Langmuir 2002, 18, 2337-2342
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Composition of Sodium Glycocholate Micellar Solutions Emilio Bottari,* Maria Rosa Festa, and Magda Franco Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy Received July 20, 2001. In Final Form: November 21, 2001 Composition and existence range of aggregates formed by sodium glycocholate in aqueous solutions were studied. Electromotive force measurements provided hydrogen, sodium, and glycocholate ion free concentrations. Lead(II) glycocholate solubility measurements yielded the free concentration of glycocholate ions, as well. Experimental data obtained at 25 °C and at three different concentrations of N(CH3)4Cl, used as a constant ionic medium, can be explained by assuming the presence of aggregates with different compositions, depending on reagent and ionic medium concentrations. The distribution of the species found at the same concentration of ionic medium and close to neutrality remains constant. As expected, the size of the aggregates increases by increasing ionic medium and reagent concentrations. A dimer is the prevailing species at low concentrations, and all of the found species have even anion aggregation numbers. In solutions with the highest concentration and ionic strength, aggregate multiples of eight are present at a high percentage. These results agree with structural investigations that proposed as building blocks of sodium glycocholate micellar aggregates a dimer and an octamer. A strong analogy with the composition of taurococholate aqueous solutions is observed. The comparison with deoxycholate, glycodeoxycholate, and taurodeoxycholate shows wide differences.
Introduction Sodium salts of cholic and deoxycholic acids and their conjugates with glycine and taurine are present in human bile.1 They are natural steroids with surface-active and detergent-like properties and play an important role in a lot of physiological and biological systems. They are capable of forming micellar aggegates and solubilizing in water many compounds, such as cholesterol and lecithin.1-4 Many studies have been performed on the structure, size, and shape of bile salt micelles, their aggregation numbers, and their critical micellar concentration (cmc).4-16 Natural bile acids are C24 saturated and belong to the cholanic acid series. The most important of them contain two (dihydroxycholanic) or three (trihydroxycholanic) OH groups in the positions 3R and 12R (deoxycholic, chenodeoxycholic, and their conjugates) or 3R, 7R, and 12R (cholic and its conjugates). (1) Hoffmann, A. F.; Small, D. M. Am. Rev. Med. 1967, 18, 333. (2) De Haen, P. J. Am. Pharm. Assoc. 1944, 33, 161. (3) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, pp 249-356. (4) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Siovall, J., Eds.; Elsevier/North Holland Biomedical Press: Amsterdam, 1985; Chapter 13, p 345. (5) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178. (6) Oakenfull, D. G.; Fisher, L. R. J. Phys. Chem. 1977, 81, 1838. (7) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (8) Kratohvil, J. P. Hepatology 1984, 4, 855. (9) Conte, G.; Di Biasi, R.; Giglio, E.; Parretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. (10) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (11) Giglio, E.; Loreti, S.; Pavel, N. V. J. Phys. Chem. 1988, 92, 2858. (12) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12 (5), 1180. (13) D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998, 14 (5), 4776. (14) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15 (8), 2996. (15) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. J. Phys. Chem. B 1999, 103, 4986. (16) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. Langmuir 2000, 16 (26), 10436.
Earlier investigations dealt with the behavior of some dihydroxycholanic salts, that is, sodium deoxycholate (NaDC), glycodeoxycholate (NaGDC), and taurodeoxycholate (NaTDC). Structural studies showed that helical models described satisfactorily aqueous solutions of micellar aggregates.12,13,15 It was verified that the structures of the fibers drawn from NaGDC and NaTDC aqueous micellar solutions are helical.12 Prevailing aggregation numbers, stability of the formed species, and their range of existence were studied in aqueous solutions by means of electromotive force measurements (emf) as a function of hydrogen ion and reagent concentrations and ionic strength. Reagent activity coefficients were kept constant, despite a wide change of concentrations, using the constant ionic medium method.17-20 More recently, structures and compositions of some trihydroxycholanic salts, that is, sodium taurocholate (NaTC) and glycocholate (NaGC), were investigated. NaTC14 and NaGC16 structural studies showed that dimers and octamers can be the building blocks of their micellar aggregates. Studies of NaTC aqueous solution composition confirmed the structural model.14,21 Moreover, the solubility of glycocholic acid (HGC) and the protonation constant of glycocholate ion (GC-) were determined.22 NaDC,18 NaGDC,19 and NaTDC20 emf measurements were explained by assuming the presence of several species with different aggregation numbers and with the presence of species formed with the uptake of protons. For such bile salts and particularly for NaTDC, a trimer, observed at all ionic medium concentrations, seems to constitute the building block of the micellar aggregates, which in most cases have aggregation numbers that are multiples of three. (17) Biedermann, G.; Sille`n, L. G. Ark. Kemi 1953, 5, 425. (18) Bottari, E.; Festa, M. R.; Jasionowska, R. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 443. (19) Bottari, E.; Festa, M. R. Mh. Chemie 1993, 124, 1124. (20) Bottari, E.; Festa, M. R. Langmuir 1996, 12 (7), 1777. (21) Bottari, E.; Festa, M. R.; Franco, M. Analyst 1999, 124, 887. (22) Bottari, E.; Festa, M. R. Chem. Speciation Bioavailability 1999, 11, 77.
