J. Phys. Chem. 1983, 87,5015-5019
For TmC13 we obtain (reaction 8) AH298 = -7 f 12 kJ/mol AS298
= -76 f 17 J/(mol K)
Our calculations depend on the assumption that only monolanthanide complexes are formed with considerable amounts in the temperature range investigated. From eq 26 and 25 one obtains the enthalpy and entropy of formation of a gaseous complex like Gd2C16formed by 2 mol of GdC13: AH298 = 303-405 kJ/mol ASpg8 = 275 J/(mol K) Hastie et al.31found for the sublimation of La2C1, AS1o3o = 230 J/mol K AS298
-
251 J/(mol K) (with AC, = -15 J / K (estimated))
The agglomeration of 1 or 2 mol of aluminum chloride should not raise the stability of a dimer lanthanide chloride so that considerable amounts of the complex form compared with the monolanthanide complexes. The best information should come from mass spectrometric investigations. Schaefer et have investigated the system NdC13/A1C13with a mass spectrometer and did not find any fragments of a lanthanide dimer, but their experiments were limited to high temperatures and low pressures.
5015
Using our experimental data we have done some calculations to interpret our data by assuming the nonexistence of a LnA14Cl16complex and other calculations assuming that only one of the smaller complexes-LnA1,Clg and LnA1C16, respectively-did exist. We find out that our experimental data are best interpreted by assuming the existence of four different gaseous lanthanide chloridealuminum chloride complexes.
Conclusion The method described is useful for the investigation of volatile inorganic complex species, in particular systems which cannot be measured by spectrometric techniques. Furthermore, it is a convenient method that yields a large quantity of data.24 For precise thermodynamic characterization of the complexes 800-1000 experimental points are available in the temperature range from 500 to 1000 K which allows the determination of complexes with lower concentration in the presence of the main complexes. From our experiments we suggest the existence of four different gadolinium chloridealuminum chloride and four thulium chloride-aluminum chloride complexes and determined the thermodynamic properties. Acknowledgment. We appreciate the financial support of the "Bundesministerium fuer Forschung und Technologie". Registry No. A1Cl3, 7446-70-0; GdC13, 10138-52-0; TmC13, 13537-18-3.
Studies of Model Bile Solutions Using Surfactant Ion Electrodes Kyoo Ryu, James M. Lowery, D. Fennel1 Evans, and E. L. Cussler' Department of Chemical Eng/ne8rlng, University of Minnesota, Minneapolis, Mlnnesota 55455 (Received: January 24, 1983; I n Final Form: April 19, 1983)
Surfactant ion electrode measurements at 25 "C show that sodium taurodeoxycholate in water forms small aggregates with little counterion binding. The critical micelle concentration determined with the electrodes agrees closely with that found by other methods. Adding sodium chloride or lecithin enhances formation of these aggregates, although sodium chloride make aggregation more gradual. In contrast, sodium taurocholate aggregates much less except in the presence of lecithin.
This paper reports measurements of bile salt anion activities as a function of bile salt concentration. The bile anion selective electrodes used depend on the emf cell shown in Figure 1. The key aspect of this cell is a liquid membrane.' The bile salt anions and an equal number of hexadecyltributylammonium cations are present in this membrane, but aggregates and small ions have much lower solubilities. As a result, the emf measured in this cell is influenced almost totally by the bile salt anion, the only ion which can move in and out of the membrane. Phrased in more thermodynamic terms, measurements of emf are proportional to bile salt anion activity uB. RT emf = KO - - In uB (1)
RT
emf = KO - - In [B] F
(2)
If the bile salt is highly dilute, then this becomes
where KO is a constant and [B] is the concentration of the bile salt anion. In other words, the emf is proportional to the logarithm of the bile salt concentration. When this is true, the emf should change 59 mV for every decade change in bile concentration. In many other solutions, this is observed experimentally.2 In more concentrated solutions the slope of the emf vs. the logarithm of concentration can change sharply. These changes are a measure of solute-solute and solutesolvent interactions in the solution. More specifically, decreases in the slope's magnitude are indications of solute aggregation, and increases in this magnitude are taken as solute hydration. The decreases occur because multiply charged
(1) T. J. Gilligan, E. L. Cussler, and D. F. Evans, Biochim. Biophys. Acta, 497, 627 (1977).
