Journal of Chromatography A, 1151 (2007) 65–73 Solvent systems for countercurrent chromatography: An aqueous two phase liquid system based on a room temperature ionic liquid Maria Jose Ruiz-Angel, Veronica Pino 1 , Samuel Carda-Broch 2 , Alain Berthod ∗ Laboratoire des Sciences Analytiques, Universit´ e de Lyon, Bat. CPE, 69622 Villeurbanne, France Available online 12 December 2006 Abstract A new aqueous two phase liquid system (ATPS) based on the ionic liquid 1-butyl-3-methyl imidazolium chloride (BMIM Cl), potassium dibasic phosphate (K 2 HPO 4 ) and water was recently proposed in the literature. The full phase diagram of this ATPS was prepared and some tie lines were fully determined. It was compared to classical ATPSs based on polyethylene glycol with an average molecular mass of 1000 (PEG 1000) and 10,000 (PEG 10000) and K 2 HPO 4 . Two countercurrent chromatography (CCC) columns, a hydrostatic Sanki and a J type hydrodynamic CCC columns were used to test the liquid phase retention of these ATPSs in all possible configurations. It was found that the BMIM Cl ATPS liquid phases were much easier to retain in the two CCC columns than the PEG 1000 ATPS phases. Using protein and alcohol solutes, it was established that the BMIM Cl ATPS has a polarity completely different from that of the PEG 1000 ATPS. For example, ovalbumin partitions equally between the two phases of the PEG 1000 ATPS (K D = 1.4) when it is completely located in the BMIM Cl upper phase of the ionic liquid ATPS (K D = 180). The discrimination factor of the ionic liquid system and its intrinsic hydrophobicity were respectively found three times higher and ten times lower than the respective values of the PEG 1000 ATPS. © 2006 Elsevier B.V. All rights reserved. Keywords: Countercurrent chromatography; Biphasic liquid system; Dual-mode; Ionic liquid; Hydrophobicity; Polarity 1. Introduction Room temperature ionic liquids (RTILs) are a new class of fashion solvents that demonstrated their interest in organic synthesis, catalysis, electrochemistry and analytical chemistry [1–5]. Basically they are just salts whose melting point is close to room temperature. As salt, they should not be considered exactly like regular molecular solvents. They are necessarily made by an anion associated with a cation. This binary and/or dual nature explains many properties of the RTILs [6]. Since RTILs may form biphasic liquid systems with numer- ous solvents, including water, it was important to consider them as possible liquid candidate in countercurrent chromatography (CCC). In previous works, it was found that RTILs could not ∗ Corresponding author. Tel.: +33 472 431434; fax: +33 472 431078. E-mail address: berthod@univ-lyon1.fr (A. Berthod). 1 On leave from Universidad de La Laguna, Departamento de Qu´ ımica Anal´ ıtica, 38206 La Laguna, Spain. 2 On leave from Universidad Jaume I, Departamento de Ciencias Experimen- tales, 12071 Castell ´ on de la Plana, Spain. be used directly in CCC due to their high viscosity. Addition of an organic solvent such as a short chain alcohol or acetonitrile greatly reduced the ionic liquid phase viscosity [7] and limited applications were possible [8]. Aqueous two phase liquid systems (ATPS) were introduced by Albertson [9]. They come from the observation that a con- centrated solution of an organic polymer such as polyethylene glycol (PEG) and an inorganic salt such as dibasic sodium or potassium phosphate (K 2 HPO 4 ) separates in two liquid layers: the upper layer contains most of the PEG and the lower layer contains most of the inorganic salt. The two liquid layers are aqueous solutions. ATPSs were found extremely useful to sep- arate and/or purify biological material and especially proteins [9]. They were early used in CCC for this purpose [10–12]. They were found very useful for protein or biological material purification and today, an impressive number of works associat- ing the CCC technique with ATPS can be found in the literature [13–20]. It was recently mentioned that a particular fully water soluble ionic liquid, 1-butyl-3-methyl imidazolium chloride (BMIM Cl), was able to form two aqueous phases when 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.072