Partitioning and Concentrating Biomaterials in Aqueous Phase Systems

Göte Johansson; Harry Walter    Department of Biochemistry, University of Lund, S-22100, Lund, Sweden
Aqueous Phase Systems, Washington D.C. 20008

I Introduction

A Immiscible Phases

Aqueous phase separation generally occurs when structurally distinct water-soluble macromolecules are dissolved, above certain concentrations, in water. First described by Beijerinck about one hundred years ago (1896), two-polymer aqueous-aqueous two-phase systems were applied by Albertsson, starting in the 1950s, to partitioning biomaterials (Albertsson, 1986; Walter et al., 1985; Walter and Johansson, 1994; Zaslavsky, 1995). Thus, differential partitioning of components of a mixture between or among immiscible liquid phases, one of the classical separatory methods, became available for the separation and fractionation of labile biomaterials: macromolecules, membranes, organelles, and even cells.

B Partitioning between Liquid Phases and Its Use in the Separation of Mixtures

The separation of components of a mixture can be effected when they have different solubilities in the top and bottom phases of an immiscible phase system to which the mixture is added. The separation of two soluble components, for example, can be induced by mixing the phases, letting the phases settle and then physically separating them. Each phase, top or bottom, will be enriched with respect to the component which has a greater solubility in it. When the solubility of each component differs greatly in the two phases (i.e., one component is essentially soluble only in one phase and the other component only in the other) a virtually complete separation is obtained in a single, or bulk, extraction step. Smaller differences in the solubility of components in the two phases still permit their separation but require that multiple extraction steps be carried out [as, for example, in countercurrent distribution (CCD) (Åkerlund and Albertsson, 1994)]. In this procedure each of the phases, top and bottom, is reextracted, respectively, with bottom and top phases. The number of sequential extraction steps required to effect a separation is determined by the relative solubilities of the components in the two phases. The distribution of each material between the phases is quantitatively described by a partition coefficient, K, which is defined as the concentration of the material in the top phase/ concentration of material in the bottom phase.

C Aqueous Two-Phase and Multiphase Systems

Aqueous two-phase systems are formed, as indicated above, when two structurally distinct polymers are dissolved in water above certain concentrations. Immiscible aqueous multiphase systems can be obtained by increasing the number of different polymers used.

1 Partitioning of Biomaterials

The most widely used and studied aqueous two-phase systems contain dextran and poly(ethylene glycol) (PEG). These have been found to be mild and nondeleterious to biological materials and often even exhibit protective effects on materials partitioned in them. They have very low interfacial tensions; moderately low viscosities; densities close to that of water; and can be buffered and rendered isotonic, if necessary (Albertsson, 1986; Walter et al., 1985; Walter and Johansson, 1994).

Both soluble and particulate materials can be partitioned in these systems. While soluble materials partition according to their relative solubilities in the top and bottom phases, the partitioning of particulates depends on their relative affinity for the bulk phases and the interface (for discussion, see Walter et al., 1992). The partitioning of macromolecules and small particulates (e.g., viruses) is influenced by both surface properties and surface area. The partitioning of particulates with surface areas larger than about 0.2 μm2 appears to depend predominantly on surface properties (Walter et al., 1990). While soluble materials and small particulates partition between the bulk phases, particulates tend to partition, with increasing size, among the two bulk phases and the interface and, finally, between one bulk phase and the interface.

The partitioning of larger particulates is time-dependent and is thus measured at the shortest time after mixing deemed adequate for virtually complete phase settling. The time required for phase separation depends, among other things, on phase composition (e.g., polymers and salt used and their concentrations), on the volume ratio (top to bottom phase), and on the presence of the particulates themselves. The partitioning is usually described as the quantity of particulate in a bulk phase as a percentage of total particulate added, P.

The physical properties of the phases can be manipulated (as described below) so as to involve non-charge related, charge-associated, or biospecific groups as determinants of the partitioning behavior of the added biomaterial.

2 Concentration by Partitioning between Phases

Any material which, due to a change in the properties of the system, changes its partitioning from being distributed in both phases to being primarily in one phase (by altering partitioning conditions) will cause it to be concentrated. Concentration also results when a partitioning material is forced from a larger into a smaller phase. If the volume ratio is extreme and the collecting phase is small, concentration of several hundredfold can be obtained.

II Aqueous Phase Systems

The separation of a solvent such as water into two liquid phases by addition of two soluble polymers is a very general phenomenon. The polymers must however, distinctively differ in their chemical structure. The concentrations of polymers necessary decrease with increasing molecular weights. In the dextran-PEG systems, the concentration of water is normally 92–98% in the top phase and 80–90% in the bottom phase. With some polymer pairs, two-phase systems with up to 98% water in each phase have been obtained (Albertsson, 1986; Tjerneld and Johansson, 1990).

A Macromolecular Compositions and Aqueous Phase Separation

The formation of two phases in water has been observed for a great number of polymer pairs (Tjerneld and Johansson, 1990). Two synthetic polymers like PEG and polyvinylalcohol (PVA), which both consist of linear molecules, generate two phases at relatively high concentrations. The most used systems have been composed of one synthetic polymer, typically PEG, and one polysaccharide, e.g., dextran. The popularity of these systems is mainly due to phase formation at moderate concentration of polymers and the relatively rapid settling of the phases. Several systems based on two polysaccharides are known. Dextran and modified dextran, e.g., hydroxypropyl dextran, benzoyl dextran, or valeryl dextran, phase separate. Also the same derivative of dextran, e.g., benzoyl dextran, but with differing degrees of substitution may, when mixed, yield phase separation (Lu et al., 1994).

Proteins are known to form liquid-liquid two-phase systems (at certain concentrations) with a number of polymers. Synthetic polymers which have been used for protein precipitation, e.g., PEG, PVA, and poly(vinylpyrrolidone) (PVP), generate, at moderate concentrations, liquid systems with many proteins and protein mixtures (Johansson, 1996). Polysaccharides may also form two-phase systems with proteins (see chapter by Tolstoguzov, this volume). Certain proteins give rise to protein–protein aqueous two- phase systems (see chapter by Tolstoguzov, this volume) while others form aqueous two-phase systems composed of a protein-rich and a protein-poorphase (see chapter by Clark and Clark, this volume).

A third polymer added to a two-phase system will give rise to a third phase if the concentrations of polymers are high enough. When a third polymer is introduced at low concentration into a PEG-dextran solution that is sufficiently dilute to form a single phase, it may cause the phases to separate. At low concentration, the third polymer will distribute between the two...