importance of the
steroidal hydroxyl groups, as demonstrated by the studies reported above, support the suggested model of the micelle. If these groups were in the interior of the micelle they would have no effect on water solubility and probably little effect on micelle size. Assuming, then, that the fundamental micelle is as shown in Fig. 12a, growth of the dihydroxy bile salt-tetradecyl-trimethylammonium bromide mixed micelles may be attributed to three possible mechanisms: (i) The micelle may expand, resulting in the bimolecular leaflet becoming very large (short cylindrical shape), as shown in Fig. 12b; (if) the small fundamental micelles may interact with each other through the opposite charges on the micelle surface, forming chain-like aggregates (rod shape), as depicted in Fig. 12c; or (iii) aggregation of the small micelles may occur because of hydrophobic interactions between the portions of the water-facing side of the steroid nucleus not covered by hydroxyl groups. In solutions that contain approximately equimolar concentrations of dihydroxy bile salt and alkyltrimethylammonium bromide, and therefore where the micelle is effectively electro-neutral, a three-dimensional
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BARRY AND GRAY
aggregate similar to that shown in Fig. 12d may be formed. Mechanism (i) is unlikely because a large number of quaternary ammonium groups would be concentrated in the center of the surface of the micelle, causing a high electrical free energy at this point. Such a micelle would also be very asymmetric and the viscosity studies show that this is not the case. Mechanism (ii) would also lead to asymmetric aggregates. Of equal importance is that neither mechanism (i) nor (ii) explains the differences observed between trihydroxy and dihydroxy systems. The fundamental mixed micelle shown in Fig. 12a is approximately spherical (1) and aggregation by mechanism (iii) would form a nearly spherical agglomerate. Furthermore, aggregation by mechanism (iii) may account for the dependence of micelle size on the number and position of the hydroxyl groups on the steroid nucleus. The sodium cholate molecule contains three hydroxyl groups which are regularly-spaced over the water-facing hydrophilic side of the steroid nucleus, thus preventing aggregation of the micelles by mechanism (iii). Examination of molecular models shows that the hydroxyl groups of the 3a, 12a dihydroxy compound (sodium deoxycholate) are situated along the same side of the hydrophilic face of the steroid nucleus in such a manner as to allow hydrophobic bonding between adjacent micelles. However, the hydroxyl groups of the 3a, 7a dihydroxy compound (sodium chenodeoxycholate) are situated on opposite sides of the hydrophilic face of the steroid nucleus, thus decreasing the possibility of hydrophobic bonding between adjacent micelles, although not completely preventing it. In this way, the differences in solubilities and aggregation numbers between alkyltrimethylammonium bromide-bile salt mixed systems, in which the number and position of the hydroxyl groups on the steroid nucleus are different, may be simply explained. Small (9) attributed the differences in aggregation numbers of dihydroxy and trihydroxy bile salt micelles in aqueous salt solutions to dehydration of the weaker nonionic moeity of the dihydroxy bile salts by
inorganic salts, resulting in hydrogen bonding between the hydroxyl groups of adjacent dihydroxy bile salt micelles. For the mixed surfactant systems described above, this would lead to an aggregate of micelles identical to that formed by mechanism (iii) above. However, in our systems, no inorganic salt, other than the counter-ions of the surfactants, was added and salting-out of the hydroxyl groups in this case is unlikely. It is possible, however, that hydrogen bonding between hydroxyl groups on adjacent micelles in the interior of alkyltrimethylammonium-dihydroxy bile salt agglomerates occurs as a result of aggregation of the basic micelles. The aggregate shown in Fig. 12d would therefore be much more stable than that illustrated in Fig. 12c because of the additional hydrophobic and hydrogen-bonding modes. If we consider the possibility that the mixed micelle is of a more usual long-chain paraffin