Separation of mixtures by aqueous two-phase systems

An aqueous two-phase system useful for the separation and purification of biochemicals and optical isomers. The two-phase system can be formed with water soluble polymers as one phase, and chiral compound as the other phase.

BACKGROUND OF THE INVENTION 
1. Field of the Invention: 
This invention is directed to a method of separation and extraction of 
organic compounds, inorganic compounds, biomolecules and biomaterials by 
partition using an aqueous two-phase solvent system. 
2. Discussion of the Background: 
The separation of a mixture by distribution between two immiscible liquids, 
either by bulk extraction or by liquid-liquid partition chromatography is 
known. Especially well known in this regard are systems containing 
immiscible organic solvents and water. Also, advantage has been taken of 
the phase separation that frequently occurs when solutions of two 
structually different water-soluble polymers are mixed above critical 
concentrations. These systems spontaneously separate into two immiscible 
liquid phases, each phase enriched with respect to one of the polymers. 
Such aqueous two-phase systems are suitable for separation of labile 
materials such as enzymes, cells and organelles. Aqueous phase systems 
containing two polymers, most commonly polyethylene glycol (PEG) and 
dextran have found wide application for the separation of biological 
materials. The phases have low osmotic pressure and high water content. 
Salts and other solutes can be included to provide buffering capacity. 
Systems containing a single polymer and a high concentration of some 
particular salt, e.g., PEG and a phosphate, have also proved useful in the 
separation of macromolecules. However, these systems have not been 
suitable for the separation of optical isomers. A description of 
two-polymer systems and polymer-salt systems as applied to separation of 
biological materials can be found in Walter, H. et al, "Partitioning in 
Aqueous Two-Phase Systems" (Academic Press, 1985) and Albertsson, P. et al 
"Partition of Cell Particles and Macromolecules" (Wiley, 1986). 
Existing methods to separate optical isomers are based on chromatographic 
techniques, selective enzymatic reactions, and fractional crystallization 
of diastereomeric complexes formed with chiral resolving agents. The most 
common technique used on an industrial scale is fractional 
crystallization. For example Manghisi et al, U.S. Pat. No. 4,533,748 
teaches the use of L-lysine to form diastereomeric salts with a racemic 
propionic acid derivative followed by fractional crystallization. 
Fahnenstich et al, U.S. Pat. No. 3,980,665, discloses the use of L-lysine 
to convert D,L-penicillamine to D-penicillamine. Chibata et al, U.S. Pat. 
No. 4,519,955, discloses a method of optical resolution of .alpha.-amino 
acids and .alpha.-phenylethane sulfonic acids by fractional 
crystallization. Optical resolution of a D,L-amino acid is disclosed in 
Yukawa et al, U.S. Pat. No. 4,610,820, which comprises reacting the 
mixture with an optically active N-acylaspartic acid, followed by 
fractional crystallization. 
A method to resolve stereoisomers which relies on a two-phase solvent 
system is disclosed by Empie, U.S. Pat. No. 4,636,470. The method relies 
upon the preferential enzymatic hydrolysis of one enantiomer of 
D,L-phenylalanine. The racemate is dissolved in a substantially water 
immissible organic material which is a solvent for the amino acid racemate 
but not for the resolved amino acid. 
McCloud (Dissertation Abstract B 1969, 29 (7), 2357-8) discloses the 
resolution of D,L-camphoric acid, D,L-dibromobutanediol, and 
D,L-isohydrobenzolin isomers by solvent extraction using water and 
D-tartrate esters in a two-phase system. 
A method of separation of optical isomers based on stereospecific 
interactions with asymmetric sorbents and solvents was disclosed by Buss 
et al, Ind. Eng. Chem., V60 (8), p. 12-28, 1968, however asymmetric 
solvents are prohibitively expensive. The extensive reviews of chiral 
adsorbents for analytical separation of optical isomers can be found in 
Lough, W. J. et al, "Chiral Liquid Chromatography" (Blackie and Son, 1989) 
and Allenmark, S.G., "Chromatographic Enantioseparation: Methods and 
Applications" (Ellis Horwood Limited, 1988). 
