Aqueous Biphasic Systems Based on Ionic Liquids for Extraction, Concentration and Purification Approaches

  • Isabel M. Marrucho
  • Mara G. Freire
Part of the Green Chemistry and Sustainable Technology book series (GCST)


During the past decade, ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been the focus of a significant amount of research, and excellent and comprehensive reviews are nowadays available. Rather than focusing on the phase equilibria and the phase separation mechanisms of ABS, this chapter provides an assessment of the current status of implementation of this liquid-liquid approach in the extraction, concentration and purification of biomolecules/solutes/products from real matrices.

Examples on the successful use of IL-based ABS in the extraction/purification of value-added compounds from plant matrices and from extracellular media are provided. The use of IL-based ABS as an extraction and concentration methodology is also discussed, focusing on two main approaches: control of environmental samples and monitoring of human health or abuse of drugs. Finally, and due to the rapid development and design of extraction processes based on ILs, the use of IL-based ABS for the treatment of aqueous effluents contaminated with ILs is also presented.


Aqueous biphasic systems Ionic liquids Real samples Extraction Purification Concentration Recovery 



Isabel M. Marrucho gratefully acknowledges the Fundação para a Ciência e Tecnologia (FCT) for a contract under the FCT Investigator 2012 Programme and Ciência Sem Fronteiras programme for the project 145/2012. The authors also acknowledge FCT for the projects PTDC/QUI-QUI/121520/2010 and PEst-C/CTM/LA0011/2013. Mara G. Freire acknowledges the European Research Council (ERC) for the Starting Grant ERC-2013-StG-337753.


  1. 1.
    Albertsson PA (1958) Partition of proteins in liquid polymer-polymer two-phase systems. Nature 182:709–711CrossRefGoogle Scholar
  2. 2.
    Albertsson PA (1986) Partitioning of cell particles and macromolecules, 3rd edn. Wiley-Interscience, New YorkGoogle Scholar
  3. 3.
    Gutowski KE, Broker GA, Willauer HD, Huddleston JG, Swatloski RP, Holbrey JD, Rogers RD (2003) Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations. J Am Chem Soc 125:6632–6633CrossRefGoogle Scholar
  4. 4.
    Plechkova NV, Seddon KR (2008) Applications of ionic liquids in the chemical industry. Chem Soc Rev 37:123–150CrossRefGoogle Scholar
  5. 5.
    Freire MG, Cláudio AFM, Araújo JMM, Coutinho JAP, Marrucho IM, Canongia Lopes JN, Rebelo LPN (2012) Aqueous biphasic systems: a boost brought about by using ionic liquids. Chem Soc Rev 41:4966–4995CrossRefGoogle Scholar
  6. 6.
    Freire MG, Louros CLS, Rebelo LPN, Coutinho JAP (2011) Aqueous biphasic systems composed of a water-stable ionic liquid + carbohydrates and their applications. Green Chem 13:1536–1545CrossRefGoogle Scholar
  7. 7.
    Freire MG, Pereira JFB, Francisco M, Rodríguez H, Rebelo LPN, Rogers RD, Coutinho JAP (2012) Insight into the interactions that control the phase behaviour of new aqueous biphasic systems composed of polyethylene glycol polymers and ionic liquids. Chem Eur J 18:1831–1839CrossRefGoogle Scholar
  8. 8.
    Domínguez-Pérez M, Tomé LIN, Freire MG, Marrucho IM, Cabeza O, Coutinho JAP (2010) (Extraction of biomolecules using) aqueous biphasic systems formed by ionic liquids and aminoacids. Sep Purif Technol 72:85–91CrossRefGoogle Scholar
  9. 9.
    Pereira JFB, Rebelo LPN, Rogers RD, Coutinho JAP, Freire MG (2013) Combining ionic liquids and polyethylene glycols to boost the hydrophobic-hydrophilic range of aqueous biphasic systems. Phys Chem Chem Phys 15:19580–19583CrossRefGoogle Scholar
  10. 10.
    Wang L, Weller CL (2006) Recent advances in extraction of nutraceuticals from plants. Trends Food Sci Technol 17:300–312CrossRefGoogle Scholar
  11. 11.
    Tan Z, Li F, Xu X (2012) Isolation and purification of aloe anthraquinones based on ionic liquid/salt aqueous two-phase system. Sep Purif Technol 98:150–157CrossRefGoogle Scholar
  12. 12.