salt shape, with the bile salt molecules interdigitated between the paraffin chains, we see that this model could not explain the differences in behavior between dihydroxy and trihydroxy-mixed systems at concentrations close to the phase-separation point. Furthermore, systems in which long-chain paraffin salt-type micelles are present, and which are close to phase-separation boundaries, tend to become viscoelastic due to formation of asymmetric micelles (19, 20). No viscoelasticity was observed in the present systems. The following conclusions may therefore be drawn for the sodium cholate-tetradecyltrimethylammonium bromide mixed micelles in water. In mixtures in which there is no large excess of the quaternary ammonium bromide the mixed micelles are similar to that proposed for the alkyltrimethylammonium cholates, shown in Fig. 12a. Aggregation of these micelles does not occur. Elementary calculations, based on the micelle molecular weights and assuming 6-8 A for the crosssectional diameter of the sodium cholate molecule (14) and 40 A2 for the area occupied by each quaternary ammonium group (21) at the micelle surface, show that the maximum ratio of cationic to bile salt that will allow a
Journal of Colloid and Interface Science, Vol. 52, No. 2, A u g u s t 1975
BILE SALT-CATIONIC MIXED MICELLES complete circumference of bile salt molecules is about 2:1. At higher cationic to bile salt ratios, however, it is not evident from the present data whether there is a mixture of micelles, i.e., mixed micelles as shown in Fig. 12a, with the remainder of the tetradecyltrimethylammonium bromide forming normal long-chain paraffin salt micelles, or whether the mixed micelle assumes this latter form, with the bile salt molecules interposed between the hydrocarbon chains, perhaps with hydrogen-bonding between pairs of bile salt molecules, as suggested by Dervichian for the bile salt-lecithin mixed micelle in organic solvents (22). The conclusions derived from the data for the sodium deoxycholate-tetradecyl-trimethyla m m o n i u m bromide mixed systems are similar to those obtained for the system just described, except that, in the former case, as the micelles approach the situation where there is an equal number of positive and negative ions in the micelle, i.e., the fundamental micelles are approaching electro-neutrality and are therefore less likely to repel each other through electrostatic interactions, aggregation of these micelles occurs primarily through hydrophobic bonding, and then through hydrogen and electrostatic bonding between adjacent micelles. This finally causes coacervation. REFERENCES 1. BARRY, B. W., Aim GRAY, G. M. T., J. Colloid Interface Sci., 52, 314 (1975).
339
2. HOFMANN, A. F., in "New Biochemical Separations" (L. ]. Morris and A. T. James, Eds.), p. 261. Van Nostrand, London, 1964. 3. SUNDARAM,G. S., AND SODttI, H. S., J. Chromatogr.
61, 370 (1971). 4. SUNDARAM,G. S., SINGH, H., AXn) SODHI, H. S., Clin. Chim. Acta 34, 425 (1971). 5. BARRY, B. W., AND RUSSELL, G. F. ]., J. Colloid Interface Sci. 40, 174 (1972). 6. DEBX% P., Ann. N. Y. Acad. Sci. 51, 575 (1949). 7. TY~zYo, K., Kolloid-Z. 175, 40 (1960).
8. DAVIES, J. T., AXD RII)EAL, E. K., "Interfacial Phenomena," p. 435. Academic Press, New York, 1963. 9. S~ALL,D. M., Advan. Chem. Ser. 84, 31 (1968). 10. HOFMANN,A. F., Biochem. Y. 89, 57 (1963). 11. CAI~Y, M. C., Aim SMALL, D. M., Arch. Intern. Med. 130, 506 (1972). 12. MYSELS,K. J., ANDOTTER,R. J., J. Colloid Sci. 16, 462 (1961). 13. SHANKLAND, W., J. Colloid Interface Sci. 34, 9 (1970). 14. SMALL,D. 1VL,in "The Bile Acids" (P. P. Nair and D. Kritchevsky, Eds.), Vol. 1, p. 249. Plenum Press, New York, 1971. 15. ANACKER,E. W., J. Colloid Sci. 8, 402 (1953). 16. BOOTH,F., Proc. Roy. Soc., Set. A. 203, 533 (1950). 17. COHEN,I., ANDVASSILIADES,T., J. Phys. Chem. 65,
1774 (1961). 18. BARRY,B. W., AND GRAY, G. M. T., J. Pharm. Sci.
63, 548 (1974). 19. COHEN,I., ANDVASSlLIADES,T., J. Phys. Chem. 65, 1781 (1961). 20. BARKER, C. A., SAUL, D., TIDDY, G. J. T., WHEELER, B. A., Aim WILLIS, E., J. Chem. Soc., Faraday Trans. I 70, 154 (1974). 21. WEINER, N. D., Aim ZOORAFI,G., J. Pharm. Sci.
54, 436 (1965). 22. DERVICHIAN,D. G., Advan. Chem. Ser. 84, 78 (1968).
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