These methods to separate optical isomers are expensive and require several 
steps. They are highly specialized procedures which must be developed 
specifically for the optical isomer mixture to be resolved. For example, 
conditions which effect the selective crystallization of a diastereomeric 
mixture must be carefully controlled with respect to concentration and 
mass transfer effects. In addition, these systems often do not yield the 
high purity product one desires. 
Therefore a need still exists for inexpensive and generally applicable 
methods for separating stereoisomers and biomolecules especially as 
applied to industrial processes. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of this invention to provide a novel process 
for the facile and efficient separation of stereoisomers of organic and 
inorganic compounds. 
It is another object of this invention to provide an improved process for 
the separation of organic and inorganic compounds, and biomaterials such 
as peptides, proteins, cells and cell particles. 
Another object of this invention is to provide an improved process for 
affinity partitioning and for partition affinity ligand assays. 
Another object is to provide a novel aqueous two-phase system that can be 
used for analytical, preparative and large scale commercial separations in 
combination with known methods such as counter-current distribution, 
cross-current extraction and counter-current extraction. 
These and other objects which will become apparent from the following 
description have been achieved by the present invention which is discussed 
in detail below. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In the present invention, separation of mixtures including the resolution 
of stereoisomers is achieved by partition between two phases of an aqueous 
two-phase system comprising (a) a water soluble polymer, (b) water, (c) a 
previously undisclosed group of phase-forming compounds which cause the 
spontaneous separation of the system into two immiscible liquid phases; 
and (d) the mixture to be separated. Soluble and insoluble materials added 
to these systems distribute themselves between the bulk phases and the 
interphase. In the case of the separation of racemic mixtures component 
(c) may act as a chiral resolving agent. 
It has been discovered that a solution containing a water soluble polymer 
plus an amino acid, protein, peptide, monosaccharide, disaccharide, or 
chiral compound surprisingly will form aqueous two-phase systems. These 
systems are capable of resolving mixtures of optical isomers such as other 
amino acids, for example. These novel two-phase systems are also capable 
of separating cell particulates, such as mitochondria, chloroplasts, 
membranes, liposomes, chromosomes; macromolecules such as proteins, 
nucleic acids and water soluble polymers; cells such as bacteria, fungi, 
algae, erythrocytes, lymphocytes, leukocytes and cancer cells; organic and 
inorganic compounds, such as metals recovery, actinides and lanthanides 
from nuclear wastes, organic pollutants from waste water, and americium 
and plutonium recovery from nitric acid waste streams. 
Mixtures of amino acids, alcohols, amines, halides, acids, aldehydes, 
ketones, fats, amides, peptides, carbohydrates, etc., containing chiral 
atoms will interact with the chiral resolving agent thereby forming 
diastereomeric complexes. If the chiral material is a mixture, e.g., a 
racemic mixture, differential partition of the diastereomeric complexes 
formed occurs between the phases. 
It has been found that monosaccharides, disaccharides, peptides, proteins, 
antibodies and optically active amino acids can fulfill the requirements 
of the phase-forming agent (c) described above. Optically active salts, 
acids, bases and solvents can also act as the chiral resolving agent (c). 
Suitable amino acids include the D- or L-naturally occurring .alpha.-amino 
acids, such as alanine, lysine, serine, proline, etc., as well as other 
chiral amino acids. By "chiral amino acids" is meant any amino acid which 
is substantially soluble in water and which contains at least one chiral 
carbon atom. Chiral amino acids include the natural and non-natural amino 
acids generally having fewer than 50 carbon atoms and, preferably fewer 
than 20 carbon atoms. The chiral amino acids may be substituted with one 
or more functional groups such as hydroxy, carboxylic acid, amino, 
substituted amino, thiol groups, etc., to enhance water solubility and 
chirality of the compound. 
Although glycine is not a chiral amino acid, it can form the aqueous 
two-phase systems of the present invention to be used for the separation 
of organic compounds, inorganic compounds, biomolecules and biomaterials. 
A chiral additive selected from the group of amino acids, peptides, 
proteins, monosaccarides, disaccharides, antibodies, chiral salts, acids, 
bases and solvents as described in this invention can modify the 
polymer-glycine-water system to effect resolution of stereoisomers. In 
addition, glycine can be modified to result in a chiral compound. 
