In 1896, Beijerinck first noted an 'incompatibility' in solutions of agar, a water-soluble polymer, with soluble starch or gelatine. Upon mixing, they separated into two immiscible phases. Subsequent investigation led to the determination of many other aqueous biphasic systems, of which the polyethylene glycol - dextran system is the most extensively studied. Other systems that form aqueous biphases are: PEG - sodium carbonate or PEG and phosphates, citrates or sulfates. Aqueous biphasic systems are used during downstream processing mainly in biotechnological and chemical industries.
The two phases
It is a common observation that when oil and water are poured into the same container, they separate into two phases or layers, because they are immiscible. In general, aqueous solutions, being polar, are immiscible with non-polar organic solvents and form a two-phase system. However, in an ABS, both immiscible components are water-based. The formation of the distinct phases is affected by the pH, temperature and ionic strength of the two components, and separation occurs when the amount of a polymer present exceeds a certain limiting concentration.
PEG–dextran system
The "upper phase" is formed by the more hydrophobic polyethylene glycol, which is of lower density than the "lower phase," consisting of the more hydrophilic and denser dextran solution. Although PEG is inherently denser than water, it occupies the upper layer. This is believed to be due to its solvent 'ordering' properties, which excludes excess water, creating a low density water environment. The degree of polymerization of PEG also affects the phase separation and the partitioning of molecules during extraction.
Advantages
ABS is an excellent method to employ for the extraction of proteins/enzymes and other labilebiomolecules from crude cell extracts or other mixtures. Most often, this technique is employed in enzyme technology during industrial or laboratory production of enzymes.
They provide mild conditions that do not harm or denature unstable/labile biomolecules
The interfacial stress is far lower than water-organic solvent systems used for solvent extraction, causing less damage to the molecule to be extracted
The polymer layer stabilizes the extracted protein molecules, favouring a higher concentration of the desired protein in one of the layers, resulting in an effective extraction
Specialised systems may be developed to favour the enrichment of a specific compound, or class of compounds, into one of the two phases. They are sometimes used simultaneously with ion-exchange resins for better extraction
Separation of the phases and the partitioning of the compounds occurs rapidly. This allows the extraction of the desired molecule before endogenousproteases can degrade them.
These systems are amenable to scale-ups, from laboratory-sized set-ups to those that can handle the requirements of industrial production. They may be employed in continuous protein-extraction processes.
Specificity may be further increased by tagging ligands specific to the desired enzyme, onto the polymer. This results in a preferential binding of the enzyme to the polymer, increasing the effectiveness of the extraction. One major disadvantage, however, is the cost of materials involved, namely high-purity dextrans employed for the purpose. However, other low-cost alternatives such as less refined dextrans, hydroxypropyl starch derivatives and high-salt solutions are also available.
Thermodynamic Modeling
Besides the experimental study, it is important to have a good thermodynamic model to describe and predict liquid-liquid equilibrium conditions in engineering and design. To obtain global and reliable parameters for thermodynamic models usually, phase equilibrium data is suitable for this purpose. As there are polymer, electrolyte and water in polymer/salt systems, all different types of interactions should be taken into account. Up to now, several models have been used such as NRTL, Chen-NRTL, Wilson, UNIQUAC, NRTL-NRF and UNIFAC-NRF. It has been shown that, in all cases, the mentioned models were successful in reproducing tie-line data of polymer/salt aqueous two-phase systems. In most of the previous works, excess Gibbs functions have been used for modeling.
