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ANALYTICAL

BIOCHEMISTRY

197,

l-18

(19%)

REVIEW
Partitioning in Aqueous Two-Phase Systems:
Recent Results
Harry Walter,* G&e Johansson,? and Donald E. Brooks$
*Laboratory
of Chemical Biology, Veterans Affairs Medical Center, Long Beach, California 90822; TDepartment
Chemical Center, University of Lund, S-221 00 Lund, Sweden; and SDepartments
of Pathology and Chemistry,
University of British Columbia, Vancouver V6T 1 W5, British Columbia, Canada

From analytical to commercial scale, aqueous twophase systems have a niche in the purification, characterization, and study of biomaterials (1). Initially described by Beijerinck toward the end of last century,
such phase systems were rediscovered and first employed in the 1950s by Albertsson for partitioning biomaterials (2,3). Aqueous two-phase systems are generally composed of a water solution of two structurally
distinct hydrophilic polymers or of one polymer and
certain salts (e.g., alkali phosphates). Above critical
concentrations
of these components, spontaneous
phase separation takes place with each of the two resulting phases enriched with respect to one of the components.
The replacement of organic by aqueous solutions in
immiscible two-phase systems has permitted the application of the classical separatory method of resolving
components of a mixture by partitioning not only to
labile macromolecules but also to cells, membranes, and
organelles. Two-polymer aqueous phase systems can be
buffered and, if necessary, rendered isotonic. Appropriate selection of polymers [the most widely used being
dextran (Dx) and polyethylene glycol (PEG)] results in

i Abbreviations
used: Ab, antibody;
CCD, countercurrent
distribution; Cross-point
(isoelectric
point),
obtained
by cross-partitioning,
i.e., the pH at which two plots of Kvs pH for a material
intersect
when
the Ks are measured
in phase systems containing
different
salts but
the same polymer
concentrations;
DEAE-,
diethylaminoethyl-;
DMSO,
dimethylsulfoxide;
Dx, dextran;
EHEC,
ethylhydroxyethylcellulose;
FACS, fluorescence-activated
cell sorter; FA-PEG,
polyethylene glycol fatty acid esters; Fi, Ficoll; G, apparent
partition
coefficient (or ratio), obtained
from the location
of the peak in a CCD curve
= r-l(n
- rma.), where r,, is the tube (or cavity) number of the peak
of the distribution
and n is the number
of transfers;
HPD, hydroxypropyldextran;
HPS, hydroxypropylstarch;
IDA, iminodiacetic
acid;
IO, inside out; K, partition
coefficient
(used with materials
that partition between
the two bulk phases)
= concentration
of material
in top
phase/concentration
of material
in bottom
phase; P, partition
ratio
(used with materials,
e.g., cells, which partition
between
a bulk phase
0003-2697191
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in any form

of Biochemistry,

phase systems which are mild and nondeleterious to


most biomaterials partitioned in them. These phases
have interfacial tensions several orders of magnitude
lower than those of aqueous-organic or organic-organic
systems. The manipulation of polymer and ion composition and concentration determines the physical properties of the phases which, in turn, effect separations of
biomaterials based on different physical parameters.
For example, even though Dx and PEG are themselves
nonionic, certain salts (e.g., phosphates, sulfates) have
different affinities for the two phases, a phenomenon
which gives rise to a Donnan potential between the
phases and causes them to be charge-sensitive. Other
salts have equal affinities for the two phases and give
rise to non-charge-sensitive systems. The incorporation
of a ligand bound to one of the phase-forming polymers
(or one which partitions or is made to partition extremely into one of the phases) permits affinity partitioning to be carried out.
Partitioning of biological particulates has proved to
be an extremely sensitive method for their separation
and fractionation. The partitioning of larger particulates depends predominantly on their surface properties. By carrying out multiple extractions [e.g., countercurrent
distribution
(CCD)] even subtle surface
alterations of cells that accompany normal and abnormal in uiuo processes (e.g., differentiation, maturation,
aging, metastatic potential) or in vitro treatments can
be traced as these are often reflected in altered partition
ratios, i.e., P values (l-3). Membranes and organelles
can be fractionated, plasma membranes can be purified,
and membrane domains charted. Right-side out and inside out vesicles can be separated. Proteins, including
isoenzymes, have been efficiently segregated while afand the interface)
= quantity
of material
in bulk phase as a percentage of total material
added, PS, photosystem;
PVA, polyvinyl
alcohol;
PVP, polyvinylpyrrolidone;
RO, right-side
out; TMA-,
trimethylamino.
1

Inc.
reserved.

WALTER,

JOHANSSON,

finity partitioning
has permitted the specific extraction
of a multitude of proteins including dehydrogenases and
kinases and the fractionation
of nucleic acids on the
basis of base- and sequence-specificity
(l-4).
The aqueous phases obtained with one polymer,
usually PEG, and certain salts, which are not isotonic,
have found their main use in the rapidly expanding biotechnology of protein purification
(4). Pa~icularly
advantageous is the fact that extractions can easily be
scaled up. Because the high salt concentration
of such
systems often precludes affinity partitioning
and because of the prohibitively
high cost of dextran for commercial applications,
other polymers (i.e., Dx substitutes) or polymer combinations
are being examined for
use in two-polymer aqueous phases. Other biotechnological applications of aqueous two-phase systems include
extractive bioconversion
processes and the concentration of biomaterials
such as viruses.
Since the parameters that determine the partition coefficient, I( (or the partition ratio, P), of materials are
exponentially related to the K (or P) value, partitioning
often yields separations and information
on physical
properties of biomaterials
not readily obtained by other
means.
Here we relate some of the rapid advances in and
novel applications of aqueous phase pa~itioning
during
the past 5 years, i.e., since the previous review (l), as
well as theoretical aspects of phase separation and partitioning.
It is gratifying that some of the prospects
we noted (1) have come to pass. These include: finding
inexpensive polymers with favorable properties, development of biospecific extraction
methods (also for
cells), new apparatuses for more rapid processing of materials, use of highly efficient columns for protein (and
nucleic acid) fractionation
based on two-polymer
aqueous phases, development of systems for use at high
and low temperatures,
addition of small quantities of
water-soluble
organic solvents to aqueous phase systems to permit partitioning
of materials with low solubility in water, and use of systems composed of more than
two phases.
THEORY

OF PETITIONING

PHENOMENA

Considerable
advances in the theory of two-phase
systems and partitioning
phenomena have been made
since this subject was last treated (5). These advances
derive largely from the impetus provided by the potential of partitioning
in biotechnology,
particularly
as one
or more steps in the medium to large scale isolations of
genetically engineered proteins. The chemical engineering community
has been dealing with liquid-liquid
extraction problems for decades (6) and has found in
aqueous phase partitioning
a fertile area for investigation. Experience in the industrial application of extraction has shown clearly that deeper understanding
leads

AND

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to more efficient separations and more profitable processes.


The goal of many of the groups involved is to secure a
sufficient understanding
of macromolecular
partitioning to allow prediction of K for a defined material under
arbitrary conditions and hence to allow optimization
of
separation. In some of the approaches currently being
pursued achievement of this goal implies an ability to
predict phase behavior of the systems, i.e., to predict
phase diagrams, on the basis of a minimum
number of
experimental
measurements of characteristic constants
describing interactions between pairs of elements in the
system (7-10). Other groups have assumed the presence
of an invariant two-phase system and concentrated on
modeling the interactions
between phase components
which result in the observed K (11-14). In all cases to be
reviewed significant agreement with experiment exists
under some sets of conditions. However, no single approach has proven so superior that all would accept its
universality.
Due to space li~tations
we will provide
only a broad overview of the theoretical developments
since 1985. For a more detailed discussion the excellent
recent review by Abbott et al. (15) is recommended.
Prediction of Phase

Diagrams

A sufficiently detailed understanding


of the interactions which cause phase separation would obviously contribute greatly to our capacity to predict the K of added
material, particularly
if the partitioned
material was a
low molecular weight species or another polymer. Two
approaches to the prediction of phase diagrams have
been taken, one based on polymer solution theory and
the other adapted from the thermodynamic
treatments
of liquid phase equilibria. In neither case have predictions of phase behavior been made from first principles,
i.e., simply from the chemical structures of the components; liquid state theories are not available to describe
such complex systems. Rather, various theoretical constructs are applied to sets of experimental
data to evaluate the constants necessary to allow prediction of phase
diagrams of related systems. Generally, the goal is to
maximize predictive power with the minimum
number
of measured constants.
The best known polymer solution treatment
is the
lattice theory of Flory and Huggins (5) which has been
quite successful in predicting general properties of polymer solutions in unstructured
solvents. In such models
the solution is represented by a three-~mensional
lattice, each site of which is filled with a solvent molecule
or a segment of one of the polymers present. The free
energy of mixing of the solution is evaluated from the
sum of the energies of interaction between the various
segments and solvents and from the configurational
entropy associated with the numbers of ways of arranging
the components on the lattice. If in a two-polymer, one-