10.1021/la0111349 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/13/2002
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The NaTC behavior in micellar solutions was explained by assuming that a dimeric species, observed in all the samples, constitutes the building block of the micellar aggregates, which always have aggregation numbers that are multiples of two.14,21 Because both the knowledge of the composition of bile salt micellar and premicellar solutions is limited only to NaTC, and NaTC and NaGC present very similar structural properties, in this paper the NaGC behavior is studied and presented. The composition of the species formed in such solutions, their range of existence, the effect of ionic strength, and other parameters have to be explained from many points of view. The aim of this work was to establish the composition of the species formed in NaGC aqueous micellar and premicellar solutions and their relative stability. For this purpose, reagent concentrations had to be changed over a wide range. The method of constant ionic medium proposed by Biedermann and Sille`n17 was adopted in order to minimize the variation of the activity coefficients despite the changes in the reagent concentrations. All experiments were performed at 25 °C using N(CH3)4Cl aqueous solutions as the ionic medium. The ionic medium concentration, expressed in mol dm-3, will be indicated as W. By adopting the constant ionic medium method, it was possible to substitute concentrations to activities in all calculations. Previously, research on the NaTC behavior in N(CH3)4Cl at various concentrations was carried out21 by measuring the emf of suitable galvanic cells with glass electrodes sensitive to free hydrogen and sodium ion concentrations (cH and cNa) in premicellar and micellar solutions. The valuable results obtained for this system motivated us to undertake a similar investigation on NaGC in order to find the species NaqHp(GC)r present in aqueous micellar solutions and to determine the q, p, and r values and the relative existence range of the various species. Because experiments were carried out at three ionic medium concentrations, it was possible to evaluate their influence on the q, p, and r values. The results of this work were compared with those obtained for NaTC, NaDC, NaGDC, and NaTDC. Experimental Section Materials. Sodium glycocholate (Sigma) was recrystallized from acetone-water. Acetone and water in the salt were removed by keeping it in a desiccator under vacuum for 2 weeks. After this time, NaGC did not show variations of weight. Tetramethylammonium glycocholate was prepared by adding a slight excess of tetramethylammonium hydroxyde solution (Riedel de Haen) to a weighed amount of glycocholic acid (Sigma), with continuous stirring and under a stream of nitrogen. The solid lead(II) glycocholate was prepared by adding an excess of Pb(NO3)2 (twice crystallized from a C. Erba RP product) to a warm (50 °C) and stirred NaGC aqueous solution. Details of preparation and the analysis of the obtained solid are described in a previous paper.23 Work solutions of hydrochloric acid, tetramethylammonium chloride, sodium chloride, and tetramethylammonium hydroxyde were prepared and analyzed as previously described.24 Electrochemical Measurements. The emf measurements were carried out by using Radiometer model pHM4 and pHM64 and Metrohm model 654 and 605 potentiometers. The emf measurements of galvanic cells involving lead amalgam electrode were performed by means of a Keithley model 199 apparatus. Glass electrodes Metrohm No. 6.0102.000 and 6.0501.000 for H+ and Na+, respectively, were used. Preparation and behavior of the lead amalgam electrode were previously described.23 The reference electrode [Ag, AgCl/W mol dm-3 N(CH3)4Cl saturated (23) Bottari, E.; Festa, M. R.; Franco, M. Ann. Chim. (Rome), in press. (24) Bottari, E.; Festa, M. R. Ann. Chim. (Rome) 1986, 76, 405.