(2) N. Lakshminarayanaiah, 'Membrane Electrodes", Academic Press, New York, 1976.
F
0022-365418312087-5015$01.50/0
0 1983 American Chemical Society
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Ryu et al.
The Journal of Physical Chemistry, Vol. 87, No. 24, 1983
-inner filling
le51 electrode
solution
Ag/AgCI
No+CI-
~
bile anion
liquid membrane
1
-
C,6H33(C4
H81JN+
blleaiionin dichlorobenzene
surfactant solution
reference electrode A
I
No+ bile anion
NH4+ NO,:
Ag/AgCI
~
Figure 1. Emf cell for bile salts. The key aspect of this cell is the llqutd membrane, an organic solutbn of hexadecyltributylammonium-bile salt anion.
solute aggregates cannot pass into the electrode’s membrane. Thus the decreased slope is a measure of unassociated solute concentration. Conversely, the increases in the slope are consistent with solute hydration, which decreases the number of unassociated solvent molecules. In previous papers, we have investigated these changes in slope for synthetic detergents and for d ~ e s . 4 ~For detergents like sodium dodecyl sulfate, the magnitude of the slope drops very abruptly at the critical micelle concentration. This drop is associated with the formation of multiply charged micelles, which cannot dissolve in the electrode’s membrane because of their high charge. The new slope is a measure of detergent monomer concentration. For surface-active molecules like Orange 11, the slope’s magnitude drops slowly; this is a consequence of gradual aggregation of the dye’s molecules. The electrodes show that detergents like hexadecyltrimethylammonium bromide are found to dimerize in dilute solution, consistent with other experiment^.^,^ They also form micelles at higher concentrations. This paper reports and interprets similar measurements for model bile solutions. The bile salts in these solutions are of physiologic interest, for they are central to fat digestion and to the formation of cholesterol gallstones.’ They are of scientific interest because they aggregate only gradually, and because their aggregates are much smaller than those of synthetic detergent^.^^^ Studying these systems with electrodes is especially interesting because the electrodes respond to the unaggregated bile salt. In contrast, other techniques like light scattering, sedimentation, and solubilization emphasize the aggregates. Experimental Section Sodium chloride (Fisher Reagent Grade) was used as received. The two bile salts, sodium taurodeoxycholate (NaTDC) (99% Cal Biochem) and sodium taurocholate (NaTC) (96% Cal Biochem) were purified by recrystallization and gel permeation chromatography using Sephadex LH-20 (Pharmacia Fine Chemicals). After purification, graphs of surface tension gave constant values and titrations with AgNO, yielded less than 0.04% chloride. Egg yolk lecithin in 9O:lO ch1oroform:ethanol (Lipid Products) was found to be 98% pure by thin layer chromatography. It was used without further purification. Solutions were prepared by evaporating the solvents from the lecithin, adding the bile salt, dissolving the mixture in 50:50 chloroform:methanol, evaporating these solvents, and then adding sodium chloride and doubly distilled (3)K. M.Kale, E. L. Cussler, and D. F. Evans, J. Phys. Chem., 84,593 (1980). (4)K. M.Kale, E. L. Cussler, and D. F. Evans, J. Solution Chem., 11, 581 (1983). (5)E. J. Bair and C. A. Kraus, J. Am. Chem. SOC.,73, 1129 (1951). (6)J. Proust and L. Ter-Minassian-Saroga, C. R . Acad. Sci., P a m , Ser. C., 270, 1354 (1970). (7)M.C. Carey in ‘The Liver: Biology and Pathobiology”, I. Arias, H. Popper, and D. A. Shafritz, Ed., Raven Prees, New York, 1982,p 429. (8)J. P. Kratohvil and H. T. Delli Colli, Can. J. Biochem., 46,945 (1968). (9)J. P.Kratohvil, W. P. Hsu, M. A. Jacobs, T. M.Aminabhavi, and Yasuo Mukunoki, Biochern. Biophys. Acta, submitted for publication.