It has been discovered that monosaccharides and disaccharides, most of 
which are optically active compounds, can also function as the 
phase-forming agent. These sugars can form aqueous two-phase systems with 
polyethylene glycol, polypropylene glycol and other water soluble 
polymers. The phase forming monosaccharides and disaccharides include 
arabinose, glucose, galactose, dextrose d-glucose, fructose, lactose, 
levulose, maltose, mannose, xylose, sucrose and others. The natural 
alditols, inositols, aldonic acids, uronic acids, aldaric acids, 
monosaccharides and disaccharides described in Weast, R. C., "Handbook of 
Chemistry and Physics," The Chemical Rubber Co. (CRC), Cleveland, Ohio, 
63rd edition, 1982, C-760 to C-767; and Davidson, E. A., "Carbohydrate 
Chemistry," Holt, N.Y., 1967, are within the scope of the present 
invention. 
Optically active salts, acids, bases and water miscible solvents are also 
phase forming agents when added to the water soluble polymers. Examples 
include lead hydrosulfate, potassium hydrosulfate, amyl alcohol, camphor, 
cedar oil, citrol oil, ethyl malate, menthol, bromocamphorsulfonic acid, 
camphorsulfonic acid, chlorocamphorsulfonic acid, codeine sulfonic acid, 
hydroxybutyric acid, lactic acid, malic acid, mandelic acid, 
methylene-camphor, phenylsuccinic acid, tartaric acid, brucine, 
cinchonidine, cinchonine, cocaine, coniine, codeine, hydrostine, 
menthylamine, narcotine, quinidine, quinine, thebaine, strychnine, 
potassium tartrate, quinine sulfate, santonin, sodium potassium tartrate 
terpenes, tartaric acid and ascorbic acid. 
The optically active salts, acids, bases, and water miscible solvents 
described in Weast, R. C., "Handbook of Chemistry and Physics," The 
Chemical Rubber Co. (CRC), Cleveland, Ohio, 63rd edition, 1982, E404-406, 
are considered to be within the scope of the present invention. 
Proteins, peptides and antibodies which can used both as the phase-forming 
component and as the chiral resolving agent include peptides as small as 
dipeptides and include proteins having up to 1,000,000 amino acid 
residues, as long as the peptide has substantial water solubility. Amino 
acid homopolymers such as polylysine, polyglycine, etc. are also included 
as suitable peptides. Preferred peptides and proteins are those having 
from 2-1,000,000 amino acid residues with peptides and proteins having 
2-1,000 amino acid residues being particularly preferred. Specific 
examples of suitable peptides and proteins which may be used as the chiral 
resolving agent include trypsin, chymotrypsin, pepsin, lipase, cytochrome 
c, ribonuclease, lysozyme, myoglobin, rhodanese, ovalbumin, amylase, 
protease, transferrin, conalbumin, bovine serum albumin and thyroglobulin. 
Polymers which are useful for the preparation of aqueous two-phase systems 
include both synthetic polymers and purified natural polymers. Any water 
soluble polymer capable of forming a two-phase aqueous system with an 
aqueous solution of component (c), above, is within the scope of the 
invention. Preferred polymers are water soluble polymers based on sugars 
and polyalkylene glycols prepared from glycols having 2-8 carbon atoms 
based on hydrocarbon ether units. 
The polymers may also be derivatized to provide additional water solubility 
and chirality for the system and to adjust the separation factor as 
discussed below. Suitable derivatives are amino, alkylamino, carboxyl, 
aldehyde, ketone, amine, amide and carbohydrated derivatives, for example 
PEG - fatty acid esters and diethyleneaminoethyl-dextran. 
Examples of commercially available sugar polymers include the glucose 
polymer dextran, polysucrose, methyl cellulose, 
ethylhydroxyethylcellulose, hydroxypropyldextran, ficoll, sodium dextran 
sulfate, butylcellosolve, sodium carboxymethyldextran, sodium 
carboxymethylcellulose, DEAE dextran-HCl, starch and other 
polysaccharides. 