Bottari et al. with AgCl/W mol dm-3 N(CH3)4Cl] was prepared according to Brown.25 The glass electrode behavior for H+ was checked against a hydrogen electrode. A stream of N2 from a cylinder bubbled through the test solutions against the CO2 absorption from the atmosphere. All experiments were carried out in a thermostated room at 25 ( 1 °C. The emf measurements were performed in a thermostat at 25.00 ( 0.05 °C. Solubility Measurements. Solid lead(II) glycocholate was shaken with aqueous solutions at different contents in sodium, hydrogen, and glycocholate ions in the presence of the ionic medium in a thermostated room at 25 °C. On the basis of earlier research,23 it was established that 12 h was sufficient to reach equilibrium. When equilibrium was reached, solutions were filtered and analyzed for total lead(II) concentration and sodium ion, cNa, and hydrogen ion, cH, free concentrations. Solubility [i.e., total concentration of lead(II)] was determined by means of atomic absorption (AA) spectrophotometry, while cH and cNa were determined by direct potentiometry. For this purpose, an AA spectrophotometer Unicam Italia equipped with air-acetylene flame was used. Method of Investigation. In the investigation of the aggregate formation in aqueous solutions, glycocholate ions (GC-), sodium ions (Na+), and hydrogen ions (H+) were considered as independent reagents able to give the following general equilibrium:
qNa+ + pH+ + rGC- S NaqHpGCr
(1)
where q g 1, p ge 0, and r g 1. Charges are omitted for simplicity. The adoption of the constant ionic medium method allows substitution of concentrations to activities. The equilibrium constant (1) can be defined by the following expression: cNaqHpGCr ) βq,p,r cNaqcHpcGCr, where cx indicates the free concentration of the species x, and in the following, Cx indicates its total concentration. This work is focused to find q, p, and r values and the corresponding βq,p,r. To study the NaGC association in solution, two different approaches were applied. They were based on emf measurements of suitable galvanic cells and on solubility measurements of a slight soluble glycocholate salt, that is, Pb(GC)2, previously studied in the absence of sodium ions.23 Approach I: Electromotive Force Measurements. The emf of the following cells was measured at 25 °C:
(-) RE/Solution S/GE (+)
(I)
(-) RE/Solution S/NaE (+)
(II)
(-) Pb(Hg)/Solution S/RE (+)
(III)
where RE is an above-described reference electrode, GE and NaE are two glass electrodes sensitive to hydrogen and sodium ions, respectively, and Pb(Hg) is a lead amalgam electrode previously described.23 Solutions S were prepared in constant ionic medium, that is, N(CH3)4Cl at the three different concentrations W ) 0.100, 0.500, and 0.800 mol dm-3, and had the following general composition: CH mol dm-3 in H+; CPb mol dm-3 in Pb2+; CNa mol dm-3 in Na+; CGC mol dm-3 in GC-; (W - CH - 2CPb - CNa) mol dm-3 in N(CH3)4+ and (W - CGC) mol dm-3 in Cl-. At 25 °C and in millivolt units, the emf of the above-reported cells could be expressed as follows:
EI ) E°I + 59.16 log cH + Ej
(2)
EII ) E°II + Y log cNa + Ej
(3)
EIII ) E°III - 29.58 log cPb - Ej
(4)
where E°I, E°II, and E°III are constant values determined in the first part of each measurement when cH ) CH, cNa ) CNa, and cPb ) CPb, when glycocholate is absent. Ej is the liquid junction potential that in the selected experimental conditions depends (25) Brown, A. S. J. Am. Chem. Soc. 1934, 56, 646.
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Table 1. Total Concentration Values of Sodium Ions, CNa, and W in the Investigated Solutionsa W
W
CNa × 103 0.100 0.500 0.800 CNa × 103 0.100 0.500 0.800 0.5 1 1.5 2.0 2.5 3.5 5 7.5 10 a
+ + + + + + + + +
+ +
+ +
+
+
+
+
+
+
12 15 20 25 30 40 50 65 80
+ + +
+ + + + +
+ + + + + + +
Both concentrations are expressed in mol dm-3.