    Tan Z, Li F, Xu X, Xing J (2012) Simultaneous extraction and purification of aloe polysaccharides and proteins using ionic liquid based aqueous biphasic system coupled with dialysis membrane. Desalination 286:389–393CrossRefGoogle Scholar
  13. 13.
    Femenia A, Sanchez ES, Simal S, Rossello C (1999) Compositional features of polysaccharides from aloe vera (Aloe barbadensis Miller) plant tissues. Carbohydr Polym 39:109–117CrossRefGoogle Scholar
  14. 14.
    Pugh N, Ross SA, ElSohly M, Pasco DS (2001) Characterization of aloeride, a new high-molecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity. J Agric Food Chem 49:1030–1034CrossRefGoogle Scholar
  15. 15.
    Wang JB, Li HF, Jin C, Qu Y, Xiao XH (2008) Development and validation of a UPLC method for quality control of rhubarb-based medicine: fast simultaneous determination of five anthraquinone derivatives. J Pharm Biomed 47:765–770CrossRefGoogle Scholar
  16. 16.
    Ribeiro BD, Coelho MAZ, Rebelo LPN, Marrucho IM (2013) Ionic liquids as additives for extraction of saponins and polyphenols from mate (Ilex paraguariensis) and tea (Camellia sinensis). Ind Eng Chem Res 52:12146–12153CrossRefGoogle Scholar
  17. 17.
    Sparg SG, Light ME, Staden JV (2004) Biological activities and distribution of plant saponins. J Ethnopharmacol 94:219–243CrossRefGoogle Scholar
  18. 18.
    Guclu-Ustundag O, Mazza G (2007) Saponins: properties, applications, and processing. Crit Rev Food Sci Nutr 47:231–258CrossRefGoogle Scholar
  19. 19.
    Vincken JP, Heng L, de Groot A, Gruppen H (2007) Saponins, classification and occurrence in the plant kingdom. Phytochemical 68:275–297CrossRefGoogle Scholar
  20. 20.
    Han CC, Hui QS, Wang YZ (2008) Hypoglycaemic activity of saponin fraction extracted from Momordica charantia in PEG/salt aqueous two-phase systems. Nat Prod Res 22:1112–1119CrossRefGoogle Scholar
  21. 21.
    Hecke WV, Kaur G, DeWever H (2014) Advances in in-situ product recovery (ISPR) in whole cell biotechnology during the last decade. Biotechnol Adv 32:1245–1255CrossRefGoogle Scholar
  22. 22.
    Sendovski M, Nir N, Fishman A (2010) Bioproduction of 2-phenylethanol in a biphasic ionic liquid aqueous system. J Agric Food Chem 58:2260–2265CrossRefGoogle Scholar
  23. 23.
    Rosa PAJ, Ferreira IF, Azevedo AM, Aires-Barros MR (2010) Aqueous two-phase systems: a viable platform in the manufacturing of biopharmaceuticals. J Chromatogr A 1217:2296–2305CrossRefGoogle Scholar
  24. 24.
    Rosa PAJ, Azevedo AM, Sommerfeld S, Bäcker W, Aires-Barros MR (2011) Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability. Biotechnol Adv 29:559–567CrossRefGoogle Scholar
  25. 25.
    Fedeniuk RW, Shand PJ (1998) Theory and methodology of antibiotic extraction from biomatrices. J Chromatogr A 812:3–15CrossRefGoogle Scholar
  26. 26.
    Pereira JFB, Vicente F, Santos-Ebinuma VC, Araújo JM, Pessoa A, Freire MG, Coutinho JAP (2013) Extraction of tetracycline from fermentation broth using aqueous two-phase systems composed of polyethylene glycol and cholinium-based salts. Proc Biochem 48:716–722CrossRefGoogle Scholar
  27. 27.
    Liu Q, Yu J, Liu HZ (2005) Extraction of penicillin G by aqueous two-phase system of ionic liquid [Bmim]BF4 and NaH2PO4. Chin Sci Bull 50:1582–1588CrossRefGoogle Scholar
  28. 28.
    Liu Q, Yu J, Li W, Hu X, Xia H, Liu H, Yang P (2006) Partitioning behavior of Penicillin G in aqueous two phase system formed by ionic liquids and phosphate. Sep Sci Technol 41:2849–2858CrossRefGoogle Scholar
  29. 29.
    Jiang Y, Xia H, Guo C, Mahmood I, Liu H (2007) Phenomena and mechanism for separation and recovery of penicillin in ionic liquids aqueous solution. Ind Eng Chem Res 46:6303–6312CrossRefGoogle Scholar
  30. 30.