Commercially available hydrocarbon ether polymers include polyethylene 
glycol (PEG) of different molecular weights, polypropylene glycol, 
methoxypolyethylene glycol, polyvinyl alcohol and polyethylene 
glycol-polypropylene glycol copolymers. Polymers prepared from 
hydrocarbon, aromatic, aliphatic and cycloaliphatic amines such as 
polyvinylpyrrolidone are also suitable. 
PEG is a linear synthetic polymer available in a variety of molecular 
weights. PEG is quite stable in solution, in the dry powdered or flake 
form in which it is sold. Common fractions of PEG supplied by Union 
Carbide Corporation used in the present phase partitioning process include 
PEGs having a molecular weight in the range of about 300-50,000 preferably 
in the range 300-30,000 such as PEG 300, PEG 400, PEG 3,400, PEG 8,000 and 
PEG 20,000. Other commercial names for PEGs include polyglycol E (Dow 
Chemical), carbowax (Union Carbide), and pouracol E (BASF) (Wyandotte 
Corp). 
Dextran, predominantly poly(.alpha.-1,6 glucose) is commerically available 
in a range of molecular weight fractions from approximately 2,000 to 2 
million. Similar molecular weights for derivatives are also useful. 
Adding salts, such as potassium phosphate, sodium chloride, ammonium 
sulfate, magnesium sulfate, copper sulfate, sodium sulfate and lithium 
sulfate, optically active salts (as described above), monosaccharides and 
disaccharides (as described above) to the water soluble polymer (a) to 
enhance the solubility, phase separation or chirality are also considered 
to be within the scope of the present invention. 
Stock solutions of the polymers are formed using known methods such as 
described in Walter et al. and Albertsson. 
Stock solutions of dextran of 20-30% by weight are made by mixing the 
powder into a paste with an equal weight of water, then adding enough 
water by weight to yield an apparent concentration approximately 10% 
higher than desired in order to allow for water in the dextran. Dextran 
stock solutions are stirred 2-3 hours at room temperature, but the time 
may be shortened with heating. Concentrations of dextran stock solution 
can be determined by polarimetry. 
PEG stock solutions of 30-40% by weight are made up by accurate weighing of 
the powder and dissolution in water at room temperature. PEG dissolves 
readily on stirring for a couple of hours and fresh, properly stored PEG 
powder usually contains less than 0.5% water. The stock concentration can 
be measured by refractive index. 
Preferably, stock solutions of amino acids, salt, sucrose, etc. are made up 
at least four times the concentration by weight desired in the phase 
system. 
The biphasic systems can also be prepared by weighing appropriate 
quantities of PEG (in solid form), amino acid stock solution, and water 
into a beaker. Thirty grams of a system may be easily prepared. The 
resulting solution is magnetically stirred for 3 hours, after which it is 
poured into a centrifuge tube. The phase systems are allowed to 
equilibrate at 5 min. to 24 hours depending on the system. 
The aqueous two-phase system may be prepared from the stock solutions of 
polymer and concentrated amino acid solutions. The stock solutions may 
contain known buffers to control the pH and salts to increase the tonicity 
of the solutions. Preferably, potassium phosphate buffer is used to adjust 
the pH. These buffers and salts are selected according to the nature of 
the material to be separated and can be readily determined by one skilled 
in the art using the criteria set forth below. For example, since the 
water soluble polymers are generally ion-free and have large molecular 
weights, their contribution, at high concentrations, to the tonicity of a 
solution is small. If a mixture to be separated comprises a protein which 
would be denatured by low ionic strength solutions, the addition of salts 
would be indicated. The tonicity of the phases can be measured by using a 
vapor pressure osmometer. 
Additives such as salts, buffers, sugars and polymers may be added to 
enhance phase separation and separation factor. Suitable additives include 
potassium phosphate, glucose, glycerol, butylcellusolve, propylalcohol and 
sodium chloride. Additives which can increase the difference in density or 
hydrophobicity between the two phases, reduce the viscosities of either 
phase, or increase the interfacial tension between the two phases, will 
enhance phase separation and separation factor. 