1 6 14‘ CONCENTRATlOh, M O L / L
$6’
Flgure 2. Emf and taurodeoxycholate actlvlty at 25 ‘C. The squares and the scale without parentheses are for the taurodeoxycholate anion; and the triangles and the scale in parentheses are for the sodium ion. The break in taurodeoxycholate activity corresponds to the formation of aggregates.
water. This water must be freshly distilled or carefully degassed to avoid erratic electrode readings caused by small gas bubbles. The electrode assembly was similar to that described ear1ier.l~~ The reference and sodium electrodes were Orion 90-00-02 and 94-11-00, respectively. The inner filling solution of the reference electrode was Orion 92-00-02, and the outer filling solution was 0.5 We. % ammonium nitrate. The membrane electrode (Orion 92-00-00) used a porous diaphragm (Orion 92-20-04) to mechanically support the liquid membrane. This membrane was a solution of the cation hexadecyltributylammonium and the bile salt anion in the o-dichlorobenzene (Eastman) containing 0.17 M hexachlorobenzene (BDH) and 0.017 M 4-bromoacetanilide (Eastman). Hexadecyltributylammonium bromide was synthesized from recrystallized 1-bromohexadecane and tributylamine (Eastman). The inner filling solution for this electrode was an aqueous mixture of 0.01 M NaCl and bile salt. The bile salt concentration was the same as that in the outer filling solution which was being tested. Experiments were made as follows. The electrodes were placed in a 100-mL thermostated beaker, immersed in a water bath maintained at 25 OC. Fifty milliliters of water or NaCl solution at a desired concentration was added as a test solution. After thermal equilibrium was reached in about 30 min, an aliquot of a stock solution of bile salt and lecithin was added by using an Eppindorf pipette, and the potential was measured. This procedure was then repeated. Typically, 25 aliquots were added, covering a to 2 x lo-’ M bile salt. concentration range from 2 X After each addition, the electrodes take 2 min to reach a new stable value, even though the test solution is continuously stirred. A check on electrode drift was made at the end of each experiment by repeating the measurement of one dilute solution potential. The drift was typically 2 mV/h for the bile selective electrode and 1 mV/h for the sodium electrode. During the time in which the electrodes were not in use, they were stored in the inner filling solution. Results and Discussion This paper reports measurements of ion activities in solutions of two bile salts: sodium taurodeoxycholate and
Studies of Model Bile Solutions
The Journal of Fhysical Chemistry, Vol. 87, No. 24, 1983 5017
sodium taurocholate. These bile salts are chemically similar: each is based on a cholesterol residue, each is conjugated with taurine, and each has hydroxyl groups in the 3 and the 12 position of the cholesterol. The only difference is that taurocholate has a third hydroxyl group, in the 7 position, but taurodeoxycholate does not. This difference can produce major changes in the chemistry. The results for sodium taurodeoxycholate in water are shown in Figure 2. The emf measured with the sodium electrode varies smoothly with the increasing concentration of the bile salt. The slope of this variation is 52 mV per decade of concentration, less than the ideal response of 59 mV but consistent with measurements with this electrode in other solutions. These data suggest that the sodium ion behaves almost ideally across the entire range studied. To be sure, the data show minor curvature at higher concentrations. Still, for sodium, there are no surprises. The emf of taurodeoxycholate measured with the bile salt electrode shows a similarly smooth variation with a slope of 58 mV until a critical concentration of 3.4 X M. Above this value, the emf is almost constant. This type of behavior is very similar to that observed for many synthetic detergents, where this sudden break corresponds closely to the critical micelle c o n ~ e n t r a t i o n .The ~ ~ ~most recent values of critical micelle concentration measured for sodium taurodeoxycholate are 4.0 X M by light scattering and 4.6 X M by surface t e n s i ~ n . ~Older J~ values obtained by light scattering, dye solubilization, and to 3.1 X M.