Micellar bile salt solutions

Interaction in Bulk of Monoolein, Oleic Acid, and Sodium Oleate with Dilute, Micellar Bile Salt Solutions... 

Effect of Bulk pH on Behavior and Solubility of Oleic Acid in Bile Salt Solution. Figure 2 shows the effect of bulk pH on the behavior and solubility of oleic acid in 0.15M buffer (above) and in 4 mM sodium glycodeoxycholate (below). In buffer, oleic acid has an extremely low solubility, and the excess, below pH 6.8, is present as an emulsion. In micellar bile salt solution, the oleic acid is solubilized to some extent. Above pH 6.5, its solubility rises markedly, and the excess now forms a dispersed phase which probably consists of droplets of fatty acid emul-... 

Hofmann, A. F. The behaviour and solubility of monoglycerides in dilute micellar bile salt solution. Biochim. biophys. Acta (Amst.) 70, 306 (lOffB). 

For this water concentration, the micellar region for the bile salt mixture is large for all oleyl compounds except oleic acid. Oleic acid is distinguished from the other compounds in that it does not form a lyotropic liquid crystalline phase spontaneously in water and, similarly, is present as oil droplets in bile salt solution when its micellar solubility is exceeded. Figure 1 shows also that the micellar area of an equimolar mixture of monoolein and sodium oleate is considerably greater than that of an equimolar mixture of monoolein and oleic acid, indicating that fatty acid ionization also enhances micellar solubility when monoolein is present. The equimolar mixture of sodium oleate and oleic acid has a micellar area similar in size to that of monoolein, as does the equimolar combination of all three compounds. 

Compounds possessing a small micellar area (clear) form an immiscible oil phase (diagonal hatching) when present in excess in bile salt solution or in buffer in absence of bile salt. Compounds with high micellar solubility form a liquid crystalline phase when present in excess in bile salt solutions and form liquid crystalline phases in buffer alone... 

The data clearly indicate that the surface pH of the bile salt micelle is higher than the surface pH of a lauryl taurate micelle for a given bulk pH—i.e., the difference between bulk and surface pH is less with the bile salt micelle. The bile salt micelle should have a lower charge density and therefore a lower concentration of protons at the surface of the micelle. Therefore, the observed bulk pH at which micellar fatty acid ionizes is closer to the bulk pKa of molecularly dispersed fatty acid (4.9) in bile salt solution than in lauryl taurate solution. 

When a bicontinuous cubic lipid-water phase is mechanically fragmented in the presence of a liposomal dispersion or of certain micellar solutions e.g. bile salt solution), a dispersion can be formed with high kinetic stability. In the polarising microscope it is sometimes possible to see an outer birefringent layer with radial symmetry (showing an extinction cross like that exhibited by a liposome). However, the core of these structures is isotropic. Such dispersions are formed in ternary systems, in a region where the cubic phase coexists in equilibrium with water and the L(x phase. The dispersion is due to a localisation of the La phase outside cubic particles. The structure has been confirmed by electron microscopy by Landh and Buchheim [15], and is shown in Fig. 5.4. It is natural to term these novel structures "cubosomes". They are an example of supra self-assembly. 

Over 50 methods have been employed in the literature to determine CMC values of bile salt solutions (reviewed in [6]). These can be divided into two broad categories (a) methods requiring no physical or chemical additive in the bulk solution and (b) methods involving the use of an additive in the bulk solution. The former methods, also called non-invasive, include surface tension and the measurements of a variety of colligative bulk properties (conductivity, turbidimetry, osmometry, self-diffusion, refractive index, modal volumes, electrometric force) or electromagnetic bulk properties (NMR, sound velocity and adsorption, etc.), all as functions of bile salt concentration. The second set of methods, also called invasive, depends upon a change in some physical or chemical property of an additive which occurs with the formation of micelles. These include the spectral change of a water-soluble dye, micellar solubilization of a water-insoluble dye, interfacial tension at liquid-liquid interfaces, and partition coefficients between aqueous and immiscible non-polar phases. Whereas a detailed discussion of the merits and demerits of both approaches can be found elsewhere [6], non-invasive methods which are correctly utilized provide the most reliable CMC values. 

The average size of bile salt micelles and the distribution of micellar sizes around the mean value are important physical-chemical characteristics of a bile salt solution [5,6]. Because bile salt micellar growth is sensitive to total detergent concentration within the micellar phase, with temperature and ionic strength, the physical-chemical conditions must be rigorously controlled and specified [5,6]. Further, most... 

Johns WH and Bates TR., Quantification of the binding tendencies of cholestyramine I effect of structure and added electrolytes on the binding of unconjugated and conjugated bile salt anions, /. Pharm. Sci., 58,179-183 (1969). NB These values were quoted from Ekwall P, Rosendahl T and Lofman N, Bile salt solutions. I. The dissociation constants of Bie cholic and deoxycholic acids, Acta Chem. Scand., 11,590-598 (1957). They were measured at concentrations boBi above and below Bie critical micellar concentration range. 