Table 2. Total Concentration Values of Glycocholate Ions, CGC, and W in the Investigated Solutionsa W
W
CGC × 103 0.100 0.500 0.800 CGC × 103 0.100 0.500 0.800 1 5 7 10 12 15 20 a
+ + + + + + +
+ +
+ +
+
+
+ +
+
25 30 40 50 65 80
+ + + +
+ + + + +
Both concentrations are expressed in mol dm-3.
only on cH and in the employed hydrogen ion concentration range can be neglected. The dependence of EII on cNa, Y, is constant for each W. Its value is different at different W, and was found to be Y ) 59.2 mV at W ) 0.100 mol dm-3, Y ) 58.3 mV at W ) 0.500 mol dm-3, and Y ) 57.5 mV at W ) 0.800 mol dm-3. After the determination of E°I, E°II, E°III, and Y, the glycocholate concentration was gradually increased in the solution S, by keeping constant for every series of measurements cH and CNa. The measurements were interrupted when the reagent total concentrations reached the limits of concentration permitted by the use of the constant ionic medium method. The data obtained from this approach, CH, CNa, CGC, cNa, cH, and cGC (calculated from cPb according to ref 23), constitute the basis for a following elaboration in order to evaluate q, p, and r and the relative constants. Approach II: Solubility. The Pb(GC)2 solubility in the selected ionic medium was studied as a function of Na+ concentration. It was determined by equilibrating solid Pb(GC)2 with a solution of the following composition: CH mol dm-3 in H+; CNa mol dm-3 in Na+; CGC mol dm-3 in GC-; (W - CH - CNa) mol dm-3 in N(CH3)4+ and (W - CGC) mol dm-3 in Cl-. Solubility was determined on equilibrated solutions by applying AA spectrophotometry in flame. The cH and cNa values were obtained by direct potentiometry, by using cells similar to those of (I) and (II).
Results and Discussion The emf measurements (approach I) and solubility (approach II) were performed on solutions with CNa and CGC collected in Tables 1 and 2, respectively. The investigation was extended in the range 4 e -log cH e 8.5. At -log cH e 4, the glycocholic acid (HGC) precipitation occurs, and at -log cH g 8.5, hydrolytic products of lead(II) cannot be neglected. Most measurements were performed in the range 5 e -log cH e 8.5, because even within the range 4-5 HGC precipitation can occur under certain conditions, especially in solutions at high GC- concentration. As two independent approaches were accomplished, results will be treated in two different sections. However, the reagent material balances are the basis for the subsequent elaboration. They, by taking into account the mass action law, can be written as follows:
∑∑∑qβq,p,r cNaqcHpcGCr CH ) cH + ∑∑∑pβq,p,r cNaqcHpcGCr + k1cHcGC CGC ) cGC + ∑∑∑rβq,p,r cNaqcHpcGCr + k1cHcGC CNa ) cNa +
(5) (6) (7)
In (6) and (7), k1 is the glycocholate protonation constant previously determined together with the solubility of HGC in the same experimental conditions.22 In Table 3, k1 and solubility values for all the studied W are reported. Solubility Results. Solubility measurements covered the hydrogen ion concentration range 5.5 e -log cH e 8.5. As above explained, the knowledge of Pb(GC)2 solubility allowed the glycocholate free concentration to be obtained.23 Experimental data obtained at the same -log cH were grouped, and their dependence on cNa and cGC was studied according to eq 5. Conditional constants, depending on cH, could be deduced at constant cH. According to Sille`n,26 a set of species with different compositions can be obtained from the data elaboration for each W. The study of the conditional constant dependence on cH showed that the species formation occurred without proton participation, that is, p ) 0. The found species are the same as in the emf approach and are collected in Table 4. Electromotive Force Results. More data could be obtained because a wider range (4 e -log cH e 8.5) than in the case of solubility measurements was investigated. Furthermore, clear solutions could be tested before Pb(GC)2 precipitation. The material balance of sodium ion (eq 5) can be rewritten as follows:
η ) log(CNa/cNa) ) log(1 +
∑∑∑qβq,p,r cNaq-1cHpcGCr)
(8)
Previous studies on NaTC aggregates21 suggest that in a wide hydrogen ion range the aggregate formation occurred without proton participation. To verify this, several series of measurements at selected and constant -log cH and at constant and low CNa were performed by gradually increasing the glycocholate concentration. Some of the experimental data, obtained at W ) 0.100 mol dm-3, are plotted in Figure 1. It can be seen that points corresponding to different -log cH fall on the same curve. This means that η is independent of -log cH, that is, p ) 0. Consequently, in the investigated -log cH range, eqs 6 and 8 can be written in the following forms:
η ) log(1 +
∑∑qβq,p,r cNaq-1cGCr)
CH ) cH + k1cHcGC
(9) (10)
Equation 10 can be used to accurately calculate the glycocholate free concentration. Since the aggregation numbers depend on the reagent concentration and on W and it is probable that q and r assume low values at low W, data elaboration will be described very shortly in two different sections. W ) 0.100 mol dm-3 and W ) 0.800 mol dm-3 are examples with the smallest and the greatest number of species, respectively. W ) 0.100 mol dm-3. It can be seen from Tables 1 and 2 that at this W the highest CNa and CGC values were both 0.020 mol dm-3. At these concentrations, it seems reasonable to assume in first approximation the existence of (26) Sille`n, L. G. Acta Chem. Scand. 1956, 10, 186.
