Associative phase separaration
Aqueous biphasic systems occur when certain solutes cause an aqueous solution to separate into two aqueous phases. Beijerinck first noted, in 1896, the 'incompatibility' of certain
polymers in aqueous solution. In this case, two phases were
formed when agar was mixed with
soluble starch or gelatin.
Since then, many immiscible biphasic aqueous systems have
been found utilizing hydrophilic polymers in aqueous solution;
for example, poly(ethylene glycol) (PEG, HO-(CH2-CH2-O)n-H
where n is the degree of polymerization)c. The most thoroughly
investigated of these is the aqueous dextran-PEG system, where dextran forms the more hydrophilic, denser,
lower phase and PEG the more hydrophobic, less dense, upper phase. Extensive examinations
of in vitro 
and in vivo 
aqueous phase separations are available.
Phases form when limiting concentrations of the polymers
are exceeded. Both phases contain mainly water (typically
70-90% w/w 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 a two-phase system by
themselves; PEG forming the upper more-hydrophobic phase in the presence of
fairly concentrated solutions of citrates, phosphates or sulfates
or at higher temperatures (see below). Such
aqueous liquid-liquid two-phase systems are finding increasing
use in the extractive separation of labile biomolecules such
as proteins, offering mild conditions due to the low interfacial
tension between the phases (that is, 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 also have a stabilizing 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,
minimizing the harmful action of endogenous proteases. The
systems have also been used successfully for the separation
of different types of cell membranes, organelles and actinide
ions, the purification of enzymes, extractive bioconversions.
Although sometimes perceived as due to polymer incompatibilities,
the properties of these biphasic systems can be mainly attributed
to incompatibility between aqueous pools of low and higher
density water. Each phase may be considered as a different,
although aqueous, solvent with properties determined by its
structuring (see for example, ).
has a far higher concentration in the upper (low-density)
phase of such solutions in spite of its inherent density being
greater than water. This, together with the properties of
this PEG phase, encourage the belief that it creates a predominantly low-density water environment
due to its partially hydrophobic character,
in turn mainly determined by the methylene groups. Further
proof of this may be seen by use of microwave dielectric measurements,
which show the water surrounding PEG to be ordered, whereas that surrounding more hydrophilic polymers
is disordered .
Also, the dissolution of PEG is exothermic (and increasingly
exothermic with PEG size), in line with a shift in the ESCS equilibrium towards the more ordered ES structure. It is interesting and perhaps not simply fortuitous
that the diameter (4.9 Å) of the favored PEG helix (formed by trans, gauche, trans links across the C-O-C-C,
O-C-C-O, C-C-O-C bonds) is the same as the diameter of the spines of the ES
water cluster (4.7 Å) formed by pentagonal
boxes, the ether (O-C-C-O) distances (2.88 Å) are
close to the O···O distances (2.84 Å)
in water and the next ether (O-C-C-O-C-C-O) (5.6 Å)
distances are close to the next vertex distance on opposite
sides of the pentagonal boxes (5.4 Å).a
Model building shows that optimum hydrogen bonding would tend
to distort this PEG helix, however. The strongly-held hydration,
as determined by viscosity, increases from two molecules of
water per PEG monomer at very low polymerization (tetramer)
to 5 molecules of water per PEG monomer for 45-mer [576a],b showing that the extent of water clustering increases with
PEG size. The total number of water molecules associated with each PEG monomer is about 32
, or out to about about 2-3 nm radius. The partitioning of proteins into the hydrophobic
PEG phase shows great sensitivity to the protein's surface
hydrophobicity (partition increasing with surface hydrophobicity)
and also depends on the PEG size; increasing with PEG molecular
in line with the extent of water clustering. Increasing PEG
size and concentration both increase the proteins' effective
hydration as the PEG is excluded from the proteins' surface
However, when the PEG phase becomes too ordered (for example, at higher PEG size) partitioned proteins are excluded 
due to the reduced available water content.
An interesting and revealing phenomenon
occurs in PEG solutions as the temperature is raised; the solution at low
temperatures separates into two phases (PEG-rich
at higher temperature (separating at the cloud-point) and
reverts to a single phase at even higher temperatures. This is shown schematically opposite and
may be explained as the PEG creating a low-density water environment with decreased entropy .
At low temperatures a solution is formed due to the enthalpy
of hydrogen bonding between the PEG and the water more than compensating for the entropy lost
in forming the low-density water. This entropy loss is required,
due to the hydrophobicity of the methylene groups, but is
not great as the water is somewhat ordered already at lower
At the cloud point, the entropy cost is greater
as the water is no longer naturally as structured, and two
phases develop; probably involving extended chains in the one continuous phase and aggregated chains in the other cloudy phase . The stronger hydrogen bonding in D2O,
relative to H2O, is expected to raise this cloud point.