The temperature and pressure can also be manipulated to enhance phase 
separation and partition coefficient. Suitable temperatures span the range 
from where the aqueous two-phase system begins to freeze up to the boiling 
point of the water, in general from about -10.degree. C. to 100.degree. C. 
The preferred temperature is from 0.degree. C. to ambient temperature. The 
range of pressure includes all pressures which result in a two-phase 
system, in general from 0.01 to 1,000 atmospheres. The preferred pressure 
is ambient pressure. 
The two-phase system is generally prepared from the stock solutions. The 
amount of the chiral compound in the two-phase system is generally in the 
range of about 0.01-50.0 wt. % relative to the weight of the aqueous 
two-phase system. Preferably, the chiral compound is present in an amount 
of about 0.1-40 wt. %. In general, the water soluble polymers are present 
in amounts from about 2-50 wt. %, preferably 2-30 wt. % relative to the 
weight of the two-phase system, with the balance being water. Obviously, 
amounts of chiral compound and water soluble polymer may vary above or 
below these numerical ranges so long as an aqueous two-phase system is 
produced when the aqueous stock solutions of amino acid and water soluble 
polymer are mixed. All proportions of chiral compound and water soluble 
polymer which interact to form an aqueous two-phase system are considered 
to be within the scope of the present invention. 
In order to separate two components of a mixture in one or only a few 
steps, the partition behavior of the components must be manipulated in a 
manner such that one component is in one phase and the other component is 
in the other phase or at the interphase. Using the present method, 
D,L-amino acids can be separated into two fractions each partially 
enriched with one optical isomer. The separation factor (.alpha.=K.sub.A 
/K.sub.B), is defined as the ratio of partition coefficient K.sub.A of 
component A to partition coefficient K.sub.B of component B. The 
separation factor can be any value other than 1.0, and is preferably less 
than 0.98 or larger than 1.02. When .alpha.=1.0, A and B will not be able 
to be resolved. 
For mixtures that do not differ greatly in their partitioning behavior, 
i.e., a separation factor close to 1.0, single extraction steps are not 
sufficient to produce a separation. In such cases multiple extraction 
procedures such as countercurrent distribution (CCD), thin-layer 
counter-current distribution, enhanced gravity counter-current 
distribution, partition column chromatography, counter-current 
chromatography, liquid-liquid extractors, centrifugal liquid-liquid 
extractors, multi-stage cross-current extractors and counter-current 
extractors are required. The preferred method is CCD, which, with aqueous 
two-phase systems, performs a discrete number of partition steps with thin 
layers of each phase. Continuous cross-current and counter-current 
extraction methods using columns are also possible. The theory of CCD and 
the design and use of the thin-layer CCD apparatus are described by Walter 
et al. The theory and practice of cross-current and counter-current 
extraction are described by Treybal, R., "Liquid Extraction" McGraw Hill, 
1963. 
Separation with the two-phase systems of the present invention depends on 
the choice of phase composition so as to obtain appropriate partition 
coefficients for the materials of interest. There are three major ways in 
which these systems can be manipulated or adjusted so as to give phases 
with appreciably different physical properties: (1) choice of polymer, 
polymer concentration, polymer molecular weight; (2) choice of the phase 
forming agent, e.g., monosaccharide, disaccharide, chiral compound, chiral 
amino acid, peptide or protein and its concentration; (3) and chemical 
modification of the water soluble polymer by attaching a ligand for which 
receptors exist on the material of interest. In the last case the 
resulting procedure is called affinity partitioning. 
With regard to the choice of the water soluble polymer we prefer the 
polymer which gives a higher density difference from the chiral phase, 
lower viscosity, higher interfacial tension between the two phases, lower 
concentration to form two phases and lower toxicity. 
With regard to the phase-forming chiral agent we prefer the compound which 
gives higher specific rotation [a] .sup.2.sub.D.sup.5, (definition of 
specific rotation is provided in Lehninger, A. L., "Biochemistry", Worth 
Publishers, 1975, page 81) higher density difference from the other phase, 
lower viscosity, lower concentration to form two phases, less toxicity and 
higher interfacial tension between the two phases. Besides chiral amino 
acids, peptides and proteins, chiral compounds, such as monosaccharides 
and disaccharides, chiral salts, chiral solvents, chiral acids and chiral 
bases also are considered to be within the scope of the present invention. 
