Separation of mixtures by aqueous two-phase systems

• US 4,980,065 A
• Filed: 10/18/1989
• Issued: 12/25/1990
• Est. Priority Date: 10/18/1989
• Status: Expired due to Fees

• Current Assignee: Lehigh University
• Original Assignee: Lehigh University
• Inventors: Hsu, James T.
• Primary Examiner(s): Jones, W. Gary
• Application Number:
  US07/423,333
• Time in Patent Office: 433 Days
• Field of Search: 210/632, 210/634, 562/401, 435/280
• US Class Current: 210/632
• CPC Class Codes: C07C 227/34 by separation of optical isomers
• CPC Class Codes: C07C 229/36 with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton

Abstract

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.

Claims

1. A process of separating a mixture of organic compounds, inorganic compounds, biomolecules and biomaterials, comprising the steps:

(a) preparing an aqueous two-phase system consisting essentially of (1) a chiral compound selected from the group consisting of amino acids, peptides, proteins, disaccharides, chiral salts, chiral solvents, chiral acids and chiral bases (2) a water soluble polymer, in amounts sufficient to form said aqueous two-phase system and (3) water;

(b) mixing said two-phase system with said mixture to be separated;

(c) separating the two phases from each other.

2. The process of claim 1, wherein said chiral compound is a D- or L- α

-amino acid or monosaccharide, or disaccharide or chiral salt, or chiral water miscible solvent or chiral acid or chiral base.

3. The process of claim 1, wherein said water soluble polymer is a polyethylene glycol having a molecular weight in the range 300-50,000, polypropylene glycol, polyvinylpyrrolidone, polyvinylalcohol, dextran and sodium dextran sulfate.

4. The process of claim 3, wherein said polyethylene glycol has a molecular weight in the range of 300-30,000.

5. The process of claim 1, wherein said mixture is a mixture of D- and L- amino acids or other optical isomers.

6. The process of claim 1, wherein said mixture is a mixture of cell particulates, macromolecules, cells, organic molecules, inorganic molecules or biochemical compounds.

7. The process of claim 1, wherein said chiral compound is a peptide having 2-1,000,000 amino acid residues.

8. The process of claim 1, wherein said chiral compound is present in an amount of about 0.01-50.0 wt. % relative to the aqueous two-phase system.

9. The process of claim 1, wherein said chiral compound is present in an amount of 0.1-40 wt. % relative to said aqueous two-phase system.

10. The process of claim 1, wherein said water soluble polymer is present in an amount of about 2-50 wt. % based on said aqueous two-phase system.

11. The process of claim 1, wherein said mixing step is accomplished in a countercurrent distribution, or cross-current extraction or counter-current extraction or liquid partition column chromatography, or counter-current chromatography apparatus.

12. The process of claim 1, wherein an additive salt, buffer, sugar or polymer is present in said two-phase system whereby the separation factor, phase separation or solubility is enhanced, the viscosity of either phase is reduced or the interfacial tension between the two phases is increased.

13. The process of claim 12, wherein said aqueous two-phase system further consists essentially of a non-chiral phase-forming agent selected from the group consisting of non-chiral water soluble polymers, salts, glycine, acids and bases.

14. The process of claim 1, wherein said aqueous two-phase system further consists essentially of an affinity agent coupled to the water soluble polymer or chiral compound.

15. The process of claim 1, wherein said aqueous two-phase system further consists essentially of an enzyme or biocatalyst system, a substrate for said enzyme together with buffers and cofactors necessary to effect a biocatalytic reaction.

16. A process of separating a mixture of stereoisomers or optical isomers comprising the steps:

(a) preparing an aqueous two-phase system consisting essentially of (1) a chiral resolving agent selected from the group consisting of amino acids, peptides, proteins, monosaccharides, disaccharides, chiral salts, chiral solvents, chiral acids and chiral bases (2) a water soluble polymer, in amounts sufficient to form said aqueous two-phase system and (3) water;

(b) mixing said two-phase system with the mixture to be separated;

(c) separating the two phases from each other.

17. The process of claim 16, wherein said chiral resolving agent is a D- or L- α

-amino acid or monosaccharide, or disaccharide, or chiral salt, or chiral solvent, or chiral acid, or chiral base.

18. The process of claim 16, wherein said water soluble polymer is a polyethylene glycol having molecular weight in the range 300-50,000, polypropylene glycol, polyvinylpyrrolidone, polyvinylalcohol, dextran or sodium dextran sulfate.

19. The process of claim 18, wherein said polyethylene glycol has a molecular weight in the range of 300-30,000.

20. The process of claim 16, wherein said mixture is a mixture of D- and L- amino acids.

21. The process of claim 16, wherein said chiral compound is a peptide having 2-1,000,000 amino acid residues.

22. The process of claim 16, wherein said chiral compound is present in an amount of about 0.01-50.0 wt. % relative to the aqueous two-phase system.

23. The process of claim 16, wherein said chiral compound is present in an amount of 0.1-40 wt. % relative to said aqueous two-phase system.

24. The process of claim 16, wherein said water soluble polymer is present in an amount of about 2-50 wt. % based on said aqueous two-phase system.

25. The process of claim 16, wherein said mixing step (b) is accomplished in a countercurrent distribution, or cross-current extraction, or counter-current extraction or liquid partition column chromatography or counter-current chromatography apparatus.

26. The process of claim 16, wherein an additive salt, buffer, sugar or polymer is present in said two-phase system whereby the separation factor, phase separation or solubility is enhanced, the viscosity of either phase is reduced or the interfacial tension between the two-phases is increased.

27. The process of claim 26, wherein said aqueous two-phase system further consists essentially of a non-chiral phase-forming agent selected from the group consisting of non-chiral water soluble polymers, salts, glycine, acids and bases.

28. The process of claim 27, wherein said mixture is a mixture of optical isomers.

29. The process of claim 16, wherein said aqueous two-phase system further consists essentially of an affinity agent coupled to the water soluble polymer or chiral compound.

30. The process claim 16, wherein said aqueous two-phas system further consists essentially of an enzyme or biocatalyst system, a substrate for said enzyme together with buffers and cofactors necessary to effect an biocatalytic reaction.