Molecular Biotechnology

, Volume 19, Issue 3, pp 269–277 | Cite as

Aqueous two-phase systems

A general overview


Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.

Index enries

Aqueous two-phase system partition bioseparation extraction 


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  1. 1.
    Albertsson, P.-Å. (1986) Partition of Cell Particles and Macromolecules (3rd edition). John Wiley, New York.Google Scholar
  2. 2.
    Flory, P. J. (1953) Principles of Polymer Chemistry. Cornell University Press, Ithaca, NY.Google Scholar
  3. 3.
    Walter, H., Brooks, D. E., and Fisher, D., eds., (1985) Partition in Aqueous Two-Phase Systems. Theory, Methods, Uses and Applications to Biotechnology. Academic Press, Orlando, FL.Google Scholar
  4. 4.
    Baskir, J. N., Hatton, T. A., and Suter, U. W. (1989) Protein partitioning in two-phase aqueous polymer systems. Biotechnol. Bioeng. 34, 541–558.CrossRefPubMedGoogle Scholar
  5. 5.
    Abbot, N. L., Blankschtein, D., and Hatton, T. A. (1990) On protein partitioning in two-phase aqueous polymer systems. Bioseparation 1, 191–225.Google Scholar
  6. 6.
    Ananthapadmanabhan, K. P. and Goddard, E. D. (1987) Aqueous biphase formation in polyethylene oxide-inorganic salt systems. Langmuir 3, 25–31.CrossRefGoogle Scholar
  7. 7.
    Huddleston, J., Veide, A., Köhler, K., Flanagan, J., Enfors, S.-O., and Lyddiat, A. (1991) The molecular basis of partitioning in aqueous two-phase systems. Trends Biotechnol. 9, 381–388.PubMedCrossRefGoogle Scholar
  8. 8.
    Galaev, I.Yu. and Mattiasson, B. (1993) Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology. Enzyme Microb. Technol. 15, 354–366.PubMedCrossRefGoogle Scholar
  9. 9.
    Karlström, G. (1985) A new model for upper and lower critical solution temperatures in poly(ethylene-oxide) solutions. J. Phys. Chem. 89, 4962–4964.CrossRefGoogle Scholar
  10. 10.
    Walter, H. and Johansson, G., eds. (1994) Methods in Enzymology: Aqueous Two-Phase Systems. Academic Press, San Diego, CA.Google Scholar
  11. 11.
    Zaslavsky, B. Y. (1995) Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications, Marcel Dekker, New York.Google Scholar
  12. 12.
    Albertsson, P.-Å. (1956) Chromatography and partition of cells and cell fragments. Nature (London) 177, 771–774.CrossRefGoogle Scholar
  13. 13.
    Albertsson, P.-Å. (1958a) Particle fractionation in liquid two-phase systems. The composition of some phase systems and the behavior of some model particles in them. Application to the isolation of cell walls for microorganisms. Biochim. Biophys. Acta 27, 378–395.PubMedCrossRefGoogle Scholar
  14. 14.
    Albertsson, P.-Å. (1958b) Partition of proteins in liquid polymer-polymer two-phase systems. Nature (London) 182, 709–711.CrossRefGoogle Scholar
  15. 15.
    Albertsson, P.-Å, and Nyns, E. J. (1959) Counter-current distribution of proteins in aqueous polymer phase systems. Nature (London) 184, 1465–1468.CrossRefGoogle Scholar
  16. 16.
    Hatti-Kaul, R., ed. (2000) Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, Humana Press, New Jersey.Google Scholar
  17. 17.
    Hustedt, H., Kroner, K. H., and Kula, M.-R. (1985) Applications of phase partitioning in Biotechnology, in Partition in Aqueous Two-Phase Systems, Theory, Methods, Uses and Applications to Biotechnology. (Walter, H., Brooks, D. E., and Fisher, D., eds.), Academic Press, Orlando, FL, pp. 529–587.Google Scholar
  18. 18.
    Flygare, S., Wikström, P., Johansson, G., and Larsson, P.-O. (1990) Magnetic aqueous two-phase separation in preparative applications. Enzyme Microb. Technol. 12, 95–103.PubMedCrossRefGoogle Scholar
  19. 19.
    Cunha, T. and Aires-Barros, R. (2000) Large-scale extraction of proteins. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems, Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 391–409.Google Scholar
  20. 20.
    Åkerlund, H.-E. (2000) Partition by countercurrent distribution. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 55–64.Google Scholar
  21. 21.
    Hansson, Y.-B. and Wingren, C. (2000) Liquid-liquid partition chromatography. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 65–75.Google Scholar