Another embodiment of the invention is in the modification of known, 
non-chiral, aqueous two-phase systems. The chiral phase-forming component 
(component c, above) of the present invention may be added to an already 
formed aqueous two-phase system which comprises PEG-phosphate salt-water, 
PEG-dextran-water or POLYx-POLYy-water, where POLYx and POLYy represent 
different polymers, to obtain the two-phase systems with chirality for the 
present invention. 
The method of the present invention may also be used for affinity 
partitioning. When an affinity interaction takes place between a ligand to 
be separated and an affinity agent (for example an antibody) in free 
solution, a soluble complex is formed. The partition behavior of this 
complex is dependent upon the characteristics of the individual 
components. In an ideal situation an affinity agent such as an antibody 
favours one phase to such an extent that the antibody-target molecule 
complex will also partition to the same phase. The aqueous two-phase 
systems with chiral phase-forming components of the present invention have 
been found to meet the requirements of an affinity partition system very 
well. By utilizing the affinity partitioning concept, one can manipulate 
the separation factor (or partition coefficient) of target molecules by 
immobilizing an affinity agent on one of the phase components, the 
polymeric or the chiral one, to enhance separation. 
Affinity partitioning is based upon the principle that the complex should 
have a different partition pattern as compared to the free target 
molecule. When the affinity agent or ligand does not exhibit extreme 
partition behavior, it must be chemically modified prior to use. In most 
cases, chemical compounds partition into the bottom phase and the desire 
has therefore been to selectively transfer the target molecule to the top 
phase. To make the affinity agent favor the top phase, chemical coupling 
of the antibody to the top phase polymer has been used. Since 
poly(ethylene glycol) is the major top phase polymer used, this has meant 
coupling of PEG to the affinity agent. A large number of coupling 
reactions have been applied to PEG and today there are several 
alternatives available (Harris, J. M., Review in Macromol. Chem. and 
Phys., C25, 325-373 (1985)). 
The affinity partitioning takes place as follows: The modified ligand is 
mixed with the mixtures and after proper binding has taken place the phase 
system is added. After proper mixing, phase separation takes place and the 
affinity complex is then found in the top phase. A normal procedure for 
isolating a pure target molecule freed of ligand and phase components has 
involved the use of a second parition step wherein the top phase has been 
mixed with a fresh bottom phase under dissociating conditions. During 
mixing of the phases, dissociation of the complex takes place and the 
target protein will then paritition according to the spontaneous pattern, 
i.e., it will be recovered from the bottom phase. Isolation of the target 
molecule from the phase components may be done using ion exchange 
chromatography or membrane filtration. The ligand may be recovered from 
the top phase and reused. 
Affinity partitioning as described above involves the need of modifying 
each individual inhibitor or ligand to go to the top phase. This may be 
impractical and in some cases even unsuitable. Therefore, the strategy of 
applying a second separator molecule having an affinity for the ligand - 
target protein complex and a partition behavior strongly favoring the top 
phase has been developed. An example of this is the protein avidin that 
was PEG-modified, and the ligand was modified with biotin residues in a 
mild and gentle modification reaction. A similar approach was applied in 
immunoaffinity partitioning by applying protein A as the separator 
molecule to be modified for extreme partitioning. As stated above, even if 
soluble molecules may be partitioned to one phase, it is much easier and 
more predictable to use particles. Therefore, second separator particles 
were developed as well. The same approach to modify the chiral resolving 
agent (c) for affinity partitioning is considered to be within the scope 
of the present invention. 
Partition affinity ligand assay is described by Mattiasson, B. and Ling, 
T.G.I., J. Immunol. Meth. 38, 217-223 (1980), and Mattiasson, B. in 
"Separations for Biotechnology" ed. by Verrall, M.S. and Hudson, M.J., 
Ellis Horwood, page 281-283, (1987). Binding assays involve the formation 
of affinity complexes. The dominating problem in conventional 
immunological assays is to carry out separation of bound antigen from free 
antigen in an efficient, quick, and reproducible way. The separation 
principle of aqueous polymer/chiral agent/water two-phase systems meets 
these demands very well and is considered to be within the scope of the 
present invention. Also, in these applications there may also be a need 
for modifying one of the reactants. 