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Table 3. Protonation Constants of Glycocholate Ion (GC-) and Solubility of Glycocholic Acid (HGC) in the Same Experimental Conditions Selected in This Investigationa W
-log s
-log k1
-log kb
0.100 0.500 0.800
3.62 3.60 3.55
3.48 3.54 3.61
2.55 2.53 2.52
a Protonation constants are defined by the following expressions: cHGC ) k1cHcGC and ecH2GC ) kbcHcHGC. Charges are omitted.
Table 4. Proposed Values for Constants (log βq,0,r) of the Species [Naq(GC)r] at 25 °C and Different W (mol dm-3) W (mol dm-3) ) 0.100 Na(GC)2 (log β1,0,2 ) 3.41 ( 0.03) Na2(GC)4 (log β2,0,4 ) 8.20( 0.05) W (mol dm-3) ) 0.500 Na(GC)2 (log β1,0,2 ) 3.82 ( 0.05) Na3(GC)4 (log β3,0,4 ) 11.26 ( 0.07) Na3(GC)6 (log β3,0,6 ) 14.95 ( 0.10) Na6(GC)8 (log β6,0,8 ) 24.20 ( 0.13) W (mol dm-3) ) 0.800 Na(GC)2 (log β1,0,2 ) 3.70 ( 0.05) Na3(GC)4 (log β3,0,4 ) 11.20 ( 0.08) Na6(GC)8 (log β6,0,8 ) 25.52 ( 0.12) Na7(GC)12 (log β7,0,12 ) 35.84 ( 0.15) Na10(GC)16 (log β10,0,16 ) 50.15 ( 0.18) Na14(GC)24 (log β14,0,24 ) 74.3 ( 0.2)
species with very low aggregation numbers. From eq 9, it can be obtained that η depends on CNa and CGC. At this stage, conditional constants by means of graphical methods26 can be obtained for constant values of log cGC, and their dependence on cGC can be studied. Equation 9 can be written as follows:
φ ) (CNa/cNa) - 1 )
∑qγq cNaq-1
Figure 1. An example of the dependence of η (log CNa/cNa) on log cGC. Points obtained at different -log cH fall on the same curve supporting the absence of protonated species in the range 5 e -log cH e 8.5.
(11)
where
γq )
∑βq,0,r cGCr
(12)
is a conditional constant. Equation 11 can give information on q and γq. By plotting φ versus cNa, the trend of experimental points can be well approximated with a straight line and it can be deduced that q can assume the values 1 and 2. From the intercept and the slope, γ1 and γ2 can be obtained, respectively. As the ratios γ1/cGC2 and γ2/cGC4 give constant values within acceptable limits of error, it could be assumed that their averages represent β1,0,2 and β2,0,4, respectively. Therefore, the existence of Na(GC)2 and Na2(GC)4 can be assumed. This result was verified by introducing β1,0,2 and β2,0,4 in the material balance (eq 9), which can be written as follows:
ψ ) (CNa - cNa)/(cNacGC2) ) β1,0,2 + 2β2,0,4cNacGC2 Figure 2 shows the ψ versus τ plot (τ ) cNacGC2). All of the points lie on a straight line, which provides the refined β1,0,2 and 2β2,0,4 values. The good fitting supports the validity of the employed procedure and of the obtained results. W ) 0.800 mol dm-3. The procedure to elaborate the experimental data was similar to that above described, but the increased complexity of this system suggested
Figure 2. The dependence of the function ψ versus τ (where τ ) cNacGC2) at W ) 0.100 mol dm-3 N(CH3)4Cl. The points can be well fitted by a straight line.
