Kinetics of Phase Separation for Po I y eth y I ene G I yco I- P h 0s pha t e Two-Phase Systems A. Kaul, R. A. M. Pereira, J. A. Asenjo,* and J. C. Merchukt zyxwv Biochemical Engineering Laboratory, The University zyxwv of Reading, Reading, England Received December 28, 7994lAccepted June 14, 1995 zyxwvu Phase separation times for polyethylene glycol (PEG)- 4000-phosphate aqueous two-phase systems were stud- ied, for small scale (5-g) and large scale (1300-g) sys- tems, as a function of the stability ratio. Profiles of dis- persion height for both large and small scale systems were represented as a fraction of the initial height and were found to be independent of the geometrical dimen- sions of the separator. Furthermore, zyxwvutsrq by plotting time as a fraction of the initial height the total time of separation can be calculated for a given height of system at a par- ticular stability ratio. This generalization is important for the design of large scale aqueous two-phase separators. Phase separation times were also found to be dependent on which of the phases is continuous. A characteristic change in phase separation time was zyxwvutsr also observed at the phase inversion point (i.e., where the dispersed phase changes to a continuous phase and vice versa) and this point tends toward higher volume ratios as the tie-line length (TLL) is increased. Furthermore, the phase inversion point at each TLL corresponds to a fixed phos- phate concentration. zyxwvutsr 0 1995 John Wiley & Sons, Inc. Key words: polyethylene glycol phosphate phase sep- aration - kinetics INTRODUCTION Aqueous two-phase systems (ATPSs) have been studied in some detail over the past 30 years’ and, more recently, the application of ATPSs for the extraction of recombinant pro- teins has been exploited by i n d ~ s t r y . ~ Cell debris removal can be combined with an initial enrichment, thus, substi- tuting two possible steps: centrifugation or microfiltration, and adsorption (e.g., ion exchange). For industrial pur- poses, polymer-salt systems are preferential to the origi- nally proposed polymer-polymer systems due to the lower viscosity, lower cost of chemicals, and smaller phase sep- aration time. Polymer-salt systems are generated by mixing a polymer such as polyethylene glycol (PEG) with certain salts, e.g., potassium phosphate or sodium citrate, above critical concentrations. Such mixtures give rise to two liquid * To whom all correspondence should be addressed at the Centre for Biochemical Engineering and Biotechnology , Department of Chemical En- gineering, Universidad de Chile, Beauchef 861, Santiago, Chile. t Present address: Ben Gurion University of the Negev, Beer-Sheva, Israel. layers with the top phase enriched in polymer and the bot- tom phase enriched in salt. Both phases have a high water content (75% to 95% w/w) and are in equilibrium. Aqueous phase systems separate biopolymers such as proteins according to their hydrophobicity, charge, size, and biospecificity . Partition is influenced by phase system parameters such as molecular weight of the polymer, pH, type, and concentration of phase forming components and concentration of added salt (e.g., NaCl). Advantages of ATPSs over conventional techniques include a negligible mass transfer resistance (if the appropriate operation system is chosen, as explained later), amenability to continuous processing, a high capacity for handling of solids and lower investment costs. The purification of a protein product can be carried out batchwise, in a continuous one-stage extrac- tion or in a continuous multistage extraction which can be either crosscurrent, cocurrent, or countercurrent. ’ ’ Two alternative ways of operation are possible; in the first, the two phases remain always distinctly separated and solutes are transferred between them due to concentration gradient^.^ This type of operation is similar to classical extraction using immiscible phases, typical in the chemical industry. The alternative operation consists of provoking a shift on the “state plane” from the single phase region to the two-phase region. This shift is easily attained by adding one of the components of the system. The new point on the state plane is unstable and separates along a tie-line to give two phases on the binodial curve which are in equilibrium. The second mode of operation is fundamentally different from the first one and zyxwv so long as good mixing is assured no problems of mass transfer are likely to arise. The rate- limiting process in this case, however, is no longer mass transfer but phase formation. The new phases form at, or very close to, their equilibrium composition. No fundamen- tal studies have been reported on phase formation kinetics. It has been reported that if the phase components are added as concentrated solutions then equilibrium with respect to phase composition and partition can be reached rapidly that is, in <80 s.~ Studies on mechanical separation of phases have been carried out under unit gravity6 with or without superim- posed flow or accelerated by centrifugati~n,~~’~ with or without superimposed flow. It has been assumed by some authors that separation of phases by sedimentation under Biotechnology and Bioengineering, Vol. 48, Pp. 246-256 (1995) 0 1995 John Wiley & Sons, Inc. CCC 0006-35921951030246-11