*J1J2 chromatography range from 1.8 X All these other techniques measure aggregates, rather than the single surfactant ion properties measured by our electrodes. This is probably why our value of the critical micelle concentration is slightly different than that found by other methods. The electrode data can be used to calculate the taurodeoxycholate monomer and sodium activities above the critical micelle concentration. The calculation, described in detail elsewhere: uses eq 2 and the results a t very high dilution to find a value of KO. This value and the results a t higher concentration are then inserted into eq 1 to find the activity. The activities calculated for taurodeoxycholate, also shown in Figure 2, are essentially a mirror image of the emf data. These activity data can be used to estimate the fractional charge on the aggregates, using the phase separation model for formation of one size of aggregate. (The chemical equilibrium model gives equivalent results but is much more elaborate a1gebrai~ally.l~)If the phase separation model is approximately correct, then we expect PmiceUe
=
nPBS -k mPNa
(3)
where n and m are the number of bile salt ions (BS) and the number of sodium ions in the micelle, respectively. Because the micelles are a second phase, their chemical potential is constant; moreover, the standard state chemical potentials of both ions are constant. Thus eq 3 can be rewritten in terms of activities ai: In
uBS
= constant - (m/n) In aNa
(4)
Thus a plot of the logarithm of bile salt activity vs. sodium activity should be a straight line whose slope is propor(10) J. P.Kratohvil, private communication, 1983. (11) M. C. Carey and D. M. Small, J. Colloid Znterace Sci., 31, 382 (1969).
(12) H.V. Ammon and L. G. Walter, Anal. Chern., 64, 2079 (1982). (13) J. M. Lowery, M.S. Thesis, Carnegie-Mellon University, Pittaburgh, 1977.
I-
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' ' ' '
I
3
1
'
10-2
I
3
I
, , / I 1
lo-'
SODIUM ACTIVITY Figure 3. Counterion blnding for taurodeoxychoiate aggregates. I f there is no counterion binding, these data should fail along a horlzontal line; if counterion binding is 8O%, as it Is for dodecyl sulfate, these data should fall along a straight line with a slope of -0.80.
0.15 M NaCl
IO-^
10-2
10.'
CONCENTRATiON TAURODEOXYCHOLATE, MOL / L Figure 4. Emf measurement In added sodium chloride. As in Figure 2, the squares and triangles refer to taurodeoxychoiate and sodium ions, respectively. The taurodeoxychoiate data show that the addition of sodium chloride makes aggregates occur earlier. The sodium data reflect the increased fraction of the total sodium ions coming from sodium chloride.
tional to the fraction of charge (mln) bound to the aggregates. Such a plot, given in Figure 3, has a near zero slope: the activity of the bile salt changes less than 50% while the sodium ion activity changes more than 5000%. (The small but systematic deviations from zero slope are discussed in greater detail later.) This implies that these aggregates are almost completely ionized and that there is little counterion bonding in these systems. Such aggregates are an interesting contrast to micelles of synthetic detergents like sodium dodecyl sulfate and decyltrimethylammonium bromide, where about 80% of the counterions are bound to the m i ~ e l l e . This ~ , ~ difference reinforces the ideagJOthat
5018
The Journal of Physical Chemistry, Voi. 87, No. 24, 1983
Ryu et ai. I
0
0.3
0.6
0.9
I
I
I
14OI1001-
lo-'
lOO(60)-
lo-'
60(201-
10-3
201-201-
10-4
-20(-60)-
10-5
1.2
CONC. LECITHIN/CONC. B I L E SALT
Flgure 5. Taurodeoxycholate activty with added lecithin. The numbers on the curves are the bile salt concentrations in lo3 M. The addition of ledthin reduces the actMty of unaggegated taurodeoxycholate. This reduction may explain why cholesterol dissolves more slowly in blie salt-lecithin mixtures than in bile salt alone (cf. text).