consideration of groups of data where only two or three species were present. Results were refined by an iterative procedure. The best agreement was obtained assuming the existence of Na(GC)2, Na3(GC)4, Na6(GC)8, Na7(GC)12, Na10(GC)16, and Na14(GC)24 aggregates. Their relative constants were calculated. It must be stressed that the introduction of species with odd and some even (6, 10, 14, 20, 22) aggregation numbers did not provide a satisfactory agreement. To verify the validity of the procedure, species and constants were introduced in the material balance (eq 9) which can be transformed as follows:
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ξ ) log[(CNa - cNa - β1,0,2cNacGC2 - 3β3,0,4cNa3cGC4 7β7,0,12cNa7cGC12) (cNa6cGC8)-1] ) log(6β6,0,8) + log[1 + (10β10,0,16/β6,0,8)cNa4cGC8 + (14β14,0,24/6β6,0,8)cNa8cGC16] (13) The trend of ξ versus log F (F ) cNa4cGC8) is shown in Figure 3. Equation 13 can be compared with a family of normalized curves of the equation y ) log(1 + Ru+ u2), where log(6β6,0,8) ) ξ - y; log u ) log cNa4cGC8 + (1/2) log(14β14,0,24/ 6β6,0,8) and log R ) log(10β10,0,16) - (1/2) log(6β6,0,8) - (1/2) log(14β14,0,24). By superimposing the observed and calculated plots and moving one of them in parallel to the abscissa and ordinate axes in order to get the best fit, β6,0,8, β10,0,16, and β14,0,24 were obtained. The good agreement shown in Figure 3 supports this procedure. Constant refined values were obtained using the experimental data independently of graphical methods. A computer refinement program27 that uses the GaussNewton technique processed experimental emf values and reagent analytical concentrations. The refinement provided the same above-mentioned species with similar constants. Both species and constants are collected in Table 4. The error limits of the graphical method were obtained from the possible maximum shift between theoretical and experimental curves taking into account the error bars. Errors are higher than standard deviations given by the program. Results of Table 4 are relative to the range 5 e -log cH e 8.5 where the presence of protonated species can be neglected. Some trials to extend the investigation at -log cH < 5 indicate the existence of NaH(GC)2, Na2H(GC)4, and Na10H(GC)16 species, but we were not able to obtain the relative constants, because their concentrations were very low and the investigation range was very narrow owing to the slight HGC solubility. A comparison with NaTC concerning the protonated species shows that NaTC forms more species than NaGC, and solutions even at -log cH ) 3 can be prepared because taurocholic acid is soluble. As taurocholate anion protonation occurs at lower -log cH than that of GC-, it is evident that GC- has more affinity for protons than taurocholate anion. A similar comparison between GC- and glycodeoxycholate anion (GDC-) shows wide differences. Protonated species formation of GC- takes place at -log cH e 5, while for GDC- it begins already at -log cH ≈ 7.5. The species and the relative constants collected in Table 4 were used to calculate the percentage of each species as a function of the GC- aggregation number. The results can be seen in Figure 4 as a histogram. Figure 4 is divided in three parts corresponding to W ) 0.100, 0.500, and 0.800 mol dm-3. Percentages are calculated for NaGC concentrations 0.020, 0.050, and 0.080 mol dm-3. All the species have even aggregation numbers, and species with odd r are not present in appreciable concentration. The GC- percentage reaches the maximum value of 50% for W ) 0.100 mol dm-3 and decreases to 35% for W ) 0.500 mol dm-3 and to 30% for W ) 0.800 mol dm-3. The GC- percentage decreases more rapidly and arrives at 10% when its concentration increases from 0.020 to 0.080 mol dm-3. The (GC)2 dimer is always present in a remarkable amount. When the ionic medium concentra(27) De Stefano, C.; Mineo, P.; Rigano, C.; Sammartano, S. Ann. Chim. (Rome) 1993, 83, 243.
Figure 3. The trend of the points at W ) 0.800 mol dm-3 N(CH3)4Cl. The dependence of the function ξ versus log F (where F ) cNa4cGC8) and the agreement with the normalized curve support the assumption of the proposed aggregates.