RECENT

RESULTS

IN

AQUEOUS

solvent mixture the interaction


energy between unlike
polymer segments is even modestly unfavorable,
the total free energy can be lowered-at
the cost of an entropy
decrease-if
the number of such contacts is minimized
by the formation
of two phases, each rich in one of the
polymers (5). In this lattice model the parameters
which
describe the system are the molar volumes of the polymers relative to the solvent molar volume, proportional
to polymer molecular weights, and the interaction
constants, xi j, which are proportional
to the energy change
which occurs on forming from pure components
contacts between solvent (component
1) and either type of
polymer segment (xl2 or x13) or contacts between the
two-polymer
segment types (xZ3). The derivation
assumes the average polymer segment density is constant
throughout
the solution.
Given the simplicity
of the approach it has been remarkably
successful
at describing
the thermodynamic
properties
of amorphous
polymer mixtures
and solutions. The main problem with applying this theory to
aqueous polymer solution phase separation
is that no
solvent
structuring
effects are taken into account.
While some ordering of solvent in contact with polymer
can be introduced by allowing the interaction
energy to
contain a local entropy component the result is not well
suited to strongly hydrogen bonding solvents like water.
In fact, one whole class of phase separation phenomena,
known as LCST behavior, cannot be described by the
Flory-Huggins
theory (16). This occurs when solutions
of certain polymers
such as PEG [or detergents
with
PEG headgroups
(17,18)] are heated, reducing the Hbonding with water which holds them in solution. Nonetheless, the lattice theory formalism has been applied to
fill out Dx-PEG-water
phase diagrams using empirical
values for the interaction
parameters
determined
from
one set of phase compositions
and reasonable
results
were obtained (19). The values of the interaction
parameters obtained do not appear to be applicable to other
types of thermodynamic
predictions,
however [compare
xij parameters
with (ll)], which indicates the FloryHuggins theory is not accurately
describing the details
of the interactions
present.
An alternate approach is to apply what is known as
the osmotic virial expansion
in which thermodynamic
functions,
in particular
the osmotic pressure or solvent
chemical potential, are described by a power series in
the polymer
concentrations
with empirically
determined coefficients.
The constants
in the osmotic pressure expression
which multiply the second order concentration
terms,
aij, are known
as second virial
coefficients
and are related to the energies of interaction between the pairs of species represented
by i and j.
For instance, to describe two polymers in water the virial coefficients
az2, us3, and az3 would have to be evaluated if the power series is taken to second order. The
degree of compatibility
or incompatibility
between the

PHASE

PARTITIONING

polymers therefore is described by the sign and magnitude of uZ3. The method was first applied to describe
phase separation of polymer (and protein) solutions by
Edmond and Ogston (20) who found it could satisfactorily treat these systems.
Recently, more rigorous calculations
along the same
lines have been made by groups in Berkeley (9,lO) and
Arizona (7,B) as part of longer term efforts to predict
protein partition. Both groups have taken the vital step
of specifically including the effects of salts in their calculations. The two approaches differ in detail, particularly in the treatment
of salt effects and polymer molecular weight.
The Berkeley
computation
does not
address the molecular weight issue and measures individual virial coefficients for each polymer fraction used.
Cabezas et al. (7,8,21), on the other hand, have applied
Renormalization
Group ideas to allow calculations
for
all fractions of a given polymer whose molecular weights
are known. Both groups use thermodynamic
measurements (low angle light scattering, osmotic pressure, isopiestically determined activities) on two- or three-component solutions to obtain all the necessary constants;
no adjustable parameters
are utilized. Both have had
considerable
success at predicting
phase diagrams for
Dx-PEG
(9,21), Dx-methylcellulose
(21), and DxPEG-salt
(8,lO) mixtures. Their most recent published
work also includes predictions
for the partition of salts
in Dx-PEG
systems which reproduce experimental
data
slightly less well than the phase diagrams. Predictions
of PEG-salt
phase diagrams are just becoming available (22).
One unexpected prediction which should be tested experimentally
is the dependence of the Dx-PEG
phase
diagram on very low concentrations
of Na,SO, (8). Cabezas et al. claim that even 0.001% Na,SO, can shift the
binodal to much lower Dx concentrations,
arguing that
experimentally
no phase system is salt-free to this level
so that true zero salt measurements
have not been
carried out. The published Berkeley calculations
(9,lO)
do not address this issue.
Another area which deserves more consideration
is
the theoretical
effect of polydispersity
of the polymer
fractions
used, particularly
the changes in molecular
weight distribution
which occur in the two phases relative to the source material. Some relevant theoretical
work has appeared but it is difficult to apply it in the
above formalisms
(23,24). Some of the reported fractionation effects are dramatic (24).
Prediction of Protein Partition

Coefficients

The first attempt at writing a molecular-level


theory
for partitioning
of macromolecules
in a two-polymer
aqueous system again utilized the Flory-Huggins
formalism, the partitioned
material being treated as a third
polymer (5). The result, obtained by equating chemical

WALTER,

JOHANSSON,

potentials
of the partitioned
species in the two phases
and retaining only first order terms in the polymer concentration
differences,
provided an expression
for the
partition coefficient, K, in terms of the polymer concentration differences
between the phases, the molecular
weights of the two polymers and the added macromolecule and xij parameters
for the macromolecule
interacting with the solvent and each polymer:
In K = PJ(cbl - d$)O - xlp) + (4: - &)(1/p,

- x2J

+ (4: - 4:) x (l/P, - x3p)l,

111

where

Pi = (molecular

volume of component
i)l
(molecular volume of solvent)
i= 1, 2, 3, or p refers to solvent, polymer 1,
polymer 2 or protein, respectively
fraction of solution volume occupied by
component i; t or b refer to top or bottom
parameters
describing the
Xij = interaction
i-j interaction,
described above.

phase

Although it is clear that K describes the distribution


of a flexible polymer coil, the predictions
of the treatment also described most of the qualitative features observed for protein partition
in the absence of electrostatic effects (25). That is, partition
becomes more
one-sided with increasing protein molecular weight and
with increasing difference in concentration
between the
phases of either polymer. Partition
also shifts in favor
of a phase in which the polymer molecular weight is
reduced, the more so the higher the protein molecular
weight. The agreement with trends observed when polymer or protein molecular weights are changed may be
fortuitous
since the protein segments are neither uniformly distributed
nor free to occupy large numbers of
configurations,
as assumed in the derivation.
In the
limit of low molecular weight proteins, i.e., for oligopeptides, the theory is much more realistic.
A simplified version of the Flory-Huggins
expression
has been derived (26), utilizing the observation
that in
most Dx-PEG
systems [but not in all two polymer mixtures (2)] the difference in Dx concentration
between
the phases is proportional
to the difference in PEG concentrations.
This reflects the fact that the tie lines in
these systems are parallel in their phase diagrams. The
equation is
In K = A*(wi

- WE),

PI

where A* is an empirical parameter which depends on


the molecular weights of the polymers and protein and
w2 is the weight fraction of PEG in the indicated phase.
Equation [2] was found to describe the partition of dipeptides and some low molecular weight proteins quite

AND

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well, as might be expected based on the discussion


above, but the linearity in PEG concentration
difference was not retained for higher molecular weight proteins. Extending [2] to second order in (wi - w!$ allowed
the equation to fit data for a variety of proteins at pH
7.0, regardless of their isoelectric points (13). The temperature dependence expected from a literal interpretation of [l] (xij is proportional
to l/T) was also found to
hold. Hence, the polymer concentration
dependence of
the protein K seems well established.
The above correlation
is useful but the predictive
powers of Eq. [2] are limited since the constants must be
measured for every phase system, i.e., for each polymer
type and molecular weight, buffer type and concentration, etc. While [l] provides expressions
for the constants, there are fundamental
objections to its literal
application,
as we have seen. The osmotic virial coefficient approach or its variants are designed to alleviate
some of these difficulties by providing a framework
with
which to extend a limited number of measurements
on
two- or three-component
solutions to the prediction of
all the thermodynamic
properties
of the systems. This
includes, of course, predictions
of the KS of added proteins and, in principle, the effects of such additions on
the properties
of the phase systems.
With the objective of predicting protein partition effects, three groups have utilized the virial expansion
method, the two groups mentioned
above and Halls
group (14). There are some fundamental
differences in
their approaches.
Cabezas et al. (7,8,21) and Forciniti
and Hall (14) begin with a constant pressure theory initiated by Hill (27) while King et al. (9) and Haynes et al.
(10) extend the constant volume equations of Edmond
and Ogston (20). The two methods are shown to produce equivalent equations for noninteracting
solvents
(14) but there can be significant differences in the coefficients under some con,ditions (Cabezas, submitted
for
publication)
and the constant pressure approach seems
the more realistic. The basic equation, for isoelectric
proteins in the absence of salts, can be expressed as
In K = A,(m!jj

