Extraction using reversed micelles

Solvent extraction has long been established as a basic unit operation for chemical separations. Chapter 7 summarizes the effects of temperature, pH, ion pairs, and solvent selection on solvent extraction for biomolecules. Solvent extraction of fermentation products such as alcohols, aliphatic carboxylic acids, amino acids, and antibiotics are discussed. Enhanced solvent extraction using reversed micelles and electrical fields are also discussed. Solvent-extraction equipment and operational considerations are adequately covered in this chapter. 

FIGURE 4 Combined forward and back extraction involving reversed micelles. (From M. Dekker, K. Van t Riet, S. R. Weijers, J. W. Baltussen, C. Laane, and B. H. Bijisterboch, Enzyme recovery by liquid-liquid extraction using reversed micelles. Chem. Eng. J. 33, B27-33, 1986, with permission from Elsevier Science.)... 

E. L. V. Goetheer, M. A. G. Vorstman and J. T. F. Kuerentjes, Protein extraction using reverse micelles in supercritical carbon dioxide... 

KE Goklen, TA Hatton. Protein extraction using reverse micelles. Biotechnol Prog 1 69 74, 1985. 

The ease that certain protein mixtures can be separated using reverse micelle extraction was clearly demonstrated by Goklen and Hatton [46], Goklen [31], and Jarudilokkul et al. [25], who investigated a series of binary and ternary protein mixtures. In two cases, they were able to quantitatively extract cytochrome c and lysozyme from a ternary mixture of these proteins with ribonuclease A. Woll and Hatton [24] investigated the separation of a mixture of ribonuclease A and concanavalin A, and showed that the system behaved ideally and that there was no interaction between the proteins. 

Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water pool of surfactant aggregates [50,51]. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles [52]. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 

Extraction of Proteins and Amino Acids Using Reversed Micelles... 

Since most of the studies concerning protein extraction with reverse micelles have used a representative surfactant, AOT and trioctyl methyl ammonium chloride (TOMAC), to form reverse micelles, the protein extraction studies using a novel surfactant shed light on the important factors in the design of reverse micelles suitable for protein extraction. In particular, a comparative investigation concerning protein extraction efficiency with a variety of synthetic surfactants clarified the role of the hydrophobic tails of the surfactant on protein extraction [9]. 

On the protein extraction behaviour using reverse micelles, the protein-surfactant interaction between charged protein surfaces and surfactant headgroups is a dominant factor in distinguishing the target proteins. In particular, some researchers have suggested that a protein can be extracted as a hydrophobic ion complex between a protein and surfactant molecules [6,7,12-15]. Therefore, an intrinsic factor of proteins also gives considerable modulation in the extraction behaviour, in which the environmental factors were maintained. 

In this section, we will introduce the extraction behaviour of DNAs from an aqueous solution into an organic phase using reverse micelles, and show some important... 

Based on the polarity difference between CO2 and the interior of the micelles, w/c microemulsions have found many applications as extraction media. Furthermore, by modifying pressure and temperature, solvent quality may be changed and it becomes, therefore, possible to exert a real control over the extraction process uptake of solutes inside micelles may be varied. This may be of use for separations/extractions involving bio-chemicals and proteins. In conventional solvents their separation from the reaction medium can be quite complicated, involving tedious processes such as fluid-fluid extraction, decantation, chromatography column, filtration, precipitation. Use of supercritical fluid technology with extraction in reverse micelles seems advantageous for proteins (e.g. 19, 76). This process was also used for the extraction of metals (77-79) and more recently of copper from a filter paper surface (1). 

Hayadii, Y., Yoshioka, S., Aso, Y., Po, A. L. W., and Terao, T., 1994, Entrapment of proteins in poly(L-lactide) microspheres using reversed micelle solvent extraction, Pharm. Res. 11 337-340. 

One problem of using water-in-oil microemulsions for nanoparticle synthesis or chemical reaction is the separation and removal of solvent from products. A unique feature of the water-in-SCF microemulsions is that SCF reverse micelle phase stability is strongly dependent on the fluid pressure (density), at higher temperatures it is necessary to use higher pressures, or the density will be insufficient, the microemulsion will break, and phase separation can be accomplished [16]. So the reaction product can be obtained by a reduction in pressure without the need for extraction from reverse micelles as in the case of organic solvents [23]. 

Figure 3.5 Schematic representation of the extraction of proteins using reverse micellization.

Recent development of the use of reversed micelles (aqueous surfactant aggregates in organic solvents) to solubilize significant quantities of nonpolar materials within their polar cores can be exploited in the development of new concepts for the continuous selective concentration and recovery of heavy metal ions from dilute aqueous streams. The ability of reversed micelle solutions to extract proteins and amino acids selectively from aqueous media has been recently demonstrated the results indicate that strong electrostatic interactions are the primary basis for selectivity. The high charge-to-surface ratio of the valuable heavy metal ions suggests that they too should be extractable from dilute aqueous solutions. 

Using a solution of water-containing reversed micelles of di(2-ethylhexyl)phospho-rothioic acid in isooctane, hemoglobin was extracted and concentrated. Desolubilization of the protein entrapped in the reversed micelles by weak alkahne solution was realized by adding small amounts of n-octanol [167]. 

Salt type and concentration For back-extraction, increases in pH are not enough to strip the protein out from reverse micelles this is also due to the size exclusion elfect resulting from a decrease in the reverse micelle size [31,32]. This means that high salt concentration and salts that form small reverse micelles favor back transfer. Most of the work reported in the literature used KCl solution, normally 1.0 mol dm KCl coupled with a pH around 7.5. Marcozzi et al. [23] also showed that the back transfer efficiency of a-chymotrypsin depends on the salt type and concentration used in the forward transfer. 

Counterion extraction Due to the relative slowness of back extraction based on the methods above, the back-extraction of proteins encapsulated in AOT reverse micelles was evaluated by adding a counterionic surfactant, either TOMAC or DTAB, to the reverse micelles [33]. This novel backward transfer method gave higher backward extraction yields compared to the conventional method. The back-extraction process with TOMAC was found to be 100 times faster than back-extraction with the conventional method, and as much as three times faster than forward extraction. The 1 1 complexes of AOT and TOMAC in the solvent phase could be efficiently removed using adsorption onto montmorillonite so that the organic solvent could be reused. 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

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