Figure 4. Percentages of NaGC micellar aggregates, expressed as GC- concentration, as a function of anion aggregation numbers at -log cH ) 7.
tion is increased (from 0.100 to 0.800 mol dm-3), species with higher aggregation numbers appear and (GC)8, (GC)16, and (GC)24 percentages, with r being a multiple of eight, increase. Also, (GC)12 is present, but its percentage is low. It seems reasonable to suppose that the (GC)2 dimer or the (GC)8 octamer constitutes the building unit of micellar aggregates at low or high reagent and ionic medium concentrations, as it was observed for NaTC. NaDC, NaGDC, and NaTDC show a different behavior than NaGC and NaTC. Dihydroxy salts seem to be constituted by trimers, with a 3-fold rotation axis, that are the building units of 8/1 or 7/1 helices.12,13 This peculiarity of their structure is supported by emf data. NaTDC forms most aggregates with r values being a
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multiple of three,20 whereas NaGDC19 and NaDC18 show the presence of trimers up to W ) 0.75 and 0.6, respectively. On the basis of the observed species, a wide difference exists between dihydroxy (NaDC, NaGDC, and NaTDC) and trihydroxy (NaGC and NaTC) salts. Dimers and octamers of the type found in both NaTC14,28 and NaGC16,29 crystals and fibers suitably represent the building blocks of NaTC and NaGC micellar aggregates. In particular, dimers have a 2-fold rotation or a 2-fold screw axis and are arranged in structural units obtained by translation along the dimer 2-fold rotation or screw axis. Dimers and octamers, formed by an assembly of four dimers, are mainly stabilized by strong polar interactions, that involve especially the cations, as inferred from X-ray analysis, electrolytic conductance, and dielectric measurements.16 Moreover, the dimer structure provides a suitable model for the enantioselective complexation of bilirubin-IXR, in agreement with circular dichroism (CD) spectra and potential energy calculations.28 Because cations can strongly interact with dimers and octamers utilizing polar binding sites (mainly anion polar heads), a high fraction of sodium ions are bound, in agreement with the composition of the species here proposed, containing several sodium ions (q increases by increasing the aggregate size). Species of Table 4 could be written with a mechanism “core + link”. For example, an aggregate can be written as Nam[Na(GC)2]n, where different values can be assumed by m (1 or 2) and n (from 1 to 12). This suggests that the aggregates can be formed by association of [Na(GC)2] units. The number of [Na(GC)2], n, increases on increasing the NaGC or the ionic medium concentration. The bigger species can be represented also by the formula Nam′ [Na4(GC)8]n′, where m′ is 2 and n′ can be 1, 2, or 3.
Bottari et al.
species and their equilibrium constants, collected in Table 4. Measurements of all three parameters cNa, cH, and cGC of equilibrium 1 ensure high accuracy and provide experimental data suitable for determining the q, p, and r prevailing values. Table 4 sets, obtained by assuming the minimum number of species with the lowest q, p, and r values, represent the aggregate distributions necessary to satisfactorily fit the experimental data. A dimeric species, observed in all the samples, seems to be the building unit of the micellar aggregates, which in all cases have aggregation numbers which are multiples of two. It seems also that by increasing NaGC and ionic medium concentrations, four dimers further aggregate to form an octamer, which is the basic unit of bigger aggregates. Octamer multiples with aggregation numbers 16 and 24 have been found under experimental conditions favoring the formation of bigger aggregates. High q values, in agreement with conductance measurements,16 indicate that sodium ions strongly contribute to the aggregate formation and stability and have more affinity for the anion aggregates than N(CH3)4+ ions, in agreement with structural results.30 The present results confirm that NaGC and NaTC show very similar structures and properties, but their behavior markedly differs from that of NaDC, NaGDC, and NaTDC. Trihydroxy or dihydroxy salt solutions and fibers form structures with aggregation numbers being multiples of two or three, the dimer or the trimer being the basic unit. Finally, from emf and structural results of NaGC and NaTC it seems probable that the transitions [aqueous micellar solution] f [gel] f [fiber] occur without drastic structural changes and that the fiber and solution models are very similar.
Conclusion The investigation of NaGC aqueous solutions at 25 °C and in an ionic medium of N(CH3)4Cl at three different concentrations permits the determination of the existing (28) D’Alagni, M.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Scaramuzza, L. Trans. Faraday Soc. 1994, 90, 1523. (29) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367.
Acknowledgment. This work was sponsored by the Italian National Research Council (CNR) and by the Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica (MURST). LA0111349 (30) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Punzo, F. Langmuir 2001, 17, 4096.
