Analysis of the Phase Behavior of the Aqueous Poly(ethylene glycol)-Ficoll System Tristan Croll, † Peter D. Munro, ‡ Donald J. Winzor, § Matt Trau, ‡ and Lars K. Nielsen* ,† Departments of Chemical Engineering, Chemistry, and Biochemistry, University of Queensland, Brisbane, Queensland 4072, Australia The PEG-Ficoll polymer phase system is one that has been overlooked in the past for biotechnology applications because of the stability of its emulsions. However, new applications, such as emulsion coating of cells, are appearing that rely on this very property. Ficoll is highly polydisperse and multimodal with three distinct Ficoll peaks in gel permeation chromatography. As a result, the transition between one-phase and two-phase systems is blurred and the binodials obtained through turbidometric titration and tie-line analysis differ significantly. Moreover, since the three Ficoll peaks partition differently, tie-line analysis cannot be described by a simple model of the aqueous two-phase system. A simple modification to the model allowed for excellent fit, and this modification may prove well-suited for the many practical cases where aqueous two-phase systems fail to display parallel tie-lines as implicitly assumed in the simpler model. Introduction Aqueous two-phase polymer systems have been studied in some detail over the past few decades for the separa- tion of biological materials, as a result of their excellent compatibility with these materials (1). Systems of this type are characterized by extremely low surface tension between phases and solution conditions that can very closely approach those of physiological fluids, so closely, in fact, that whole cells and cell clusters can be effectively partitioned without damage (2). Basta et al. developed a novel technique for the creation of immunoisolatory barriers on pancreatic islets based on coating of the cell clusters with the Ficoll phase in a poly(ethylene glycol) (PEG)-Ficoll emulsion (2). This technique shows much promise, but an essential first step to systematic optimization is an understanding of the phase behavior, i.e., the development of a phase diagram. A feature of the PEG-Ficoll system is its ability to form very stable emulsions. Although this is essential for the microencapsulation technique, it is not very useful for bioseparations, where a very fast phase separation is optimal. For this reason, the PEG-Ficoll system has not been studied in any detail, and the sole published phase diagram (3) raises more questions than it answers, consisting as it does of a binodial and a single tie-line that appear to bear little or no relation to each other. In this paper we present an analysis of the PEG 10,000-Ficoll 400,000 aqueous phase diagram in the presence of physiological salt concentrations. We compare a binodial curve obtained by turbidometric titration (4) to the phase diagram obtained by tie-line analysis (3) and note a striking similarity to the phenomenon displayed in Albertsson (3). Theory Using common features for aqueous two-phase sys- tems, such as low polymer concentration and near identical density in the two phases, Diamond and Hsu developed a simplified partitioning model based on the Flory-Huggins theory of polymer solutions (5). According to this model polymer partition between the upper (′′) and lower (′) phases can be written where In these expressions m 1 and m 2 denote the ratio of the molar volume of the respective polymer to that of solvent and hence are constants.  01 and  02 are the Flory- Huggins parameters describing interactions between * To whom correspondence should be addressed. Tel: +617 3365 4682. Fax: +617 3365 4199. E-mail: lars.nielsen@uq.edu.au. † Department of Chemical Engineering. ‡ Department Chemistry. § Department of Biochemistry. ln K 1 ) ln ( w 1 ′′ w 1 ′ 29 ) A 1 (w 1 ′′ - w 1 ′) ln K 2 ) ln ( w 2 ′′ w 2 ′ 29 ) A 2 (w 1 ′′ - w 1 ′) (1) A 1 ) m 1 [ R 1 ( 1 m 1 - 1 + 2 01 29 +R 2 φ ( 1 m 2 - 1 +  01 +  02 -  12 29 ] A 2 ) m 2 [ R 2 φ ( 1 m 2 - 1 + 2 02 29 +R 1 ( 1 m 1 - 1 +  01 +  02 -  12 29 ] φ ) w 2 ′′ - w 2 ′ w 1 ′′ - w 1 ′ (2) 1269 Biotechnol. Prog. 2003, 19, 1269-1273 10.1021/bp025711a CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/06/2003