  22. 22.
    Berggren, K., Egmond, M. R., and Tjerneld, F. (2000) Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning. Biochim. Biophys. Acta — Prot. Str. Mol. Biol. 1481, 317–327.Google Scholar
  23. 23.
    Backman, L. (2000) Detection and analysis of interactions by two-phase partition. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 219–228.Google Scholar
  24. 24.
    Mattiasson, B. (1983) Applications of aqueous two-phase systems in biotechnology. Trends Biotechnol. 1, 16–20.CrossRefGoogle Scholar
  25. 25.
    Ling, T. G. I. and Mattiasson, B. (1983) A general study of the binding and separation in partition affinity ligand assay. Immunoassay of β-microglobulin. J. Immunol. Methods 59, 327–337.PubMedCrossRefGoogle Scholar
  26. 26.
    Hammar, L. (2000) Concentration and purification of viruses. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 143–158.Google Scholar
  27. 27.
    Walker, S. G. and Lyddiatt, A. (1999) Processing of nanoparticulate bioproducts: application and optimisation of aqueous two-phase systems. J. Chem. Technol. Biotechnol. 74, 250–255.CrossRefGoogle Scholar
  28. 28.
    Boland, M. J., Hesselink, P. G. M., Papamichael, N., and Hustedt, H. (1991) Extractive purification of enzymes from animal tissue using aqueous two-phase systems: pilot scale studies. J. Biotechnol. 19, 19–34.PubMedCrossRefGoogle Scholar
  29. 29.
    Jordan, P. and Vilter, H. (1991) Extraction of proteins from materials rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems. Biochim. Biophys. Acta 1073, 98–106.PubMedGoogle Scholar
  30. 30.
    Veide, A., Lindbäck, T., and Enfors, S.-O. (1989) Recovery of β-galactosidase from a poly(ethylene glycol) solution by diafiltration. Enzyme Microb. Technol. 11, 744–751.CrossRefGoogle Scholar
  31. 31.
    Frej, A.-K., Gustafsson, J. G., and Hedman, P. (1986) Recovery of β-galactosidase by adsorption from unclarified Escherichia coli homogenate. Biotechnol. Bioeng. 28, 133–138.CrossRefPubMedGoogle Scholar
  32. 32.
    Sánchez-Ferrer, A., Bru, R., and Garcia-Carmona, F. (1989) Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in Triton X-114. Plant Physiol. 91, 1481–1487.PubMedCrossRefGoogle Scholar
  33. 33.
    Ramelmeier, R. A., Terstappen, G. C., and Kula, M.-R. (1991) The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems. Bioseparation 2, 315–324.PubMedGoogle Scholar
  34. 34.
    Minuth, T., Thömmes, J., and Kula, M-R. (1996) A closed concept for purification of the membrane-bound cholesterol oxidase from Nocardia rhodochrous by surfactant-based cloud-point extraction, organic solvent extraction and anion exchange chromatography. Biotechnol. Appl. Biochem. 23, 107–116.Google Scholar
  35. 35.
    Sivars, U. and Tjerneld, F. (2000) Mechanisms of phase behavior and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins. Biochim. Biophys. Acta 1474, 133–146.PubMedGoogle Scholar
  36. 36.
    Cole, K.D. (1991) Preparation of plasmid and high molecular weight DNA by the use of salt-polymer two-phase extraction. Biotechniques 11, 18–24.PubMedGoogle Scholar
  37. 37.
    Johansson, H. O., Karlström, G., and Tjerneld, F. (1998) Separation of amino acids and peptides by temperature induced phase partitioning: Theoretical model for partitioning and experimental data. Bioseparation 7, 259–267.CrossRefGoogle Scholar
  38. 38.
    Chu, I.-M. and Chen, W.-Y. (2000) Partition of amino acids and peptides in aqueous two-phase systems. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Humana Press, NJ, pp. 95–105.Google Scholar
  39. 39.
    Sikdar, S. K., Cole, K. D., Stewart, R. M., Szlag, D. C., Todd, P., and Cabezas, H., Jr. (1991) Aqueous two-phase extractions in bioseparations: an assessment. Bio/Technol. 9, 253–256.CrossRefGoogle Scholar
  40. 40.
    Hatti-Kaul, R. (2000) Extractive bioconversions in aqueous two-phase systems. In: Methods Biotechnol. Vol. 11, Aqueous Two-Phase Systems. Methods and Protocols, (Hatti-Kaul, R., ed.), Human Press, NJ, pp. 411–417.Google Scholar
  41. 41.
    Ström, G.B., Mälarstig, B., and Blomquist, G. (1994) Purification of water-based cutting fluids. Methods Enzymol. 228, 672–678.PubMedCrossRefGoogle Scholar