The method of the present invention is also useful for extractive 
biocatalytic conversions. When used for such purposes, the components of 
the bioconversion system, for example, enzymes plus the required buffers, 
co-factors, etc., are mixed into the aqueous two-phase system. Upon 
addition of the appropriate substrate for the enzyme or enzyme system, 
bioconversion is effected and product compounds are produced by the 
biocatalytic or enzyme system. When the enzyme system is preferably 
soluble in the lower phase of the two-phase system and the product 
produced by bioconversion is preferably soluble in the upper phase of the 
two-phase system, a continuous extraction process is established in which 
the product obtained by bioconversion in the lower phase is continuously 
extracted into the upper phase by means of the aqueous two-phase system of 
the present invention. Product feedback inhibition of the enzyme system is 
substantially reduced since the product is continuously extracted away 
from the components of the bioconversion system. Obviously, extractive 
bioconversions may be performed when the components of the bioconversion 
system are present in either phase of the two-phase system so long as the 
product produced is soluble in the opposite phase. 
The present method of separating a mixture of organic compounds, inorganic 
compounds, biomolecules and biomaterials is flexible and inexpensive. The 
method may be applied to the separation of a wide variety of biological 
materials and is particularly interesting for the separation of 
stereoisomers. The method is also generally useful in the areas of 
extractive bioconversions and affinity partitioning.

Other features of this invention will become apparent in the course of the 
following description of exemplary embodiments which are given for 
illustration of the invention and are not intended to be limiting thereof. 
EXAMPLES 
Example 1 
Aqueous two-phase systems containing chiral amino acid-water soluble 
polymer-water are prepared as follows: 
An appropriate quantity of distilled water is weighed in a beaker. Add an 
appropriate quantity of either amino acid, monosaccharide, disaccharide, 
or chiral compound, to the water, which is stirred and dissolved. With the 
water still being stirred, add an appropriate quantity of water soluble 
polymer. Continue stirring until the polymer dissolves. Stop mixing and 
let the system settle into two phases. 
The following compositions of chiral amino acid-water soluble polymer-water 
form aqueous two-phase systems: 
(a) 23.08% L-lysine/5.77% PEG-20,000/71.15% water at 25.degree. C. 
(b) 33.33% L-proline/5.00% PEG-20,000/61.67% water at 25.degree. C. 
(c) 13.04% L-alanine/6.50% PEG-20,000/80.37% water at 51.5.degree. C. 
(d) 9.78% L-proline/51.10% PPG-425/39.12% water at 25.degree. C. 
Example 2 
Aqueous two-phase systems containing monosaccharides or disaccharides-water 
soluble polymer-water were prepared based on the same procedure described 
in Example 1. 
The following are compositions of monosaccharides or disaccharides-water 
soluble polymer-water that form aqueous two-phase systems: 
(a) 29.40% glucose/29.40% PPG-425/41.20% water at 25.degree. C. 
(b) 30.00% fructose/15.00% PPG-425/55.00% water at 25.degree. C. 
(c) 25.72% fructose/27.14% PPG-425/47.14% water at 25.degree. C. 
(d) 30.00% maltose/30.00% PPG-425/40.00% water at 25.degree. C. 
(e) 41.20% sucrose/24.15% PPG-425/34.65% water at 25.degree. C. 
(f) 44.44% sucrose/5.56% PEG-20,000/50.00% water at 73.degree. C. 
Example 3 
Aqueous two-phase systems containing chiral compounds or salts-water 
soluble polymer-water were prepared based on the procedure described in 
Example 1. 
The following are compositions of chiral acid-water soluble polymer-water 
that form aqueous two-phase systems: 
(a) 16.04% L-ascorbic acid and 8.53% NaOH/8.21% PEG-8000/67.22% water at 
25.degree. C. 
(b) 6.67% L-sodium tartrate/16.67 PEG-8000/66.66% water at 25.degree. C. 