- m$ + A,,(rni

- m,)

[31

where mi is the molality of component i in the indicated


phase and A,, and A,, are the virial coefficients to be
evaluated. Clearly, this is of the same form as [l] and [2]
above. A more complicated
form results when salts are
included and a major problem is to treat their interactions with the other components
correctly.
The virial coefficients
which describe the binary interactions
between each pair of components
can be calculated from first principles
(14), measured (9,lO) or
obtained by a combination
of these approaches. For example, molecular weight effects can be predicted given a
value for a monomer interaction
coefficient (8). Forciniti and Hall (14) have calculated the A, and A,, values

RECENT

RESULTS

IN

AQUEOUS

for PEG, Dx, and protein assuming there are no net


attractions
between any of the species. Their interactions simply consist of the repulsion associated with the
assumption
that no two molecules can occupy the same
location. The coefficients
can then be calculated from
the volume from which the centers of the two interacting molecules are excluded, which depends on the geometry assumed for the two species. This was the original
approach taken by Ogston (20) and has been applied in
studies of protein/polymer
mixtures
(28,29). Forciniti
and Hall calculated
excluded volumes assuming
the
protein was a rigid sphere and the polymers behaved as
spheres, rods, or Gaussian coils. None of these assumptions produced satisfactory
fits to their data set, however, and they concluded that attractive
interactions
among some of the components
must be included to reproduce the experimental
results.
In order to test their theoretical
predictions,
Forciniti, Hall, and Kula [ (30) and D. Forciniti, personal communication]
amassed an enormous
data set based on
four Dx fractions
and four PEG fractions
which were
combined at four compositions
each at three temperatures and four proteins partitioned
in each system at
their isoelectric point and three other pH values (the pH
dependence was studied only at one temperature).
Molecular weight distributions
were measured
for each
polymer fraction both in pure solution and in the separated phases of a number of systems as well. Once this
information
is published it will provide a valuable resource for comparison
with theoretical
predictions.
The Berkeley group has recently utilized a version of
Eq. [3] which includes salt interaction
terms and concentrations
and measures the virial coefficients as mentioned above, with the ion-related constants provided by
membrane
osmometry
and published
vapor pressure
data (10). When salts which partition
unequally
between the phases, such as SO; or H,PO;,
are present
an electrostatic
potential difference should appear between the phases and its contribution
to the distribution
of charged proteins has to be included in the calculation.
To date, computation
of the partition
behavior of bovine serum albumin as a function of tie line length in
Dx-PEG
systems containing
either NaCl (which does
not partition
strongly in these systems)
or NaH,PO,
provides predictions
which show the measured trends
but are significantly
higher than the measured KS. The
discrepancy
is felt to be due to neglect of ion pairing,
inappropriate
treatment
of protein electrostatics,
and
neglect of higher order terms in the virial expansion, necessary
to describe the phases at high tie line
length (10).
There are clearly many challenges to be taken up by
those following the above approach. Salt-single polymer
phase systems have received relatively little attention,
for instance, although some interesting
correlations
of
K with hydrophobicity
(31) and free volume (32,33)

PHASE

PARTITIONING

have been made. Prediction of phase diagrams and protein KS for such systems will test the ability of the
theory to handle concentrated
salt solutions.
Proteinprotein interactions
have not yet been addressed (except among molecules of the same substance where the
effect is likely to be small since K is usually independent
of protein concentration).
It should be possible to treat
the protein-protein-water
two-phase
systems which
have been reported (34) or protein-polymer-water
systems (20,35) to investigate
high protein concentration
behavior. It will also be of interest to see whether parameters can be identified which correlate with the hydrophobicity of distributed
material. Zaslavsky and collaborators have made an extensive investigation
of this issue
(36,37).
While there is much to be gained by using the virial
expansion approach to predict overall system behavior,
by measuring
rather than calculating
key coefficients
the physical picture of what is occurring
is deemphasized. A series of investigations
which is richer in this
regard has originated from MIT (11,12,38) in which the
lattice model is modified for a spherical protein interacting with a polymer phase. The issue of whether attraction between the protein and polymer is necessary
to describe observed results was addressed by adapting
the computationally
intensive
polymer
adsorption
theory of Scheutjens and Fleer (39) and calculating the
free energy of the particle in each phase, the difference
being proportional
to In K. The treatment
provides as
well the polymer segment distribution
profile in the vicinity of the protein.
The protein-polymer
segment
interaction
is described by x8, equal to the difference in the protein-solvent and protein-polymer
dimensionless
interaction
energies per lattice site. It was found that a small attraction between protein and either polymer was necessary to reproduce experimental
values for K as a function of PEG molecular weight. The required attraction
is small, however, and the polymer is at a lower concentration near the surface of the protein than in the bulk
due to the unfavorable
entropy loss suffered by the polymer chains when their configurations
are constricted
by
the surface. The overall free energy of the protein is
therefore increased. That is, the protein is incompatible
with both polymers,
the exclusion being least for the
polymer which predominates
in the phase to which the
protein partitions.
It was found necessary to make the
Dx-protein
x8 slightly more attractive
than the PEGprotein parameter. A similar conclusion was reached by
Forciniti and Hall (14) in attempting to rationalize their
data on the temperature
dependence of K.
This theory was further tested on data generated in
polyvinyl
methyl ether-PEG
systems for a number of
proteins
and, with appropriate
choice of parameters,
reasonable agreement was achieved (38). Hence, there
is reason to accept the general picture provided by these

WALTER,

JOHANSSON,

calculations.
The problem with applying it in a more
general sense is the difficulty in estimating
some of the
important
parameters,
particularly
xs, independently.
The lattice model also suffers from most of the same
limitations
with respect to solvent interactions
as were
discussed above for the Flory-Huggins
calculations.
A further contribution
from the MIT group (1540)
discusses the physical picture of protein-PEG
interactions in the context of the scaling theory of polymer
solutions initiated by de Gennes (41). For low molecular
weight fractions (<lO,OOO) the polymers, at the concentrations
typical of the PEG-rich
phase of a Dx-PEG
system, behave as in dilute solution and should interact
with protein as individual polymer coils. Higher molecular weight fractions
are present more as a continuous
mesh due to molecular overlap in the semidilute regime
in which they find themselves.
The transition
from dilute to semidilute regimes occurs at the overlap concentration,
equal to the average segment concentration
within a coil, which decreases with increasing molecular
weight. In the semidilute region characteristics
of the
individual PEG molecules are lost due to the coil overlap and the properties become independent of molecular
weight,
as observed for protein partition
(1525). A
scaling analysis based on these ideas was carried out for
various levels of attraction
between polymer and protein and different solvency conditions
in both polymer
concentration
regimes.
In the dilute region (low molecular weight PEG fractions; concentrations
as in top phase) the importance
of
excluded volume effects is confirmed and the impossibility of explaining experimental
trends in the presence of
strong polymer-protein
attraction
is pointed out. The
presence of a weak attraction
is consistent
with observation for the solvency conditions
which characterize
PEG (or Dx) in water but the nature of the analysis does
not allow the relative contributions
of the exclusion and
attraction
terms to be determined.
A conclusion
as to
the necessity for attraction
is therefore not possible.
In the semidilute regime, present in the top phase of
systems containing high molecular weight PEG, no molecular weight dependence of K ought to be present and
little is found. An expression
is derived for the polymer
concentration
dependence of K in this regime, again assuming weak polymer-protein
attraction.
This expression has not yet been tested experimentally
because it is
not possible to independently
vary polymer concentrations in the phases. The above considerations
dealt only
with the PEG-rich
phase for which the cross-over
concentration
between the two regimes occurs in the top
phase as the molecular weight is varied. In the Dx-rich
phase, on the other hand, all lower phase compositions
used for partitioning
are in the semidilute region for Dx
molecular weights of 40,000 and above. For example,
using experimentally
determined values for Rg, the radius of gyration (42), the cross-over
concentration,
c*

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(c* = 3M/4?rRz), is calculated to be 7.2% w/w for Dx


40,000 and 3.3% w/w for Dx 500,000, while typical bottom phase Dx concentrations
are 12% and 7.5%, respectively, for these fractions.
Hence, the bottom phase
would be well into the semidilute
region for all commonly used Dx fractions
and by the above argument
should provide very little molecular weight dependence
to K. However,
the experimental
data show strong dependence on Dx molecular weight (25). The reason for
this departure from expectation
is not obvious but may
be related to polydispersity
of the fractions or the presence of the low concentrations
of PEG in the bottom
phase, which was not considered in the above. It is also
not clear from scaling theory just how far into the semidilute or dilute regimes systems must be to exhibit the
posited behavior. As Abbott et al. have noted (40), partitioning may be occurring in a cross-over
regime in all
cases, necessitating
a more complex analysis. Regardless of these problems the scaling considerations
provide a fresh way in which to think about the mechanisms involved in partitioning,
one which is sure to
contribute
to our understanding
of these systems.
Affinity