    Jiang Y, Xia H, Guo C, Mahmood I, Liu H (2007) Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system. Biotechnol Progr 23:829–835CrossRefGoogle Scholar
  31. 31.
    Shewale JG, Sivaraman H (1989) Penicillin acylase-enzyme production and its application to the manufacture of 6-APA. Proc Biochem 24:146–154Google Scholar
  32. 32.
    Freire MG, Neves CMSS, Marrucho IM, Coutinho JAP, Fernandes AM (2010) Hydrolysis of tetrafluoroborate and hexafluorophosphate counter ions in imidazolium-based ionic liquids. J Phys Chem A 114:3744–3749CrossRefGoogle Scholar
  33. 33.
    Zatloukalova E, Kucerova Z (2004) Separation of cobalt binding proteins by immobilized metal affinity chromatography. J Chromatogr B 808:99–103CrossRefGoogle Scholar
  34. 34.
    Piergiovanni AR (2007) Extraction and separation of water-soluble proteins from different wheat species by acidic capillary electrophoresis. J Agric Food Chem 55:3850–3856CrossRefGoogle Scholar
  35. 35.
    Scopes RK (1994) Protein purification: principles and practice. Springer, New YorkCrossRefGoogle Scholar
  36. 36.
    Oppermann S, Stein F, Kragl U (2011) Ionic liquids for two-phase systems and their application for purification, extraction and biocatalysis. Appl Microbiol Biotechnol 89:493–499CrossRefGoogle Scholar
  37. 37.
    Matsumoto M, Mochiduki K, Fukunishi K, Kondo K (2004) Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus. Sep Purif Technol 40:97–101CrossRefGoogle Scholar
  38. 38.
    Chen X, Liu J, Wang J (2010) Ionic liquids in the assay of proteins. Anal Methods 2:1222–1226CrossRefGoogle Scholar
  39. 39.
    Ranke J, Molter K, Stock F, Bottin-Weber U, Poczobutt J, Hoffmann J, Ondruschka B, Filser J, Jastorff B (2004) Biological effects of imidazolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays. Ecotoxicol Environ Saf 58:396–404CrossRefGoogle Scholar
  40. 40.
    Gathergood N, Scammells PJ, Garcia MT (2006) Biodegradable ionic liquids. Part III. The first readily biodegradable ionic liquids. Green Chem 8:156–160CrossRefGoogle Scholar
  41. 41.
    Quijano G, Couvert A, Amrane A (2010) Ionic liquids: applications and future trends in bioreactor technology. Bioresour Technol 101:8923–8930CrossRefGoogle Scholar
  42. 42.
    Ventura SPM, Barros RLF, Barbosa JMP, Soares CMF, Lima AS, Coutinho JAP (2012) Production and purification of an extracellular lipolytic enzyme using ionic liquid-based aqueous two-phase systems. Green Chem 14:734–740CrossRefGoogle Scholar
  43. 43.
    Barbosa JMP, Souza RL, Fricks AT, Zanin GM, Soares CMF, Lima ÁS (2011) Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system. J Chromatogr B 879:3853–3858CrossRefGoogle Scholar
  44. 44.
    Dreyer S, Kragl U (2008) Ionic liquids for aqueous two-phase extraction and stabilization of enzymes. Biotechnol Bioeng 99:1416–1424CrossRefGoogle Scholar
  45. 45.
    Chambrlain J (1995) The analysis of drugs in biological fluids, 2nd edn. CRC Press, Boca RatonGoogle Scholar
  46. 46.
    Wey AB, Thormann W (2001) Capillary electrophoresis-electrospray ionization ion trap mass spectrometry for analysis and confirmation testing of morphine and related compounds in urine. J Chromatogr A 916:225–238CrossRefGoogle Scholar
  47. 47.
    Churley MM, Lyons TP, Robandt PV, Bruins MR (2003) Evaluation of a solid-phase extraction method for codeine and morphine in urine using Cerex Polycrom™ Clin II columns and the Speedisk™ 48 pressure processor at a high-throughput forensic drug-testing laboratory. J Anal Toxicol 27:530–532CrossRefGoogle Scholar
  48. 48.
    Li SH, He CY, Liu HW, Li K, Liu F (2005) Ionic liquid-based aqueous two-phase system, a sample pretreatment procedure prior to high-performance liquid chromatography of opium alkaloids. J Chromatogr B 826:58–62CrossRefGoogle Scholar
  49. 49.