these bile salt aggregates are much smaller than micelles of synthetic detergents, and suggests that their charges are more widely separated. Measurements of emf s in solutions with added sodium chloride are shown in Figure 4. The measuremenh with the sodium electrode are as expected: sodium activity becomes more and more constant as a greater fraction of the sodium ions comes not from the bile salt but from the added sodium chloride. The measurements with the bile salt electrode are more interesting. As before, the emf for bile salt behaves ideally in highly dilute solution, but breaks at more moderate concentration. These breaks occur at 3.4 X 2.0 X and 1.7 X M bile salt in 0.01,0.05, and 0.15 M NaC1. They agree reasonably well with the critical micelle concentrations of 2.7 X 1.9 X and 1.7 X M bile salt, which can be interpolated from surface tension measurements made in these salt solutions.s~9 However, this comparison of critical micelle concentrations obscures the fact that the breaks in emf vs. concentration are less sharp in added salt than in water. Indeed, above these critical concentrations, the emf again begins to drop significantly. These changes are consistent with the slight S shape observed in Figure 3. While we are not certain why these trends occur, we are sure that they are evidence of gradual aggregation rather than sudden micelle formation. In other words, in added salt, sodium taurodeoxycholate tends to associate more like the dye Orange I1 than like the detergent sodium dodecyl ~ u l f a t e . ~ We also studied the emf's in solutions of bile salt and lecithin, obtaining the results given in Figure 5. This system is involved in the formation of cholesterol galls t o n e ~ . ' ~The data show that adding lecithin reduces the bile salt monomer activity, presumably by enhancing the formation of micelles. The reduction is greater at high bile salt concentration. These results have significant implications for the kinetics of solubilization in bile salt-lecithin solutions. The addition of lecithin increases the solubility of cholesterol in bile, but it decreases the solubilization rate.'"'' The data in Figure 5 show why this might be so. Studies of solubilization kinetics in synthetic detergents indicate solubilization begins with the adsorption of detergent (14)M.M.Fisher, "Gallstones",Plenum, New York, 1979. (15) D. M.Small, Adu. Intern. Med., 18, 243 (1970). (16)W.I. Higuchi, S. Prakongpan, and F. Yong, Science, 178, 633 (1972). (17)J. C. Tao, E. L. Cussler, and D. F. Evans, R o c . Natl. Acad. Sci. U.S.A. 10, 3917 (1974).
10-4
10-3
10-2
10-1
CONCENTRATION, MOL /L
Figure 8. Emf and taurocholate activity at 25 O C . The squares and the scale without parentheses refer to the taurocholate anion: and the triangles and the scale with parentheses are for the sodium ion. There is no sharp break in taruocholate activity, suggesting no sudden formation of micelles. 120
\
100
0.01 M NaCS 00
60 40
20
0 -20
401
-40
-60
E0 20
10-4
103
10-2
10-1
CONCENTRATION TAUROCHOLATE, M O L / L
Flgure 7. Emf measurements in added sodium chloride. As in Figure 6, the squares and triangles refer to taurocholate and sodium ions, respectively. The curvature of the taurocholate results is in the opposite direction to that expected for aggregation.
monomers on the solid surface.'*-20 If this is true for cholesterol dissolving in bile, then the reduction of mo(18)A. F. Chan, E. L. Cussler, and D. F. Evans, AIChE J., 22, 1006 (1976). (19)J. L. Shaeiwitz, A. F. Chan, E. L. Cussler, and D. F. Evans, J. Colloid Interace Sci., 84, 47 (1981). (20) C. T. Huang, D. F. Evans, and E. L. Cussler, J. Colloid Interace Sci., 82, 499 (1981).
