Reverse micellar solution

Product recoveiy from reversed micellar solutions can often be attained by simple back extrac tion, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solu-bihzation, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, for example, to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or sihca gel has also been found to cause a precipitation of the protein from the organic phase. 

Investigation of water motion in AOT reverse micelles determining the solvent correlation function, C i), was first reported by Sarkar et al. [29]. They obtained time-resolved fluorescence measurements of C480 in an AOT reverse micellar solution with time resolution of > 50 ps and observed solvent relaxation rates with time constants ranging from 1.7 to 12 ns. They also attributed these dynamical changes to relaxation processes of water molecules in various environments of the water pool. In a similar study investigating the deuterium isotope effect on solvent motion in AOT reverse micelles. Das et al. [37] reported that the solvation dynamics of D2O is 1.5 times slower than H2O motion. 

A further possibility is the formation of liquid crystals on contact with body fluids at the site of application. The initially applied drug solution interacts with body fluids such as plasma, tears, or skin lipids and undergoes a phase transition into a mono-or multiphasic system of liquid crystals (Fig. 15). For example, oily solutions of reverse micellar solutions of phospholipids, which solubilize additional drug, trans-... 

Mueller-Goymann, C.C., and Hamann, H.-J., Sustained release from reverse micellar solutions by phase transformation into lamellar liquid crystals, J. Contr. Rel., 23 165-174 (1993). 

Friedrich I. and Muller-Goymann C.C., Characterization of solidified reverse micellar solutions (SRMS) and production development of SRMS-based nanosuspensions, Eur. J. Pharm. Biopharm., 56, 111, 2003. 

Dungan et al. [186] have measured the interfacial mass transfer coefficients for the transfer of proteins (a-chymotrypsin and cytochrome C) between a bulk aqueous phase and a reverse micellar phase using a stirred diffusion cell and showed that charge interactions play a dominant role in the interfacial forward transport kinetics. The flux of protein across the bulk interface separating an aqueous buffered solution and a reverse micellar phase was measured for the purpose. Kinetic parameters for the transfer of proteins to or from a reverse micellar solution were determined at a given salt concentration, pH, and stirring... 

Although the thermodynamic model for protein solubihzation in - and water distribution over - the reverse micellar solutions presented here is consistent with the experimental results, it must be recognized that significant improvements can be made in the model formulation. Obvious extensions include a... 

An organic phase can be used several times provided the sample feed (fermentation broth) does contain cells or cell debris. Presence of such contaminants may render it necessary to regenerate the organic phase for its prolonged use. A literature survey suggests that the knowledge available on the recovery and reuse of surfactants is very little. However, the removal of surfactants from the stripping aqueous solution can be achieved by filtration and then can be recycled [10]. Use of ultrafiltration was also shown to be a successful technique for the separation of surfactants from reverse micellar solution [203]. 

In order to obtain a thermodynamically stable micro emulsion, the analysis of the phase behaviour is indispensable. With bovine serum albumin instead of an enzyme (because of the cost of the bio-catalyst) phase behaviour studies are shown in Fig. 2. A strong shift of the phase boundary is observed, yielding a system that solubilises much less water in the presence of the protein. In case of hydrophobic enzymes, the addition of dry lyophilised protein to an already prepared reverse micellar solution can also work well [53]. 

The importance of the particular compartmentation in this field is made apparent by a series of interesting and partly still unexplained effects. For example, when the amount of water is varied in the reverse micellar solution, the maximum enzyme activity - even in the case of hydrolases - is not observed with higher water-content values, but with relatively low amounts of water. In addition, the local pH - due to the constraints of the water pool - is anomalous with respect to the pH value in water (El Seoud, 1984 Luisi and Straub, 1984). 

Figure 9.13 Dynamic-light-scattering size distribution (angle 120°) of a CgPC reverse micellar solution, containing aqueous DNA solution, wq = 5. (a) 0.5 mg mD DNA (b) 4 mg ml DNA. In (b) three size distributions are plotted, referring to 15 min (—) 1 d (- -) 6 d (-0-) from the preparation of the micellar solution (from Ousfuri et al, 2005) i and iii are empty micelles ii and iv are DNA-containing micelles.

The intermicellar exchange process, governed by the attractive interactions between droplets, can be modified by changing the bulk solvent used to form reverse micellar solution (26). This is due to the discrete nature of solvent molecules and is attributed to the appearance of depletion forces between two micelles (the solvent is driven off between the two droplets) (26). When the droplets are in contact forming... 

The influence of the addition of cetyl trimethyl ammonium chloride, CTAC, to the reverse micellar solution affects the droplet size and micellar interactions, as demonstrated by the DQLS experiment (64). Addition of CTAC to micellar system at a given water content leaves the droplet size unchanged, whereas a decrease in the intermicellar attraction has been observed. This decrease is more important for high CTAC concentrations. This has been interpreted to steric repulsion induced by the long hydrocarbon tail of CTAC (C ft). Thus, the addition of this compound to CdS synthesis could modify the nucleation and/or growth process. The experiments were performed by solulization of CTAC in the micellar solution containing either sodium sulfide or Cd(AOT)2. 