    Freire MG, Neves CMSS, Marrucho IM, Canongia Lopes JN, Rebelo LPN, Coutinho JAP (2010) High-performance extraction of alkaloids using aqueous two-phase systems with ionic liquids. Green Chem 12:1715–1718CrossRefGoogle Scholar
  50. 50.
    Essig D, Costill DL, Van Handel RJ (1980) Effects of caffeine ingestion on utilization of muscle glycogen and lipid during leg ergometer cycling. Int J Sports Med 1:86–90CrossRefGoogle Scholar
  51. 51.
    Flenker U, Schänzer U (2001) Caffeine in doping control: determination of 13C in urinary excreted caffeine. Eur J Sport Sci 2:1–5CrossRefGoogle Scholar
  52. 52.
    Louros CLS, Cláudio AFM, Neves CMSS, Freire MG, Marrucho IM, Pauly J, Coutinho JAP (2010) Extraction of biomolecules using phosphonium-based ionic liquids + K3PO4 aqueous biphasic systems. Int J Mol Sci 11:1777–1791CrossRefGoogle Scholar
  53. 53.
    White NJ (1992) Antimalarial pharmacokinetics and treatment regimens. Br J Clin Pharmacol 34:1–10CrossRefGoogle Scholar
  54. 54.
    Huston M, Levinson M (2006) Are one or two dangerous? Quinine and quinidine exposure in toddlers. J Emerg Med 31:395–401CrossRefGoogle Scholar
  55. 55.
    Jansson Å, Gustafsson LL, Mirghani RA (2003) High-performance liquid chromatographic method for the determination of quinine and 3-hydroxyquinine in blood samples dried on filter paper. J Chromatogr B 795:151–156CrossRefGoogle Scholar
  56. 56.
    Samanidou VF, Evaggelopoulou EN, Papadoyannis IN (2005) Simultaneous determination of quinine and chloroquine anti-malarial agents in pharmaceuticals and biological fluids by HPLC and fluorescence detection. J Pharm Biomed Anal 38:21–28CrossRefGoogle Scholar
  57. 57.
    Flieger J, Czajkowska-Żelazko A (2015) Aqueous two phase system based on ionic liquid for isolation of quinine from human plasma sample. Food Chem 166:150–157CrossRefGoogle Scholar
  58. 58.
    He C, Li S, Liu H, Li K, Liu F (2005) Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt. J Chromatogr A 1082:143–149CrossRefGoogle Scholar
  59. 59.
    Carroll MF, Temte JL (2000) Proteinuria in adults: a diagnostic approach. Am Fam Physician 62:1333–1340Google Scholar
  60. 60.
    Sacks DB, Bruns DE, Goldstein DE, Maclaren NK, McDonald JM, Parrott M (2011) Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Diabetes Care 34(6):e61–e99CrossRefGoogle Scholar
  61. 61.
    Loghman-Adham M (1998) Evaluating proteinuria in children. Am Fam Physician 58:1145–1152Google Scholar
  62. 62.
    Du Z, Yu YL, Wang JH (2007) Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system. Chem Eur J 13:2130–2137CrossRefGoogle Scholar
  63. 63.
    Kumar SS, Chouhan RS, Thakur MS (2010) Trends in analysis of vitamin B12. Anal Biochem 398:139–149CrossRefGoogle Scholar
  64. 64.
    Berton P, Monasterio RP, Wuilloud RG (2012) Selective extraction and determination of vitamin B12 in urine by ionic liquid-based aqueous two-phase system prior to high-performance liquid chromatography. Talanta 97:521–526CrossRefGoogle Scholar
  65. 65.
    Han J, Wang Y, Kang W, Li C, Yan Y, Pan J, Xie X (2010) Phase equilibrium and macrolide antibiotics partitioning in real water samples using a two-phase system composed of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and an aqueous solution of an inorganic salt. Microchim Acta 169:15–22CrossRefGoogle Scholar
  66. 66.
    Li CX, Han J, Wang Y, Yan YS, Xu XH, Pan JM (2009) Extraction and mechanism investigation of trace roxithromycin in real water samples by use of ionic liquid–salt aqueous two-phase system. Anal Chim Acta 653:178–183CrossRefGoogle Scholar
  67. 67.
    Wang Y, Han J, Xie XQ, Li CX (2010) Extraction of trace acetylspiramycin in real aqueous environments using aqueous two-phase system of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and phosphate. Cent Eur J Chem 8:1185–1191Google Scholar
  68. 68.