The Journal of Physical Chemistry, Vol. 87,
Studies of Model Bile Solutions
No. 24, 1983 5019
7
u [ 1
0.40
Id’ i0-2
I
16’
SODiUM ACTiVlTY
Flgure 8. Taurocholate vs. sodium activity. I f taurocholate behaved Ideally, without any hydration or aggregatlon. then the data should fall along the straight line shown. If taurocholate set aggregates like those in dodecyl sulfate, the data would have a slope of -0.8; if it formed aggregates like those in taurodeoxycholate, the data would have a slope of zero.
nomer concentration caused by adding lecithin would reduce the kinetics of solubilization. In summary, sodium taurodeoxycholate behaves almost ideally until the critical micelle concentration. Above this, bile salt activity is nearly constant, with little counterion bonding. Adding salt makes bile aggregation more gradual; adding lecithin makes it occur sooner. The data for sodium taurocholate given in Figure 6 are different, both from sodium taurodeoxycholate and from synthetic detergents. As before, the emf measured with the sodium electrode varies smoothly across the entire concentration range with a slope of 51 mV/decade concentration, and that measured with the bile salt electrode varies with a slope of 60 mV/decade concentration in dilute solution. At higher concentrations, the data do not exhibit the sharp breaks characteristic of most detergent ~ y s t e m s . ~They , ~ do show a slight change in slope near 1.2 X M, which is close to the value of the “critical micelle concentration” reported recently for this bile salt,1p9,10 but less than 3 X M reported earlier.11$21v22The emf of sodium taurocholate is not altered dramatically by the addition of sodium chloride as shown in Figure 7. However, if aggregation of sodium taurocholate does cause the small change in slope in Figure 6, that aggregation is considerably less significant than that for sodium taurodeoxycholate shown in Figure 2. In addition, the variation of taurocholate activity with sodium activity, (21) K. Fontell, Kolloid 2.2.Polym. 244, 253 (1971). (22) N. A. Mazur, R. F. Kwasnick, M. C. Carey, and G. B. Benedek
in “Micellization Solubilization and Microemulsions”, K. L. Mittal, Ed., Plenum, New York, 1977, p 383.
0.2 0.4 CONC. L E C I T H I N CONC. B I L E S A L T
Flgure 9, Taurocholate activity with lecithin. The bile salt concentrations given are in M. The addition of lecithin does reduce the taurocholate activity, so that aggregates do form in this system.
shown in Figure 8, is totally different than that in Figure 3. It shows a positive slope close to unity, in contrast with the near-zero value observed before. This positive slope is inconsistent with that predicted by eq 4 micelle formation. Taurocholate activities are reduced by the addition of lecithin, as shown in Figure 9. This is consistent with micelle formation as a means of lecithin solubilization. The reductions are somewhat greater in concentrated bile salt, which is the same as that observed in Figure 5. However, the systematic trends observed before do not continue here.
Conclusions Using bile salt electrodes, we have measured the activities of solutions of sodium taurodeoxycholate, sodium taurocholate, sodium chloride, and lecithin. These electrodes respond to the unaggregated bile salt, but not to bile salt aggregates. The measurements show that sodium taurodeoxycholate forms micelles with very little counterion binding. The critical micelle concentration determined here from monomer activity is close to that reported elsewhere from micellar measurements. The addition of sodium chloride causes aggregates to form more gradually, but a t lower taurodeoxychlolate concentrations. Lecithin enhances aggregation. In contrast, sodium taurocholate apparently aggregates very little, except in the presence of lecithin. Acknowledgment. This work was supported by National Science Foundation Grants CPESO-17376, 80-14567, and 80-25304; by the National Institute of Arthritis, Metabolic and Digestive Diseases Grant SR01-AM16143; and by the Department of Defense Grant DAAG29-81-K099. Registry No. Sodium taurodeoxycholate, 1180-95-6; sodium taurocholate, 145-42-6; sodium chloride, 7647-14-5.