At low water content from vv = 2 to 5.5, a homogeneous reverse micellar solution (the L2 phase) is formed. In this range, the shape of the water droplets changes from spheres (below ir = 4) to cylinders. At tv — 4, the gyration radius has been determined by SAXS and found equal to 4 nm. Syntheses in isolated water-in-oil droplets show formation of a relatively small amount of copper metallic particles. Most of the particles are spherical (87%) with a low percentage (13%) of cylinders. The average size of spherical particles is characterized by a diameter of 12 nm with a size polydispersity of 14%. 

Pulsed gradient spin-echo (PGSE) NMR techniques have also been employed to study the structure of the oil phase [12]. This gives an idea of the mobility of each component in the HIPE, and showed that, for stable emulsions and HIPEs, the oil phase was indeed a reverse micellar solution which solubilises water. Further work using PGSE NMR has shown that water can diffuse between aqueous droplets in concentrated emulsions [101]. Presumably this involves solubilisation of the water molecules by the micellar oil phase. 

In case of non-ionic surfactants in water, the behaviour of the water structure outlines three main concentration regions, which closely coincide with the three phases intersected by the experimental isotherms. In the micellar solution phase, no significant changes in the water structure are indicated, while, in the lamellar phase, rapid destruction of the tetrahedral hydrogen bond network occurs due to the confinement of the water between the hydrophilic surfaces of the lamellae. The dehydration of the surfactant head groups was found to start near the border between the lamellar and the reverse micellar solution phases. At higher concentrations, water demonstrates its trend to form clusters of tetrahedrally bonded molecules even at the very low content in the system. The results with surfactant solutions have been obtained by Raman spectroscopy (Marinov el al., 2001). 

For small amounts of solubilized water, as a polar additive, the stability of the micelle is markedly increased, as shown by a decrease in the CMC. On the other hand, large amounts of water as a polar additive decrease the stability of the micelle. It is known that a solution of AOT in iso-octane solubilized up to 50 moles of water per mole of surfactant. As the concentration of water increases, the isotropic reverse micellar solution changes to a water-in-oil microemulsion. A clear understanding of the complex analyte-micelle-water pool interactions, especially analyte concentration and pH at the head group interfacial region, is under intensive study (Cline Love and al., 1984 Little and Singleterry, 1964 Luisi and Straub, 1984 Mclntire, 1990). 

Reverse micelles are self-organized aggregates of amphiphilic molecules that provide a hydrophilic nano-scale droplet in apolar solvents. This polar core accommodates some hydrophilic biomolecules stabilized by a surfactant shell layer. Furthermore, reverse micellar solutions can extract proteins from aqueous bulk solutions through a water-oil interface. Such a liquid-liquid extraction technique is easy to scale up without a loss in resolution capability, complex equipment design, economic limitations and the impossibility of a continuous mode of operation. Therefore, reverse micellar protein extraction has great potential in facilitating large-scale protein recovery processes from fermentation broths for effective protein production. 

This shows that haemoglobin is recovered from a reverse micellar solution only at higher pH values than pl and high salt concentrations to inhibit the electrostatic... 

FIGURE 14.7. Effect of the addition of alcohol on the lithium ion leakage from DOLPA reverse micelles during back extraction. A back-extraction operation was performed by contacting the reverse micellar solution extracting lithium in advance and the fresh recovery of the aqueous solution. The observed amounts of lithium ion in each phase were measured using an ICP analyser. 

The ratio of the water content in the reverse micellar solution after back extraction to that before back extraction. 

The functionalization of the reverse micelles will create a novel application in bioseparation processes in the analytical and medical sciences. It is therefore important to reveal the recognition mechanism of proteins at the liquid-liquid interface in reversed micellar solutions. DNA is also successfully extracted in a few hours by reversed micelles formed by cationic surfactants in isooctane. The driving force of the DNA transfer is the electrostatic interaction between the cationic surfactants and the negatively charged DNA. Another important factor is the hydrophobicity of the cationic surfactants. Doublechain type cationic surfactants are found to be one of the best surfactants ensuring the efficient extraction of DNA. These results have shown that reverse micellar solutions will become a useful tool not only for protein separation, but also for DNA separation. 

Kinugasa T, Tanahashi S, Takahashi S, and Takeuchi H. Transport of proteins through reversed micellar solution layer as a liquid membrane. In Proceedings of ISEC 90, 16-21 July 1990 Kyoto, Japan, Vol. 13 pp. 1839-1846. 

Priedrich 1, Reichl S, MuUer-Goymann CC (2005) Drug release and permeation studies of nanosuspensions based on sohdified reverse micellar solutions (SRMS). hit J Pharm 305 167-175. 

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