  42. 42.
    Huddleston, J. G., Willauer, H. D., Boaz, K. R., and Rogers, R. D. (1998) Separation and recovery of food coloring dyes using aqueous biphasic extraction chromatographic resins. J. Chromatogr. B 711, 237–244.Google Scholar
  43. 43.
    Rogers, R. D., Willauer, H. D., Griffin, S. T., and Huddleston, J. G. (1998) Partitioning of small organic molecules in aqueous biphasic systems. J. Chromatogr. B 711, 255–263.Google Scholar
  44. 44.
    Quina, F. H. and Hinze, W. L. (1999) Surfactant-mediated cloud point extractions: An environmentally benign alternative separation approach. Ind. Eng. Chem. Res. 38, 4150–4168.CrossRefGoogle Scholar
  45. 45.
    Kopperschläger, G. and Birkenmeier, G. (1990) Affinity partitioning and extraction of proteins. Bioseparation 1, 235–253.PubMedGoogle Scholar
  46. 46.
    Kamihira, M., Kaul, R., and Mattiasson, B. (1992) Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation. Biotechnol. Bioeng. 40, 1381–1387.CrossRefPubMedGoogle Scholar
  47. 47.
    Suzuki, M., Kamihira, M., Shiraishi, T., Takeuchi, H., and Kobayashi, T. (1995) Affinity partitioning of protein A using a magnetic aqueous two-phase system. J. Ferment. Bioeng. 80, 78–84.CrossRefGoogle Scholar
  48. 48.
    Mattiasson, B. and Ling, T. G. I. (1986) Efforts to integrate affinity interactions with conventional separation technologies. Affinity partition using biospecific chromatographic particles in aqueous two-phase systems. J. Chromatogr. 376, 235–243.CrossRefGoogle Scholar
  49. 49.
    Kwon, Y. J. and Hatti-Kaul, R. (1999) Protein separation using metal ion-bound particles in aqueous two-phase system. Biotechnol. Techn. 13, 145–148.CrossRefGoogle Scholar
  50. 50.
    Fernandes, S., Johansson, G., and Hatti-Kaul, R. (2001) Purification of recombinant cutinase by extraction in aqueous two-phase system facilitated by a fatty acid substrate. Biotechnol. Bioeng. 73, 465–475.PubMedCrossRefGoogle Scholar
  51. 51.
    Kula, M.-R. (1990) Trends and future prospects of aqueous two-phase extraction. Bioseparation 1, 181–189.PubMedGoogle Scholar
  52. 52.
    Enfors, S-O., Köhler, K., and Veide, A. (1990) Combined use of extraction and genetic engineering for protein purification: Recovery of β-galactosidase fused proteins. Bioseparation 1, 305–310.PubMedGoogle Scholar
  53. 53.
    Berggren, K., Tjerneld, F., and Veide, A. (2000) Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems. Bioseparation 9, 69–80.PubMedCrossRefGoogle Scholar
  54. 54.
    Van Berlo, M., Luyben, K. Ch. A. M., and van der Wielen, L. A. M. (1998) Poly(ethylene glycol)-salt aqueous two-phase systems with easily recyclable volatile salts. J. Chromatogr. B 711, 61–68.Google Scholar
  55. 55.
    Greve, A. and Kula, M.-R. (1991) Cost structure and estimation for the recycling of salt in a protein extraction process. Bioproc. Eng. 6, 173–177.CrossRefGoogle Scholar
  56. 56.
    Vernau, J. and Kula, M.-R. (1990) Extraction of proteins from biological raw material using aqueous polyethylene glycol-citrate phase systems. Biotechnol. Appl. Biochem. 12, 397–404.Google Scholar
  57. 57.
    Nguyen, A. L., Grothe, S., and Luong, J. (1988) Applications of pullulan in aqueous two-phase systems for enzyme production. Appl. Microbiol. Biotechnol. 2, 341–346.Google Scholar
  58. 58.
    Harris, P. A., Karlström, G., and Tjerneld, F. (1991) Enzyme purification using temperature-induced phase formation. Bioseparation 2, 237–246.PubMedGoogle Scholar
  59. 59.
    Patrickios, C., Abbott, N. L., Foss, R., and Hatton, T. A. (1992) Synthetic polyampholytes for protein partitioning in two phase polymer systems. AIChE Symp. Ser. 88, 80–88.Google Scholar
  60. 60.
    Johansson, H.-O., Persson, J., and Tjerneld, F. (1999) Thermoseparating water/polymer system: A novel one-polymer aqueous two-phase system for protein purification. Biotechnol. Bioeng. 66, 247–257.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2001

Authors and Affiliations

  1. 1.Department of Biotechnology, Center for Chemistry & Chemical EngineeringLund UniversityLundSweden

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