Liquid-liquid extraction is a well established technology in chemical processing and in certain sectors of biochemical processing. However, the use of organic solvents has limited application in the processing of sensitive biologicals. Aqueous two-phase systems, on the other hand, have a high water content and low interfacial surface tension and are regarded as being biocompatible (Mattiasson and Ling, 1987).
Two-phase aqueous systems have been known since the late nineteenth century, and a large variety of natural and synthetic hydrophilic polymers are used today to create two (or more) aqueous phases. Phase separation occurs when hydrophilic polymers are added to an aqueous solution, and when the concentrations exceed a certain value two immiscible aqueous phases are formed. Settling time for the two phases can be prolonged, depending on the components used and vessel geometry. Phase separation can be improved by using centrifugal separators (Huddlestone et al, 1991), or novel techniques such as magnetic separators (Wikstrom et al, 1987).
Many systems are available:
(i) Non-ionic polymer/non-ionic polymer/water, e.g. polyethylene glycol/dextran.
(ii) Polyelectrolyte/non-ionic polymer/water, e.g. sodium carboxymethyl cellulose/polyethylene glycol.
(iii) Polyelectrolyte/polyelectrolyte/water, e.g. sodium dextran sulphate/sodium carboxymethyl cellulose
(iv) Polymer/low molecular weight component/water, e.g. dextran/propyl alcohol.
The phases distribution of a solute species between the is characterized by the partition coefficient, and ■"'influenced by a number of factors such as tempera-'iiie polymer (type and molecular weight), salt concen-n then, ionic strength, pH and properties (e.g. molecule weight) of the solute. As the goal of any extraction ■occss is to selectively recover and concentrate a solute, affinity techniques such as those applied in ■hmnatographic processes can be used to improve -I. clivity. Examples include the use of PEG-NADH iL-inatives in the extraction of dehydrogenases, p-.unmobenzamidine in the extraction of trypsin and iih.icron blue in the extraction of phosphofructokinase. 11 is possible to use different ligands in the two phases leading to an increase in selectivity or the simultaneous recovery and separation of several species (Cabral and Nik-Ban-os, 1993).
I wo phase aqueous systems have found application in ihe purification of many solutes; proteins, enzymes (Gonzalez et al., 1990; Guan et al, 1992), cells and subcellular particles, and in extractive bioconversions. Several aqueous two-phase systems for handling large-scale protein separation have emerged, the majority of which use PEG as the upper phase forming polymer with either dextran, concentrated salt solution or hy-droxypropyl starch as the lower phase forming material (Mattiasson and Kaul, 1986). Hustedt et al. (1988) demonstrated the application of continuous cross-current extraction of enzymes (fumarase and penicillin acylase) by aqueous two-phase systems at production scale.
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