Partitioning

The theory of the effect on K of a material binding a


ligand which partitions preferentially
into one phase
was given previously (5). It was a generalization for the
whole ligand concentration range of the treatment provided by Flanagan and Barondes (43). Recently the
theory has been extended to include effects of more
than one type of binding site (44), an extension made
necessary in order to fit experimental data. It has also
been modified by Suh and Arnold to include the effect of
competitive inhibitors on affinity ligand binding and
partitioning for materials bearing one or two classes of
ligand binding sites (45). The affinity of PEG-derivatized Cu(I1) chelates for histidine residues on the surfaces of proteins was used in an experimental test of
their treatment. Knowing the number of binding sites
for several proteins and using H+ as an inhibitor, the
theory was successfully tested. K was increased by a
factor of up to about 2.5 at the highest pH (lowest inhibition) by addition of ligand to whale myoglobin (five binding sites) at ligand concentrations well below the dissociation constant in either phase. The dissociation
constant was found to differ by a factor of about two
between the phases, the association being stronger in
the Dx-rich phase. Hence, the theory was shown to apply, but well below saturation.
The outstanding question regarding affinity partitioning of macromolecules is why the predicted behavior is not followed nearer saturation. At saturation for
one class of binding sites (5),

K,/K
where

= [K(L)P,I~],

141

RECENT

K,IK
K(L)
K;IK;

RESULTS

IN

AQUEOUS

= ratio of partition
=
=
n=_-

coefficients
in the presence
and absence of affinity ligand
affinity ligand partition
coefficient
ratio of ligand association
constants
in top
and bottom phase
number of binding sites.

This equation produced considerable


excitement when
it was originally published because of the power n which
suggested that enormous effects could be obtained for
modest values of K(L) if n was much larger than 1. This
dependence has never been observed, however (44,46),
presumably
because [4] assumes ideal behavior for the
bound and free species. Once one polymer-derivatized
ligand is bound, further binding may well be sterically
hindered, introducing
negative cooperativity
into the
problem and invalidating
the analysis. It would be of
interest to investigate
this idea by, for instance, performing systematic
tests of [4] with ligands bearing different molecular weight polymers.
A number of studies indicate that the affinity constant for a PEG-derivatized
affinity ligand is lower in
the top, PEG-rich
phase than in the bottom, Dx-rich
phase (5,44,45). This is expected if the ligand is to some
degree removed from contact from the phase polymers
when it is bound (5). The effect is due to the greater
decrease in free energy when the ligand reduces its contact with the dextran-rich
phase, with which it is incompatible, compared to the PEG-rich
phase. This effect
was in clear evidence in the binding of PEG-fatty
acids
to cell surfaces
(5) where it is likely that the PEG is
partially buried in the surface glycoprotein
layer on the
cell surface. With a simple protein substrate,
as in the
work described above, it has not been so obvious why
differences in K would be observed. The work of Baskir
et al. (11) has provided an explanation
in the form of
spherical
lattice calculations
that indicate there is a
polymer concentration
gradient near the protein surface, with the region near the protein reduced in concentration
due to excluded volume effects. Accumulation of PEG-ligand
in this region would allow the
differences in K measured.
Cell and Particle Partitioning
The outstanding
problem in theoretical
understanding of cell partition is the fundamental
relationship
between the predictions
of thermodynamics
and the observed dependence of P on measured surface properties.
The situation, which has been described in more detail
elsewhere
(5,47), can be summarized
as follows. Cells
generally partition
between one phase and the liquidliquid interface,
rather than between
the two bulk
phases, because adsorption in the interface significantly
lowers the free energy of the system by reducing the
interfacial
area. The free energy difference,
AG, between particles immersed completely in one phase and

PHASE

PARTITIONING

those adsorbed in the interface (where they are in contact with both phases) can be calculated by measuring
the interfacial tension of the liquid-liquid
boundary and
the contact angle formed between the interface and the
cell surface (5). Thermodynamic
equilibrium
implies
that K ought to depend exponentially
on the ratio AGo/
kT, where kT, Boltzmanns
constant X absolute temperature, is the average thermal energy of the cell. The
problem is that although the exponential dependence is
approximately
followed, the slopes of In K - AG plots
are orders of magnitude lower than the predicted value,
IlkT (5,48). A related problem has been identified in
studies on the distribution
of BaSO, particles between
the oil/water
interface and one of the bulk phases (49),
so the discrepancy
is not unique to systems with low
interfacial
tension.
It has been proposed that this discrepancy
is due to
fluid shear forces removing cells from the interface during settling of the phases after they have been mixed
(47). Some support for this idea is found in the results of
cell separations
using Dx-rich
phases immobilized
on
beads past which the PEG-rich
phase flows (50,51).
When mixtures
of two types of cells were adsorbed to
the liquid-liquid
interface between the beads and the
PEG-rich
phase, then eluted with a PEG-fatty
acid affinity ligand, a more efficient separation
was achieved
than in the equivalent partition
step carried out in the
conventional
manner. That is, in the different hydrodynamic environment
in the column, the P values were
closer to the predictions
of thermodynamic
equilibrium
than were Ps measured in single tube partitions.
Another approach to investigating
dependence of P
on the hydrodynamic
environment
during demixing is
to perform partitioning
in the absence of settling, utilizing the low gravity environment
of the Space Shuttle.
To date it has been found that Dx-PEG
phases demix
more slowly in space than on the ground and the final
disposition
of the phases is determined by which wets
the container
wall (52) but cell Ps have not yet been
measured.
The predicted dependence of P on particle surface
area has been tested with liposomes of different sizes
but the same composition
(53). It was found that above
a diameter of approximately
0.25 pm, P became independent of area whereas theory predicts In P should be proportional
to this parameter.
Since fluid shear forces
would be expected to be larger for larger particles, which
also adsorb at the interface more strongly, these results
support the qualitative
explanation
for failure of the
thermodynamic
theory given above.
APPARATUSES

AND

TECHNIQUES

For large-scale applications of two-phase extraction


in general, as well as for multistep partitioning, a vast
number of apparatuses has been developed. Many such

WALTER.

JOHANSSON,

apparatuses,
originally constructed
for other kinds of
two-phase
systems, e.g., water-organic
solvent, can also
be used for aqueous systems. In some cases the viscosity
of the phases and the long settling times may prevent
the appropriate
functioning
of an apparatus.
Commercial mixer-settler
systems and centrifugal
separators
have been successfully
applied in processes
with
aqueous two-phase
systems (3).
The main device for multistep partitioning
on a laboratory scale has been the thin-layer
CCD apparatus designed by Albertsson
(2,3). This equipment
has been
further developed by Akerlund
(54). Instead of using
chambers with low height, the time for phase separation
is reduced by centrifugation
at 1OOg after each equilibration step. The operating unit of the machine consists of
an outer ring with 60 cavities for the lower phases and
an inner plate with corresponding
cavities for the upper
phases. Both parts are made of perspex (Plexiglas).
After mixing, by shaking on a platform in the machine,
the plates start to rotate to achieve the centrifugal force.
While still rotating the transfers
of all upper phases
with respect to the lower phases are carried out by a
rotational
movement of the inner plate relative to the
ring. With this machine CCD can also be carried out
with systems of high viscosity or when the difference in
density of the phases is quite low. Another CCD apparatus which provides for the stepwise addition and withdrawal of one of the phases (elution
CCD) has been
described (55). Such equipment permits alterations
in
phase system parameters
during a CCD run and facilitates the use of gradients
(e.g., of salt, pH, affinity ligands) .
A number of apparatuses
for countercurrent
chromatography based on the coil planet centrifugation
principle have been developed by Ito et al. and have just been
reviewed (56). Two recently introduced apparatuses
specially constructed
for use with aqueous systems are (a)
an eccentric multilayer
coil planet centrifuge for semipreparative
separations
(mg quantities),
and (b) a multistage mixer-settler
planetary centrifuge for separations
in gram quantities.
A system containing
a centrifuge
rotor constructed
by stacking circular, disk-type
partition cells and used for high-performance
centrifugal
partition chromatography
(HPCPC)
has been described
(57). It is designed in several sizes suitable for the processing of 2.8 to 107 liters (corresponding
to a productivity of 15 g to 1.5 kg/hr).
A new and interesting
possibility
to enhance phase
separation
entails making one of the phases magnetic
by including ferric particles with extreme partitioning
to one of the phases (58). An apparatus containing a set
of eight chambers
equipped with mechanical
mixing,
moveable permanent
magnets,
and pumping devices
has been devised. This setup permits automatic mixing
of the system (with the magnets removed), very rapid
phase separation
caused by the magnetic field and,