Example 4 
D and L phenylalanine separation 
The following L-lysine/PEG8000/H20 system was prepared at 25.degree. C.: 
28.57% w/w L-lysine, 10.71% w/w PEG8000, 60.72% w/w water. 10 ml of the 
phase system was poured into 15 ml, polypropylene centrifuge tubes. 6 mg 
of L-phenylalanine was added to one tube, 6 mg of D-phenylalanine to a 
second, while the third tube was left blank. The contents of the tubes 
were mixed, and then permitted to settle over a period of time. The 
partition coefficients were determined by diluting the phases 1/5 and 
measuring absorbance at 254 nm versus an appropriately diluted phase 
blank. The results were as follows: 
______________________________________ 
Partition Coefficient, K 
Sample 1 
Sample 2 Sample 3 
______________________________________ 
L-Phenylalanine 0.979 0.974 0.968 
D-Phenylalanine 1.094 1.061 1.053 
##STR1## 1.11 1.09 1.09 
______________________________________ 
L-phenylalanine gives a lower partition coefficient than D-phenylalanine 
and is, therefore, enriched in the L-lysine (Lower) phase. 
Example 5 
.beta.-lactoglobulins A and B separation 
.beta.-lactoglobulin concentration was 1.0 mg/ml, and 10 ml of two-phase 
system with composition of 28.57% L-lysine, 10.71% PEG 8000 and 60.72% 
water was utilized. The separation factor (.alpha.) is 1.94 in favor of 
.beta.-lactoglobulins A in upper phase. 
Example 6 
D and L tryptophan separation 
D and L tryptophan were partitioned in the aqueous two-phase system with 
composition of 28.57% L-lysine, 10.71% PEG 8000 and 60.72% water. The 
separation factor .alpha. is 1.24 in favor of D-tryptophan in the upper 
phase. 
Example 7 
D and L tryptophan separation 
D and L tryptophan were partitioned in the aqueous two-phase system with 
composition of 14.60% L-serine, 11.00% PEG 20,000 and 74.4% water. The 
separation factor .alpha. is 1.19 in favor of D-tryptophan in the upper 
phase. 
Example 8 
D and L tryptophan separation 
D and L tryptophan were partitioned in the aqueous two-phase system with 
composition of 36.59% L-proline, 9.76% PEG 8000 and 53.65% water. The 
separation factor .alpha.=1.06 in favor of D-tryptophan in the upper 
phase. 
Example 9 
D and L tryptophan separation 
D and L tryptophan were partitioned in the aqueous two-phase system with 
composition of 36.59% L-proline, 9.76% PEG 8000, 53.65% water and 0.008 M 
copper sulfate at 22.degree. C. The separation factor .alpha.=1.18 in 
favor of D-tryptophan in the upper phase. 
Example 10 
D and L tryptophan separation in PPG/Sucrose/H.sub.2 O aqueous two-phase 
system 
D and L tryptophan were partitioned in the aquous two-phase system with 
composition of 41.20% sucrose, 24.15% polypropylene glycol-425, 34.65% 
water. The separation factor .alpha.=0.90 in favor of D-tryptophan in the 
lower (sucrose) phase. 
Example 11 
D and L tryptophan separation in PEG/Potassium phosphate/L-lysine/water 
system 
Stock solutions composed of 9.42% polyethylene glycol/11.37% potassium 
phosphate/79.21% water were prepared. 20 gm of top and bottom phase was 
weighted into a 100 ml beaker. 10 gm L-lysine was then added, and stirred 
until the lysine dissolved. 10 ml of the phase system was then poured into 
each of three polypropylene centrifuge tubes. 3 mg of D-tryptophan was 
added to the first tube, 3 mg of L-tryptophan was added to the second, 
while the third was left blank. After allowing the tryptophan to dissolve, 
the systems were settled at room temperature. The top and bottom phases 
were collected and the concentration of either D and L tryptophan were 
determined by UV spectrophotometer at 280 nm. The separation factor 
(K.sub.D /K.sub.L) was 1.03. 
Example 12 (Comparative) 
The process of Example 11 was repeated with the omission of L-lysine. The 
separation factor of the PEG/potassium phosphate/water system without 
L-lysine was 1.0. 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein.