Aqueous biphasic systems

The 'incompatibility' of certain polymers in aqueous solution was first noted by Beijerinck in 1896. In this case two phases were formed when agar was mixed with soluble starch or gelatine. Since then, many two phase aqueous systems have been found; the most thoroughly investigated being the aqueous dextran-polyethylene glycol system (e.g., 10% polyethylene glycol 4000/2% dextran T500), where dextran forms the more hydrophilic, denser, lower phase and polyethylene glycol the more hydrophobic, less dense, upper phase. Aqueous three phase systems are also known.

Phases form when limiting concentrations of the polymers are exceeded. Both phases contain mainly water and are enriched in one of the polymers. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH, ionic strength and temperature of the solution. Some polymers form the upper hydrophobic phase in the presence of fairly concentrated solutions of phosphates or sulphates (e.g., 10% polyethylene glycol 4000/12.5% potassium phosphate buffer). A drawback to the useful dextran/polyethylene glycol system is the high cost of the purified dextran used. This has been alleviated by the use of crude unfractionated dextran preparations, much cheaper hydroxypropyl starch derivatives and salt-containing biphasic systems.

Aqueous biphasic systems are of considerable value to biotechnology. They provide the opportunity for the rapid separation of biological materials with little probability of denaturation. The interfacial tension between the phases is very low (i.e., about 400-fold less than that between water and an immiscible organic solvent), allowing small droplet size, large interfacial areas, efficient mixing under very gentle stirring and rapid partition. The polymers have a stabilising influence on most proteins. A great variety of separations have been achieved, by far the most important being the separation of enzymes from broken crude cell material. Separation may be achieved in a few minutes, minimising the harmful action of endogenous proteases. The systems have also been used successfully for the separation of different types of cell membranes and organelles, the purification of enzymes and for extractive bioconversions (see Chapter 7). Continuous liquid two-phase separation is easier than continuous solid/liquid separation using equipment familiar from immiscible solvent systems, for example disc-stack centrifuges and counter-current separators. Such systems are readily amenable to scale-up and may be employed in continuous enzyme extraction processes involving some recycling of the phases.

Cells, cell debris proteins and other material distribute themselves between the two phases in a manner described by the partition coefficient (P) defined as: 

               (2.4)

where C_t and C_b represent the concentrations in the top and bottom phases respectively. The yield and efficiency of the separation is determined by the relative amounts of material in the two phases and therefore depends on the volume ratio (V_t/V_b). The partition coefficient is exponentially related to the surface area (and hence molecular weight) and surface charge of the particles in addition to the difference in the electrical potential and hydrophobicity of the phases. It is not generally very sensitive to temperature changes. This means that proteins and larger particles are normally partitioned into one phase whereas smaller molecules are distributed more evenly between phases. A partition coefficient of greater than 3 is required if usable yields are to be achieved by a single extraction process. Typical partition coefficients for proteins are 0.01-100 whereas the partition coefficients for cells and cell debris are effectively zero.

The influence of pH and salts on protein partition is complex, particularly when phosphate buffers are present. A given protein distributes differently between the phases at different pH's and ionic strength but the presence of phosphate ions affect the partition coefficient in an anomalous fashion because these ions distribute themselves unequally resulting in electrostatic potential (and pH) differences. This means that systems may be 'tuned' to enrich an enzyme in one phase, ideally the upper phase with cell debris and unwanted enzymes in the lower phase.

An enzyme may be extracted from the upper (polyethylene glycol) phase by the addition of salts or further polymer, generating a new biphasic system. This stage may be used to further purify the enzyme. A powerful modification of this technique is to combine phase partitioning and affinity partitioning. Affinity ligands (e.g., triazine dyes) may be coupled to either polymer in an aqueous biphasic system and thus greatly increase the specificity of the extraction.