AND

BROOKS

thereby, fast and effective transfers


of the nonferric
phase in a countercurrent
manner. Column-type
arrangements
have also been developed for magnetically
enhanced phase separation
(58,59).
An even simpler way of reducing phase settling times
is the application of an electric field over a mixed phase
system. A field of 26 V cm- increased the rate of formation of bulk phases by up to 5.5-fold (60).
Continuous
countercurrent
extractions
with aqueous
two-phase
systems have recently been investigated.
Joshi et al. (61) studied spray columns (in which the PEG
phase is dispersed
into fine droplets
by passing it
through a nozzle at the lower end of a tube filled with the
other phase); columns packed with Raschig rings, glass
beads, or wire-mesh;
and York-Scheibel
columns with
alternating
zones of mixing and coalescence. This simple, effective and pilot-plant-scale
equipment is of great
interest for industrial
extractions.
Column partition chromatography
based on two-polymer systems has been developed by Miiller et al. (62).
The Dx phase is adsorbed in a carrier consisting of porous particles.
Specially developed beads made from
vinyl polymers are marketed by Merck under the name
LiParGel. Excellent results have been reported by using
beads grafted with polyacrylate
to exclude the PEG
phase (with which it is highly incompatible)
and retain
the Dx phase. The height of a theoretical plate in these
columns is impressive: 0.5-10 mm for proteins. They are
therefore effective separation
devices in which the efficiency and the separation of individual proteins can be
predicted from their K values obtained in a single partition. Excellent
separations
of nucleic acid fragments
have been achieved by using salt gradients. The technique has recently been expanded by inclusion of affinity ligands in the mobile phase (63). Use of PEG-bound
Procion red HE-3B resulted in a much-reduced
elution
volume for formate dehydrogenase
while other proteins
in the extract from Candida boidinii remained unaffected.
BIOTECHNOLOGY

One of the main costs of producing proteins by gene


technology is the isolation and purification of the target
protein. Two-phase systems have found considerable
application in this area, especially as the first fractionation step after cell disintegration, i.e., in the removal of
cell debris from the protein solution (3). For this purpose the PEG-salt systems are preferred and processes
on a scale of up to 100 m3 have been reported (64). Much
work has focused on the search for systems which are
economical and have favorable technological and environmental properties. Economics requires both low
price per mass unit and phase separation at low polymer
concentrations. Technology depends on phases possessing low viscosity and relatively high difference in den-

RECENT

RESULTS

IN

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sity to achieve rapid phase settling. Biodegradable


polymers and environment-friendly
salts are desired to meet
waste management concerns. Two-phase
systems have
been used as reactors with enzymes or microorganisms
restricted
to one phase and the other phase employed
for product extraction
(65). Plant cells, e.g., Nicotiana
tubacum, have been cultivated in Dx-PEG
systems containing growth medium (66). While the cells partition
into one phase the produced secondary metabolites can
be removed by extraction
into the other phase and
thereby enhance the growth rate.
Extraction
of proteins
and further
separation
has
been carried out in both PEG-salt
systems and in systems based on PEG and another polymer. The effectiveness of the extraction is determined by a number of parameters
(e.g., polymer concentration,
structure,
and
molecular weight; concentration
and type of added salt;
pH and temperature).
Much attention has been given to
enhanced selective partitioning.
This can be achieved by
introducing
affinity ligands for specific proteins in one
of the phases (affinity partitioning)
(67). The kind of
interaction
between the (usually polymer-bound)
ligand
and the protein determines whether a polymer-polymer
or a polymer-salt
system is to be preferred.
If the ligand-protein
binding is a consequence of electrostatic
interactions,
affinity partitioning
is usually sensitive to
salt concentration.
When the binding is of a hydrophobic nature, high concentrations
of salt enhance ligandprotein interaction
and polymer-salt
systems can be
used.
In many cases good separation can be achieved in simple (i.e., Dx-PEG
or PEG-salt)
systems. An example is
the isolation of penicillin-G
which was obtained from
fermentation
broth by extraction
into the PEG-phase
(PEG-salt
system) with a K value of 10 while the cell
mass was concentrated
at the interface and in the lower
(salt) phase which also contained the phenylacetic
acid
(68). Dehydrogenases
and kinases have been extracted
by use of PEG-linked
textile dyes (63,69-72).
Such ligands are inexpensive,
surprisingly
specific, and easy to
attach to the polymers
because of the dyes reactive
groups. The ligand-PEG,
together
with the normal
PEG, can be effectively recovered (up to 96%) by addition of salt (e.g., phosphate)
(69). This provides a basis
for continuous
recycling processes (73).
Another kind of affinity ligands with great potential
are metal chelates. Iminodiacetic
acid (IDA) has been
bound to PEG and loaded with divalent ions of the transition elements such as Cup+, Zn2+, or Ni2+ (67,74-76).
The polymer-bound
copper chelate effectively extracts
proteins with clustered
histidine
residues. The introduction of a few juxtapositioned
histidines on a genetically engineered protein therefore
allows its effective
extraction
from a homogenate.
In PEG-salt
systems most proteins partition strongly
into the lower salt-rich
phase. A striking
exception is

PHASE

PARTITIONING

P-galactosidase
which has strong affinity for the PEGcontaining
phase. Enfors et al. have used P-galactosidase fused to other proteins as a means to recover the
latter in the PEG phase (77). Since the high affinity of
,!I-galactosidase
for the PEG phase seems related to the
large number of tryptophan
groups on its surface, the
same researchers
suggest that an oligo-tryptophan
chain genetically linked to a target protein could be used
for its selective extraction.
The partitioning
of biomacromolecules
can also be
steered by introducing
ionic or hydrophobic
groups on
either of the phase-forming
polymers
or on another
polymer included in the system. Heavily charged polymers, e.g., diethylaminoethyl
(DEAE)-Dx
or Dx-sulfate, have recently been used to concentrate
enzymes in
one phase. These polyelectrolytes
can be directed toward either the top or bottom phase by change of salt
composition
in a Dx-PEG
system. This simple adjustment can result in a change in K value of proteins of
lOO,OOO-fold (78).
Analogous
salt-directed
steering to one phase has
been reported for some ligand-polymers.
When the ligand carries charged groups and the degree of substitution is relatively high, the net charge causes the ligandpolymer itself to partition
in a manner sensitive to the
kind of salt included in the phase system (79).
The degree of phase hydrophobicity
can be adjusted.
This is of importance for separation of proteins with low
solubility in water. Such proteins are found, for example, in plant seeds and can be extracted with the aid of
organic solvents (e.g., ethanol). They can be fractionated in phase systems in which part or all of the water
has been replaced by a water-soluble
organic solvent.
Dimethylsulfoxide
(DMSO)
and dimethylformamide
have been incorporated
in systems based on PEG-hydroxypropyldextran
(HPD)
or PEG-Ficoll
(Fi) (80).
The former system (free of water) has been used in a
countercurrent
process for the partial fractionation
of
zein from corn. In a number of cases the ratio of water to
solvent was the same in the two phases. Substances of
low molecular weight, e.g., benzoic acid, showed nearly
the same Kvalue irrespective
of the water/solvent
ratio.
The polymer concentrations
in such systems still influence protein partitioning.
Antibodies
as ligands of high specificity
have been
studied by several groups [see Refs. (81-83) and below].
Their binding properties change gradually with the number of PEG chains attached to them.
Chiral separation
of D- and L-kynurenine
has been
achieved by including serum albumin (which itself partitions primarily
into the bottom phase) in a Dx-PEG
system. The enantiomers
were fairly well separated by a
CCD process consisting of only eight transfers
(84). Instead of albumin, cyclodextrin
has also been used as a
chiral-separating
agent in the systems.
Use of several ligands in a three-phase
system, con-

10

WALTER,

JOHANSSON,

taining PEG, Fi, and Dx, for the separation


of plasma
proteins, has been demonstrated
(85).
The effectiveness
of a ligand in binding and extracting a protein depends on the structure
of the polymer
which carries the ligand and on the nature of the phaseforming polymers
(86). The polymers
Fi, hydroxypropylstarch
(HPS), and Dx, when used as ligand carriers,
yield more effective
affinity
extractions
than PEG
which had been the traditional
carrier of choice.
Dx-PEG
systems can be used for the concentration
and purification
of viruses (2). A major advantage of
partitioning
over centrifugation
procedures
is that the
glycosylated
envelope proteins (i.e., the viral antigens)
can be almost totally recovered (87).
OTHER