    Han J, Wang Y, Yu CL, Yan YS, Xie XQ (2011) Extraction and determination of chloramphenicol in feed water, milk, and honey samples using an ionic liquid/sodium citrate aqueous two-phase system coupled with high-performance liquid chromatography. Anal Bioanal Chem 399:1298–1304CrossRefGoogle Scholar
  69. 69.
    Bogusz MJ, Hassan H, Al-Enzai E, Ibrahim Z, Al-Tufail M (2004) Rapid determination of chloramphenicol and its glucuronide in food products by liquid chromatography–electrospray negative ionization tandem mass spectrometry. J Chromatogr B 807:343–356CrossRefGoogle Scholar
  70. 70.
    Akhtar MH, Danis C, Sauve A, Barray C (1995) Gas chromatographic determination of incurred chloramphenicol residues in eggs following optimal extraction. J Chromatogr A 696:123–130CrossRefGoogle Scholar
  71. 71.
    Kubala-Drincic H, Bazulic D, Sapunar-Postruznik J, Grubelic M, Stuhne G (2003) Matrix solid-phase dispersion extraction and gas chromatographic determination of chloramphenicol in muscle tissue. J Agric Food Chem 51:871–875CrossRefGoogle Scholar
  72. 72.
    Han J, Wang Y, Yu C, Li C, Yan Y, Liu Y, Wang L (2011) Separation, concentration and determination of chloramphenicol in environment and food using an ionic liquid/salt aqueous two-phase flotation system coupled with high-performance liquid chromatography. Anal Chim Acta 685:138–145CrossRefGoogle Scholar
  73. 73.
    Shahriari S, Tomé LC, Araújo JMM, Rebelo LPN, Coutinho JAP, Marrucho IM, Freire MG (2013) Aqueous biphasic systems: a benign route using cholinium-based ionic liquids. RSC Adv 3:1835–1843CrossRefGoogle Scholar
  74. 74.
    Pereira JFB, Kurnia KA, Cojocaru OA, Gurau G, Rebelo LPN, Rogers RD, Freire MG, Coutinho JAP (2014) Molecular interactions in aqueous biphasic systems composed of polyethylene glycol and crystalline vs liquid cholinium-based salts. Phys Chem Chem Phys 16:5723–5731CrossRefGoogle Scholar
  75. 75.
    Passos H, Sousa ACA, Ramiro Pastorinho M, Nogueira AJA, Rebelo LPN, Coutinho JAP, Freire MG (2012) Ionic-liquid-based aqueous biphasic systems for improved detection of bisphenol A in human fluids. Anal Methods 4:2664–2667CrossRefGoogle Scholar
  76. 76.
    Braun JM, Kalkbrenner AE, Calafat AM, Yolton K, Ye X, Dietrich KN, Lanphear BP (2001) Impact of early-life bisphenol A exposure on behaviour and executive function in children. Pediatrics 128:873–882CrossRefGoogle Scholar
  77. 77.
    Kang JH, Kondo F, Katayama Y (2006) Human exposure to bisphenol A. Toxicology 226:79–89CrossRefGoogle Scholar
  78. 78.
    Deng YF, Long T, Zhang DL, Chen J, Gan SC (2009) Phase diagram of [amim]Cl + salt aqueous biphasic systems and its application for [amim]Cl recovery. J Chem Eng Data 54:2470–2473CrossRefGoogle Scholar
  79. 79.
    Li CX, Han J, Wang Y, Yan YS, Pan JM, Xu XH, Zhang ZL (2010) Phase behavior for the aqueous two-phase systems containing the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and kosmotropic salts. J Chem Eng Data 55:1087–1092CrossRefGoogle Scholar
  80. 80.
    Neves CMSS, Freire MG, Coutinho JAP (2012) Improved recovery of ionic liquids from contaminated aqueous streams using aluminium-based salts. RSC Adv 2:10882–10890CrossRefGoogle Scholar
  81. 81.
    Wu B, Zhang YM, Wang HP (2008) Phase behavior for ternary systems composed of ionic liquid + saccharides + water. J Phys Chem B 112:6426–6429CrossRefGoogle Scholar
  82. 82.
    Wu B, Zhang YM, Wang HP (2008) Aqueous biphasic systems of hydrophilic ionic liquids + sucrose for separation. J Chem Eng Data 53:983–985CrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Instituto de Tecnologia Química e Biológica António Xavier, ITQBUniversidade Nova de LisboaOeirasPortugal
  2. 2.CICECO - Aveiro Institute of Materials, Department of ChemistryUniversity of AveiroAveiroPortugal

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