Fig. 9. Phase equilibria for the bile salt (bile acid)-fatty acid-water system at constant water concentration in relation to temperature (see Fig. 5). Six mixtures varying in molar ratios of bile salt (bile acid) and palmitic acid with total concentration of micellar bile acid plus palmitic acid equal to 40 mM were examined. Fatty acid has a finite solubility in the micellar bile acid solution, the excess being crystalline at body temperature. At 50-60 C, there is a marked increase in micellar solubility, and the fatty acid melts. At higher fatty acid/bile acid ratios, the micellar solubility is exceeded, and an immiscible oil phase occurs. The melting point of fatty acid in the presence of water is nearly identical to that in the anhydrous state (38), in contrast to the behavior of monoglyceride (Table I). As shown in Fig. 3, the size of the micellar area decreases with increasing chain length. Unsaturated fatty acids (not shown) behave similarly to saturated fatty acids, but their micellar solubility is greater, and at most experimental temperatures a crystalline phase will not occur.

MLC uses micellar mobile phases with classical RPLC columns. This chapter expands the field to include some mobile phases that can be considered close to micellar phases, such as normal and reverse microemulsions, bile salt solutions, and surfactant solutions in supercritical fluids. Also, this chapter rapidly surveys the use of micellar mobile phases with non-RPLC stationary phases such as size exclusion or gel permeation polymer phases. Allied techniques using micellar phases such as ion-exchange chromatography and capillary electrophoresis are also briefly presented. 

Chapter 2 discussed microemulsion structure. These organized media are stable and transparent. They are possible candidates for mobile phases in chromatography. Bile salt solutions are another kind of special micelles with chiral properties that can be used in MLC as well. Supercritical fluids (SF) were also used as surfactant solvents to perform micellar SFC, a variation of MLC. 

Hinze was the first to investigate the capabilities of micellar bile salt mobile phases [11, 12]. He found that a significant amount ( 5% v/v) of a long chain n-alcohol (pentanol, hexanol or heptanol) was useful to minimize the bile salt adsorption on the C18 stationary plmse. A wide range of solutes could be separated by these phases, PAHs, quinones, steroids, indoles, polar and lipophilic vitamins. These phases were also able to resolve optically big enantiomers such as binaphthyl derivatives [12]. Such compounds are... 

Ekwall and Baltcheffsky [265] have discussed the formation of cholesterol mesomorphous phases in the presence of protein-surfactant complexes. In some cases when cholesterol is added to these solutions a mesomorphous phase forms, e.g. in serum albumin-sodium dodecyl sulphate systems, but this does not occur in serum albumin-sodium taurocholate solutions [266]. Cholesterol solubility in bile salt solutions is increased by the addition of lecithin [236]. The bile salt micelle is said to be swollen by the lecithin until the micellar structure breaks down and lamellar aggregates form in solution the solution is anisotropic. Bile salt-cholesterol-lecithin systems have been studied in detail by Small and coworkers [267-269]. The system sodium cholate-lecithin-water studied by these workers gives three paracrystalline phases I, II, and III shown in Fig. 4.37. Phase I is equivalent to a neat-soap phase, phase II is isotropic and is probably made up of dodecahedrally shaped lecithin micelles and bile salts. Phase III is of middle soap form. The isotropic micellar solution is represented by phase IV. The addition of cholesterol in increasing quantities reduces the extent of the isotropic... 

Preparation of monodisperse vesicles with variable size by dilution of mixed micellar solutions of bile salt and phosphatidylcholine, Biochim. Biophys. Acta, 775. 111-114. 

Micellar electrokinetic capillary chromatography (MECC), in contrast to capillary electrophoresis (CE) and capillary zone electrophoresis (CZE), is useful for the separation of neutral and partially charged species [266,267]. In MECC, a surfactant, usually sodium dodecyl sulfate (SDS), is added to the buffer solution above its critical micellar concentration to form micelles. Although SDS is certainly the most popular anionic surfactant in MECC, other surfactants such as bile salts have proved to be very effective in separating nonpolar analytes that could not be resolved using SDS [268]. 

The initial concentration, C0, in square centimeters, was determined directly from the area under the curve of a synthetic boundary trace. Although usual practice is to place solvent in one cell and the solution in the other, the solvent used in this work was a solution of bile salt slightly above its critical micellar concentration, CMC (15), usually 0.5 gram per 100 ml. The solution, which was placed in the other cell, was a more concentrated solution (1.5 to 5.0 grams per 100 ml.). Therefore, since both the solvent and the solution were saturated with monomers, C0 represents an initial concentration difference of bile salts in the micellar phase. The pertinent data for each equilibrium ultracentrifugation experiment are presented in Table I. 

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