SYSTEMS

Besides the standard two-phase systems, Dx-PEG


and PEG-salt, a number of others have been used and
proposed (35). PEG can, in some systems, be replaced
by polyvinyl alcohol (PVA) or polyvinylpyrrolidone
(PVP). Two-polymer aqueous systems have also been
obtained without use of any polysaccharide, e.g., with
PEG-PVA. Dx has been replaced by pullalan, dextrin,
maltodextrin,
HPS, and ethylhydroxyethylcellulose
(EHEC). These polymers are all inexpensive and potentially useful for large-scale operations. Some of these
polysaccharides, e.g., HPS and EHEC, phase-separate
with one another.
Interesting acrylate copolymers have been synthesized and studied by Hughes and Lowe (88). Groups of
weak acids and bases were incorporated in the polymer
chain giving rise to a polymer which could be precipitated by small adjustments in pH. The polymer was
combined with PVA to yield two-phase systems. After
recovery of the acrylate phase (with its contents of extracted material) the phase polymer was precipitated
and collected.
PVA and PEG form phase systems with many proteins (35). For biotechnological purposes the bulk proteins can thus be used as one of the phase-forming components in the first partitioning
step, replacing (or
strongly reducing the required quantity of) Dx. When
proteins are precipitated by moderate concentrations
of PEG or PVA a viscous but obviously still liquid protein phase is observed.
Copolymers of ethylene oxide and propylene oxide of
various compositions are commercially available. Some
of these copolymers have an upper cloud point in water,
i.e., they will be excluded from the water at higher temperatures. This property makes it possible to recover
the polymers after use in a two-phase system (P. Harris
and F. Tjerneld, submitted for publication). Higher temperatures can also be used to achieve two-phase systems
with PEG and some salts, e.g., NaCl, which do not exclude PEG at room temperature (89). The two phases

AND

BROOKS

which form when a water solution of Triton X-114 is


warmed above its cloud point (17,18) have been used to
extract and separate integral membrane proteins. This
method differs from one in which Triton X-100 is added
to a system, e.g., Dx-PEG, primarily as a solubilizing
agent. Systems composed of Triton X-100 and ammonium sulfate have been used for partitioning lymphocyte lysates (90). The K values of the solubilized membrane proteins could be adjusted by change in the
concentration of ammonium sulfate.
The previously popular PEG-sodium phosphate system (3), especially for large-scale extractions, has the
unwanted side-effect of waste phosphate which increases expenditures due to environmental concerns.
Phosphate has, therefore, been replaced by sodium sulfate, magnesium sulfate (32), potassium carbonate, (91)
and recently also by citrate (92), all of which have lower
disposal costs.
SYNTHESIS
OF POLYMER-LIGANDS
FOR AFFINITY
PARTITIONING

A large number of methods for linking groups to polymers has previously been summarized (3). The main
polymer carrier for affinity ligands and other groups has
been PEG. PEG has two hydroxyl groups which can be
used for the attachment. When only one group is to be
derivatized, methoxy-PEG, with one hydroxyl group,
can be used. Lately attention has been given to the polyhydroxypolymers where a higher degree of substitution
is feasible. Such polymers include Dx, Fi, HPS, EHEC,
and PVA. For polysaccharides a diversity of linkage
methods are known from the preparation of adsorbent
beds for affinity chromatography.
An excellent way to activate PEG as well as other
polymers with hydroxyl groups is tresylation, i.e., reacting the polymer under anhydrous conditions with tresyl
chloride. Dx can be tresylated by dissolving it in DMSO.
The activated polymers react readily with amino groups
and tresylation has been employed both for attaching
affinity ligands, e.g., naloxone and naltroxone (93), and
proteins (94). Recently, tresylated monomethoxy-PEG
was reacted with topoisomerases while they were covalently attached to DNA. This permitted the extraction,
in a PEG-phosphate system, of protein-bound DNA
which, apparently, becomes linked during induced cell
differentiation
(95). Cyanuric chloride (1,3,5-trichlorotriazine) has also been used to attach methoxy-PEG to
proteins, e.g., antibodies (96). The biotin-avidin
complex has been used in affinity partitioning with antibodies. The protein (antigen) to be extracted forms a conjugate with its antibody to which biotin has been attached.
These complexes, in turn, bind to avidin linked to PEG
chains. The PEG-avidin
can, consequently, be used
with any biotinylated antibodies.
NADH has been bound to PEG by employing an ele-

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gant method well suited for large-scale


production
[Buckmann
et al. in (97)]. NAD was reacted with ethyleneimine giving N(l)-(2-aminoethyl)-NAD
which was
linked to succinylated
amino-PEG
using carbodiimide
as condensing
agent. After reduction
with dithionite
and purification
(by formation
of a two-phase
system
between the product and sodium carbonate)
a Dimroth
rearrangementwascarriedoutyieldingPEG-N-(2-aminoethyl)-NADH.
A simple and effective method for attaching ATP to Dx based on activation
of the polysaccharide
with
1,4-butanediol
diglycidylether
in the
presence
of sodium borohydride
has been described
(98). The activated Dx was reacted with ATP for about
12 h, unreacted epoxide groups were deactivated,
and
the product was precipitated
with ethanol. Unbound
ATP was removed by ultrafiltration.
All the bound ATP
molecules (up to 34 per Dx molecule, M, 40,000) were
available for enzyme reactions. Coupling methods based
on the presence of a reducing end group for derivatizing
Dx have been described by Yalpani and Brooks (99).
IDA has been introduced
as a metal chelator in either
of two ways: chloromethoxy-PEG
was reacted with IDA
or aminomethoxy-PEG
was reacted with an excess of
bromoacetic
acid (67).
Another
possibility
is to include functional
groups
during the polymerization
process. In this way a metal
chelator, 8-hydroxyquinoline,
has been incorporated
in
PEG giving a polymer which phase-separates
with Dx
and also extracts a number of ions of the transition
elements (100). The coacrylates
mentioned earlier (88) are
also examples of tailor-made
polymers.
The main problem in the preparation
of ligand-polymers often lies in their purification.
The quantity of
unreacted ligand and reaction biproducts
might be considerable and the purification
may involve problems
which increase with the volume of the product mixture.
Purification
procedures
vary from polymer to polymer.
Methods used include precipitation
with salt or organic
solvent, dialysis or ultrafiltration,
two-phase
partitioning, exclusion by increased temperature,
gel filtration,
and adsorption
to ion exchanger
(86). One common
problem is the removal of free ligand which, when it
consists of a large aromatic system, associates with the
polymer-bound
ligand, i.e., aromatic stacking.
BIOLOGICAL

PARTICULATES

The partitioning of particulates (cells, membranes,


organelles) is a particularly sensitive method for (a)
their separation and (b) subfractionation; (c) tracing or
detecting surface alterations which occur as a function
of in uiuo processes or in vitro treatments; (d) establishing correlations between their surface and other biological properties; (e) studying cell-cell or cell-molecule interactions; (f) detecting very small surface differences
between closely related cell populations; and (g) ex-

PHASE

PARTITIONING

11

tracting specific cells, membranes, and organelles by the


use of ligands attached to one of the phase-formingpolymers. For a summary of earlier work see Refs. (l-3).
As the size of the partitioned biomaterial increases
(from macromolecules to cells), its distribution changes
from between the two bulk phases to the two bulk
phases and the interface and finally to between one bulk
phase (in most of the cases described here the top
phase) and the interface. The partition coefficient, K,
which reflects the partitioning behavior of soluble materials is replaced by the partition ratio, P, which delineates the partitioning behavior of larger particulates.
Furthermore, size (or surface area), which is one of the
parameters reflected in the partitioning of macromolecules and small particulates, appears to have no discernible role in the partitioning behavior of particulates with
surface areas larger than 0.2 pm2 where surface properties per unit area are more determinant than surface
area per se (53).
Cells
Mammalian Cells
Red blood cells--A
model for cell partitioning
behavior. Particulate partitioning is a dynamic process in which some cells attach to droplets of one phase
suspended in the other after mixing, in a cell- and
phase-specific manner, and are delivered on the droplets to the bulk interface (101). Cell partitioning is thus
a time-dependent process which, it has recently been
found, can go on for hours during which the quantity of
total cells in the top phase diminishes while the percentage of cells with higher P values increases (102). It may
therefore be possible to select a time which balances
desired relative cell purity and yield. More efficient separations are attained with high rather than with thinlayer phase columns [e.g., in a Craig rather than an Albe&son CCD unit] (102), presumably because the
faster phase settling in the latter case interferes with
the cell partitioning mechanism. Thus, apparatuses (see
above) designed to speed phase separation and shorten
separation times, which are suitable for macromolecules or small particulates, may be counterproductive
when used for cell segregation. Reports of enhanced cell
separations by thin-layer CCD using long settling times
[e.g., (103)] are likely due to the superimposition of cell
sedimentation effects on partitioning. Since sedimentation can also, depending on the relative P values and
sizes of cells to be separated, counteract cell fractionation by partitioning (lOa), it is preferable to use high
phase columns (or at least shorter settling times).
The original affinity ligands used for cell partitioning
were either charged (i.e., DEAE-Dx,
trimethylamino(TMA-) or sulfonyl-PEG) or hydrophobic [e.g., esters of
fatty acids and PEG (FA-PEG)]
(1). Currently being
explored are the species-specific differences in metal