At higher temperatures still, the water possesses excess energy
and cannot be structured by the PEG.
This reduces the entropic cost, so allowing a solution to
form once more.
When small PEG molecules are used, for example, in PEG400 , the PEG may aggregate rather than form a second aqueous phase but the behavior otherwise is similar.
Other work, however, indicates that water competes successfully for hydrogen bonding to ethylene glycol compared with other ethylene glycol molecules , so the situation may be finely balanced and depend on temperature, concentration and molecular size.
Anions have a distinct effect on the cloud point in line
with the Hofmeister Series (cloud
point lowering: SCN- < I- < Br- < Cl- < F- < OH- < SO42- < HPO42- < CO32- < PO43-);
the greater lowering of the cloud point is in line with greater
surface charge density ,
stronger hydration, greater tendency to avoid low-density
water and the greater destruction of the natural structuring
of the water. A oppositely-ordered compensating effect on
the cloud point has been recognized due to binding of the
anions to the polymer surface .
This tends to raise the cloud point at lower salt concentrations
as the bound salt increases the polymer net charge and, hence,
solubility. The relative effect of the ions is the reverse
of the Hofmeister series just given with weakly hydrated ions
binding best, i.e. SCN- having the greatest
effect and ionic kosmotropes below Cl- having negligible
effect. The Hofmeister effects on the phase separation process of poly(propylene oxide) has been described by a balance between thse interactions .
Cations have a lesser but opposite effect to anions with chaotropes (for example, NH4+)
tending to lower the cloud point but kosmotropes (for example, Li+) raising it. Exceptionally,
however, some di- and trivalent cations such as Mg2+ and Zn2+ act counter to their normal Hofmeister
behavior, due presumably to their specific chelation to oxygen
atoms in the PEG molecules .
Anions and cations distribute themselves differently between
the phases depending on their affinity for low or higher density
water but with the requirements that the phases be electrically
neutral and iso-osmotic, so producing an interfacial potential
difference, which may aid the partitioning of charged biomolecules.
Thus sulfate and phosphate ions prefer the bottom phase and,
as a consequence, negatively charged proteins are partitioned
into the upper PEG phase, so allowing more sulfate or phosphate ions to partition
into their preferred lower phase. Preference for the PEG-rich
phase is related to the Hofmeister
Series for the structuring ability of the salts, particularly
the anions (for example, preference for PEG-rich
phase: I- > Br- > Cl- > F- > SO42-; Cs+ > Na+ > Ba2+ > Ca2+; preference for PEG-poor
phase: SO42- > F- >
Cl- > Br- > I-)
. A similar Hofmeister
Series effect is noticed intensifying the incompatibility
between two polymers such as polyethylenimine-PEG,
or dextran-PEG, by increasing the concentration
of strongly hydrated (CS-forming)
anions, such as sulfate .
Recently, ionic liquidsd have proven to be alternative to polymers in the formation of aqueous biphasic systems. Addition of common aqueous salt solutions induce the formation of ionic-liquid-based aqueous biphasic systems, with the salting-out ability of the anions and cations following their Hofmeister
Associative phase separation
A different form of aqueous biphasic system can be formed by associating molecules. This occurs where a water soluble molecule self associates but remains soluble, thus forming a soluble polymeric mass that behaves as a distinct aqueous phase .
a Note that the polymers formed with
either one (-O-C-O-C-O-) or three (-O-C-C-C-O-C-C-C-O-) methylene
groups between the oxygen atoms are both insoluble in water.
The reason however is not so much that the O···O
distances (2.18 Å and 4.79 Å respectively) fit
less well with the water cluster spacing but rather that the
molecules form almost-linear extended (rather than helical)
chains with a pronounced hydrophobic character that have strong
intra-molecular attraction. [Back]
b Dielectric studies show 3.7 hydration water molecules per monomer residue [576b]. [Back]
c Poly-(ethylene glycol) (PEG, n HOCH2-CH2OH PEG + n-1 H2O) and poly-(ethylene oxide) (PEO, n C2H4O PEO ) are often used interchangeably as both consist of repeating CH2-CH2-O- units with the only difference being at the polymer ends. [Back]
d Ionic liquids are salts with large ions with low surface charge density that form only short-lived ion pairs and have difficulty crystallising so forming liquids at low temperatures. Examples are ethylammonium nitrate (C2H5)NH3+·NO3- (m.pt. 12 °C) and 1-butyl-3-methylimidazolium tetrafluoroborate (m.pt. -80 °C). [Back]