Journal of Chromatography A, 1151 (2007) 115–120 Spiral coils for counter-current chromatography using aqueous polymer two-phase systems Y.H. Guan ∗ , J. Smulders, D. Fisher, I.A. Sutherland Brunel Institute for Bioengineering, Brunel University West London, Uxbridge, Middlesex UB8 3PH, UK Available online 8 January 2007 Abstract Retention properties of polyethylene glycol–phosphate aqueous two-phase systems in a spiral coil (5 mm I.D.) on Type-J synchronous counter- current chromatographic devices have been compared for the elution mode where the lower phase is the mobile phase and flows from the inside head terminal. This was achieved with the aid of digital imaging under stroboscopic illumination, an image analysis and measurement of the displaced volume of the stationary phase. For the spiral coil, high and stable stationary phase retention at mobile phase flow rates up to 64 ml/min has been obtained. Wave-like disturbance of the interface near the proximal point was observed and analyses have been made for possible use in protein separation. © 2006 Elsevier B.V. All rights reserved. Keywords: Counter-current chromatography; Aqueous two-phase systems; Spiral column holders; Polyethylene glycol; PEG; Stroboscopic imaging 1. Introduction Counter-current chromatography (CCC) is a support-free form of liquid–liquid chromatography using two immiscible liquid phases; typically with a stationary phase and a mobile phase [1,2]. For separation and purification of natural and syn- thetic products using CCC, a large array of applications has been developed and/or identified [3]. A pre-requisite for suc- cessful separation of targeted molecules using high-speed CCC (HSCCC) has been good retention of the stationary phase [4]. For biochemical and bioengineering applications, aqueous polymer two-phase systems (ATPS) have been shown to pro- vide a benign environment for separating and purifying bioactive macromolecules (e.g. protein, DNA and RNA) and bio-particles (plasmids, sub-cellular membranes) with potential to scale up as a bioengineering downstream process [5–7]. Clearly aqueous two-phase systems have potential for use in CCC [8]. However, for some time there have been difficulties in using ATPS in the most common multilayer CCC coils on Type-J HSCCC. The main concern has been the unpredictably low lev- els of retention for stationary phase [3], especially for high  ∗ Corresponding author. Tel.: +44 1895 267113; fax: +44 1895 274608. E-mail address: hugh.guan@brunel.ac.uk (Y.H. Guan). value multilayer coils [9]. Additionally, the stationary phase shows gradual loss over several hours [10]. It has been suggested that this results from the formation of an emulsion between the heavy and light phases of an ATPS, due to the low interfacial tension and high viscosity of the polymer-rich phase [8]. To reduce the problem of low stationary phase retention for ATPS mounted on Type-J synchronous CCC device, it appears that the use of coils with low  value in combination with low mobile phase flow rate might lead to an improved retention (e.g. S f ca. 30% and flow rate ca. 0.5 ml/min for I.D. 1.6 mm and  value close to 0.3 coil) [8]. Another approach to increase the retention of the station- ary phase has been to develop new CCC devices other than the Type-J and, broadly speaking, the most successful devices for such use with ATPS by Ito [11] have been those termed as X- axis CCC, where the axes of the rotation for the column holder and the revolution of the rotor cross each other. Compared with Type-J CCC, the X-axis CCC devices are more cumbersome and complicated, and are less appealing to the commercial end users, as the scaling-up of the X-axis CCC devices for large- scale separation processes would be both costly and technically complicated. The general dilemma with the use of ATPS has been that, on one hand the Type-X CCC devices have offered more success in separating proteins and enzymes than the Type- J CCC, but on the other hand, the market interests have been on 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.12.097