12

WALTER,

JOHANSSON,

chelate affinity partitioning


of red blood cells from different species (104). Immunoaffinity
methods are being
developed. This approach has the advantage over FACS
in that virtually any quantity of cells can be separated;
and it does not suffer from recovery and nonspecific
adsorption
problems common when using solid phase
adsorbents.
Rabbit anti-human
erythrocyte
antibodies
(Ab) covalently coupled to PEG (PEG-Ab),
a modification which both reduces the tendency of Ab to agglutinate cells and increases its affinity for the top phase, has
permitted
specific extraction
of, for example, human
red blood cells in a mixture of human and sheep red cells
in phase systems which gave no resolution
in the absence of the PEG-Ab
(4681). To obviate the need for
synthesizing
specific Ab-PEG
for every desired separation, a general ligand for immunoaffinity
partitioning,
namely PEG linked to mouse monoclonal
IgG specific
for rabbit IgG, has been used (82). When cells of interest
are targeted with rabbit-Ab raised against a surface antigen they can be extracted in a phase system containing
the PEG-anti-rabbit
IgG. These methods have not yet
been.used in any but model systems and it is not clear
whether minor cell subpopulations
of interest bearing
relatively few surface antigens can be extracted.
A recent study suggests that combination
of bulk extraction
and CCD may improve the purification
of minor cell
subpopulations
by immunoaffinity
partitioning
(105). A
promising
approach, based on the great incompatibility
of PEG and polyacrylamide
previously
utilized in DxPEG columns (106), may be one in which Ab is derivatized with polyacrylamide
(107). Such a polyacrylamide-ligand
tends to partition strongly in favor of the
bottom Dx-rich phase in Dx-PEG
systems. Polyacrylamide-derivatized
Ab enabled a separation by CCD, not
feasible by the PEG-Ab
methodology
outlined above
(81,82), of two sublines of transformed
mouse lymphocytes which differ solely in the number of antigen molecules per cell (8 X lo5 and 2.4 X 106).
Partitioning
is a powerful method for tracing cell surface changes that accompany in vivo processes and/or
in vitro treatments.
For example, rat red blood cells of
different
ages have characteristic
G values in both
charge-sensitive
and non-charge-sensitive
Dx-PEG
phase systems (1). By combining
5gFe labeling of rat
erythrocytes
with glutaraldehyde
fixation, different
in
vitro treatments
of the fixed cells, and CCD, aspects of
the chemical nature of the surface alterations
that accompany erythrocyte
aging could be established
(108).
Combination
of CCD with other separatory
methods
(e.g., centrifugation)
can result in segregations
not obtainable by the use of any one method alone [see also
Refs. (log-Ill)].
Thus partition
analysis of densityfractionated
rat erythrocytes
revealed a young erythrocyte population in the most dense cell fraction (112).

AND

BROOKS

Selected Separations

and Studies

Lymphocytes.
As an extension of earlier studies on
the fractionation
of human peripheral
blood lymphocytes (l), the surface heterogeneity
of T-lymphocyte
subpopulations
has now been examined by CCD (113).
The helper/inducer
T-cell subset (OKT4/Leu3+)
has a
single distribution
curve while the suppressor/cytotoxic
(OKT8/Leu2+)
is clearly heterogeneous
and gives two
peaks. The cells with higher G values form part of a
minor subpopulation
of lymphocytes
(increased in patients with rheumatoid
arthritis)
which also have a
marker of natural killer cells (HNK-1).
Tissue cells: Differentiation,
maturation,
and aging.
Differentiation
and pattern
formation
in developing
chick limb bud was examined by partitioning
(114) and
progress zones and more proximal regions resolved into
several cell populations.
In other studies extensive
changes in surface properties
of sperm during passage
through
the epididymis
were discerned by partitioning (115).
Tissue culture cells. Use of the previously
described
method for the detection of subtle surface differences
between closely related cell populations
(1) (in which
small quantities of 51Cr-labeled cells are mixed with an
excess of cells to which they are to be compared and the
mixture then subjected to CCD) has shown that no two
K-562 cell sublines examined have the same surface
properties
(i.e., overlapping distribution
curves) (116).
The
surface
properties
of seCell-Cell interactions.
lected bacterial and mammalian cells were examined by
partitioning
in phase systems containing
either TMAPEG or FA-PEG to obtain a measure of relative surface
charge or hydrophobicity,
respectively.
Such data (together with other physicochemical
measurements
of cell
surface properties)
were examined for possible correlations to relative adhesion of bacteria to certain tissues
and resistance of cells to phagocytosis
and to serum bactericidal systems. Interactions
were found to be both
specific (i.e., at a receptor level) and nonspecific (hydrophobic and charge-mediated)
(117).
Plant Cell-s
Partitioning
has been used in the chemotaxonomic
classification
of three strains each of three closely related Penicillium
species in the P. viridicatum
group
(118). The conidia from these strains were subjected to
cross-partition
analysis (1) using phase systems containing Dx-PEG-Fi
and either DEAE-Dx
or Dx-sulfate. The P value of conidia at the cross-point
and at
certain other pHs, as well as growth rate measurements
on different culture media, were determined
and statistically analyzed. The results indicate that the different
species are well separated and correlate with the species characterization
based on mycotoxin production.

RECENT

Bacterial

RESULTS

IN

AQUEOUS

Cells

Different
strains of the fish pathogen Aeromonas salmonicida which differ in their ability to produce the surface protein array known as the A layer and a smooth
lipopolysaccharide
were
examined
by partitioning
(119). Both the protein layer and the 0-polysaccharide
were found to be important
in determining
the partitioning behavior of the cells. The A layer, crucial to the
virulence of the cells, decreased the surface hydrophilicity and increased (specifically)
surface affinity for FAPEG. Thus partitioning
may be a useful tool in the analysis of surface
properties
important
in bacterial
virulence and, perhaps, in the selection of mutants with
specific surface characteristics.
Membranes

and Organelles
Animal

Materials

General.
The partition
ratios of membranes
and organelles tend to increase in the order: endoplasmic reticulum, mitochondria,
lysosomes,
Golgi membranes,
and
plasma membranes.
Such materials, purified to a high
degree, when subjected to CCD reveal additional heterogeneities (120). Thus plasma membrane vesicles can
be fractionated
into vesicles with different marker activities; mitochondria
separated into two populations
(the
ratio of which changes with rat age); and microsomal
membranes
into smooth, light rough, and heavy rough
fractions.
A general multiparameter
approach for the fractionation of plasma membranes from polar cells and tissues
has been developed which involves tissue homogenization, low speed centrifugation,
density gradient centrifugation, differential
rate sedimentation,
and partitioning. This approach takes into account the possibility
that enzyme activities
traditionally
used as plasma
membrane
markers
may not be exclusively
localized
there (109).
Separations
of at least certain (e.g., synaptic)
membranes can be significantly
improved and/or their biochemical
activities
maintained
by the incorporation
of glycerol or ethylene glycol into the phase systems
and carrying
out the partitions
at subzero temperatures (121).
Affinity partitioning
has been applied to membranes
and organelles both to effect biospecific extractions
as
well as to probe for additional heterogeneities.
Rat liver
plasma membranes can be separated from other cellular
membranes
in a few bulk extraction
steps by use of a
lectin ligand (wheat germ agglutinin)
attached to Dx.
The phase composition
was chosen such that all membranes partitioned
to the top phase in the absence of the
Dx-lectin
while its presence caused the desired membrane to be specifically pulled into the Dx-rich bottom

PHASE

13

PARTITIONING

phase (122). The heterogeneity


of a synaptosomal
preparation was established
by affinity partitioning
using
hexaethonium-PEG
and centrifugal
CCD. Three major
synaptosomal
fractions were obtained which differed in
a number of physical and biochemical properties
(123).
To foster rapidity of phase separation
and thereby reduce undesirable
time-dependent
side effects, the centrifugal CCD apparatus
(54) described above can be of
advantage when working with membranes.
A method for the analysis of the domain structure
of
membranes
(i.e., mapping their lateral heterogeneity)
has been described (124). It depends on membrane purification, fragmentation,
and CCD of the fragments
followed by quantitation
of different domain markers
in
the fractions.
Specific activities of markers
differ depending on their proximity
in the membranes,
the size
of fragments,
and location of breaks. By plotting the
concentrations
of one marker vs another for different
size classes, relations characteristic
of domain relative
proximity
are obtained.
Selected Separations

and Studies

Whole homogenates.
Since the partitioning
behavior
of biomaterials
depends to a great extent on their surface properties,
in vitro treatments
(e.g., with enzymes)
often result in changes in partition
ratios which reflect
the physicochemical
nature of the alteration (l-3). Enzyme treatments
were, thus, generally avoided if partitioning data were to be interpreted
in terms of original
surface properties.
However, a comparison
of the multiparameter subcellular fractionation
(109) of acini from
rat lacrimal gland prepared with and without the use of
enzymes (collagenase,
hyaluronidase,
DNase) pointed
up no major difference in the partitioning
properties
of
the fractions obtained (125). The use of enzyme treatments of short duration in membrane (or cell) preparation for partitioning
studies should thus be considered
on a case to case basis and need not necessarily
be
shunned.
Plasma membranes.
The lateral heterogeneity
of rat
liver plasma membranes was probed by use of the membrane domain analysis method outlined above (124).
Fragmented
membranes were fractionated
by CCD and
the distribution
pattern was analyzed by plotting specific activities of marker components
against one another. Asialo-orosomucoid
receptors were in a domain
separated
from those containing
5-nucleotidase
and
leucine aminopeptidase
by another domain devoid of
these markers (126).
Subcellular
membranes.
Three-dimensional
fractionation (involving differential centrifugation,
equilibrium density gradient centrifugation,
and partitioning)
was used to probe the subcellular
distribution
of Na/H
antiport activity in rat renal cortex. About 53% of Na+/

14

WALTER,

JOHANSSON,

H+ antiport
activity was localized in a population
of
brush border membranes. Another 26% of recovered activity was associated with membranes
that could have
originated in brush border membranes
or in the Golgi
complex involved in assembly
or recycling
of brush
border membranes.
Thus, depending on the origin of
membranes in question, not more than one-third of the
recoverable Na+/H+ antiport activity could be assigned
to cytoplasmic
membranes
of the proximal tubular epithelium (127).
Mitochondria.
Highly purified brain mitochondria
have been obtained by partitioning
in phase systems
containing
Naf, NH:, or K+ and the effects of various
ions on mitochondrial
respiration
have been examined (128).
Synaptosomes.
/3-bungarotoxin
has been found to increase the partition ratio of a discrete (30-32%) population of cerebrocortical
synaptosomes.
The affected population has virtually all of the choline acetyltransferase
(a marker for cholinergic synaptosomes)
(129).
Lysosomes.
Partitioning
of a crude mitochondria
preparation
in a Dx-PEG
system containing
ammonium chloride permitted
the separation
of lysosomes
(high partition)
from mitochondria
(low partition).
The
procedure is reproducible
and lysosomes were never contaminated with more than 16% mitochondria
(130).

Plant Materials
General.

Aqueous phase partitioning


has found one
of its greatest applications
in the study of plant materials where it has led to some unique separations.
The
isolation
by partitioning
of highly
(95%) purified
plasma membranes (without the use of detergents)
from
a large variety of plant sources has recently been expanded to include algae and microalgae [for a review see
(131)]. The method appears to be superior to sucrose
density gradient centrifugation
which yields preparations of lower purity (50-75%),
greater leakiness,
and
mixed polarity. The isolation of membrane vesicles of
opposite sidedness (i.e., right-side
out (RO) and inside
out (IO)) is important
in the characterization
of any
membrane system. Plasma membrane vesicles obtained
by partitioning,
from the top phase, are mainly RO. Part
of these vesicles can be turned IO by freezing and thawing. Since the partitioning
of membranes
depends on
their surface properties,
the sealed RO and IO vesicles
can be separated by another extraction.
Such preparations permit studies on transport,
signal transduction,
enzyme topology, etc., using plasma membrane vesicles
of either orientation
(132).
The fundamental
conversion of light energy to chemical energy that takes place in the photosynthetic
chloroplast membrane,
the thylakoid,
makes the study of
this membranes
architecture
of prime importance.
Yeda press treatment
of thylakoid membranes gives rise

AND

BROOKS

to IO and RO vesicles which can be purified by partitioning (133) and have been shown to be derived from
appressed
and nonappressed
regions,
respectively
(134). A large number of studies on the lateral and
transbilayer
distribution
and function of thylakoid protein complexes
and lipids, under various conditions,
have thereby been made feasible (135).
A novel method to fractionate
cell organelles is one in
which fragmentation
and separation are carried out concurrently.
IO vesicles obtained from thylakoids
were
sonicated while in a two-phase
system. Several sonication and partitioning
cycles yielded highly enriched
photosystem
(PS) II vesicles (and without
the use of
detergents)
[for references see (136)].
Unlike animal cells for which good fractionation
procedures have long been available, plant cells suffer from
a dearth of such methodology.
By combining partitioning (in which diverse plant fragments
display differing
affinities for the top phase, i.e., differ in partition ratios)
with centrifugation
(sucrose, sucrose gradient, glycerol
gradient, Percoll), a scheme has recently been devised
for the fractionation
of a plant (spinach) single homogenate fraction into endoplasmic
reticulum,
Golgi apparatus,
nuclei, plasma membranes,
tonoplast,
mitochondria,
chloroplasts,
and soluble supernatant
solution (110).

Selected Separations

and Studies

Thylukoid
membrane.
Membrane
domain analysis
[see above, (124)] applied to the thylakoid
membrane
(137) indicated that PS I and PS II are segregated from
one another in large domains. It was support for a model
of the thylakoid
in which PS I is located in the nonappressed stroma membranes while PS II is in the grana.
The presence of a third domain rich in cytochrome
be-f
complex was also inferred. Subsequently
found is that
both PS I and PS II are highly heterogeneous
[e.g., in
antenna size, redox properties]
(136). PS Ia, PS 10, PS
IIa, and PS II/3 are in separate domains with the a! systems being in the grana (lower partition)
and the fi systems in the stroma lamellae (higher partition).
Endoplasmic reticulum and tonoplast.
Smooth endoplasmic reticulum and tonoplast from etiolated hypocotyls have been separated. Centrifugation
(on a Fi-sucrose
density
gradient)
of a crude
microsomal
membrane fraction gave most of the smooth endoplasmic reticulum and tonoplast cobanded at the interface
between sample load and Fi layer. Partitioning
of this
mixture yielded highly purified smooth endoplasmic
reticulum
(low partition)
and tonoplast
(high partition) (111).
Leaf mitochondria.
Spinach leaf mitochondria,
virtually free of chlorophyll,
have been prepared.
The
crude preparation
containing about half mitochondrial
proteins contaminated
with thylakoid
membrane frag-

RECENT

RESULTS

IN

AQUEOUS

ments and peroxisomes


was partitioned.
Mitochondria
and thylakoid
membranes
were in the top phase in a
system near the critical point. Increasing
the polymer
concentration
(which increases the interfacial
tension
between the phases) reduced the P value of the mitochondria
and led to their ten-fold purification
(138).
Mitochondria
from Jerusalem
artichoke
tubers have
been purified; RO- and IO-submitochondrial
particles
have been prepared (by adjustment
of ionic conditions
of the disruption
medium)
and then separated,
for
study, by partitioning
(134).
CONCLUSIONS

AND

PHASE

15

PARTITIONING

In biotechnology the systems will result in extremely


rapid and selective extractions in large-scale processes
with recovery of the phase-generating components.
An interesting speculation with regard to aqueous
two- (or multi-)phase systems deals with the role they
might play in biological systems. Many analogies have
been noted between the distribution of different structures and metabolites in the cytoplasm of cells, ascribed
to its microcompartmentation,
and the partitioning
behavior of biomaterials in aqueous phase systems
(139). The future will, hopefully, provide an experimental approach to test this hypothesis.

PROSPECTS

Aqueous two-phase systems have, during the past 5


years, been increasingly used in basic biological research and in biotechnical processes. The great interest
in aqueous phase partitioning, both for separations and
in the physical characterization of biomaterials, is reflected in the search for new phase systems, the development of a variety of apparatuses for multistep procedures, the elaboration of extraction
methods with
increased selectivities by use of modified polymers, and
the evolution of theories of phase separation and of partitioning.
Probably the most difficult problem in theoretically
modeling partitioning phenomena is in developing effective ways to deal with water, particularly in the presence of salts. This problem is hardly unique to partitioning but is clearly critical if realistic treatments are
to evolve. Many of the interactions which take place
likely occur between hydrated species which may to
varying degrees shed the water molecules between them
as contact occurs. Properties such as the dielectric constant of the intervening water molecules may differ considerably from the bulk, for instance. Treating the electrostatic properties of proteins correctly is likely to be
important and will require more sophisticated models
which take into account nonuniform surface properties.
Treating proteins as rigid objects could also be relaxed.
There may well be contributions to the entropy of mixing from configurations of the protein surface which allow some mixing of the interior amino acids with external solution components. This might explain why the
Flory-Huggins expression works as well as it does in
describing protein partitioning.
The future applications of aqueous two-phase systems will include more selective affinity separations of
cells and membranes based on receptors.
Examination of the binding constant of ligand-protein pairs in the presence of different polymers should
yield new phase systems with considerably greater effectiveness in extraction (measured as A log K).
Partitioning at subzero temperatures will permit isolation of sensitive biological structures, e.g., fragile enzyme complexes and labile membrane associations.

ACKNOWLEDGMENTS
Work in Harry
Walterslaboratory
was supported
by the Medical
Research
Service of the Department
of Veterans
Affairs;
in Giite Johanssons
laboratory
by the Swedish
Natural
Science
Research
Council,
the Technical
Research
Council
of the National
Swedish
Board for Technical
Development,
and the Swedish Council
for Forestry and Agricultural
Research,
and in Donald
E. Brookss
laboratory by the Medical
Research
Council
of Canada.

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