The Effect of Guanidine Hydrochloride on Phase Diagram of PEG- Phosphate Aqueous Two-Phase System Farshad Rahimpour, and Mohsen Pirdashti Abstract—This report focus on phase behavior of polyethylene glycol (PEG)4000/ phosphate/ guanidine hydrochloride/ water system at different guanidine hydrochloride concentrations and pH. The binodal of the systems was displaced toward higher concentrations of the components with increasing guanidine hydrochloride concentrations. The partition coefficient of guanidine hydrochloride was near unity and increased with decreasing pH and increasing PEG/salt (%w/w) ratio. Keywords—Aqueous two-phase system, guanidine hydrochloride, partition coefficient, phase diagram. I. INTRODUCTION IXING two or more incompatible polymers or a polymer and a structuring salt in aqueous conditions generally forms an aqueous two-phase system (ATPS), which the percentage of water in both phases being 75-90% (v/v) [1]. These systems present a gentle, scalable and efficient procedure for separation of various biological materials such as recombinant proteins and enzymes [2]-[5]. Inclusion body refolding processes are poised to play a major role in the production of recombinant proteins. One step of general strategy used to recover active protein from inclusion bodies is solubilization of the aggregated protein with denaturant such as guanidine hydrochloride and urea [6]. Many articles have been written about applying guanidine hydrochloride in aqueous two-phase system for the initial recovery step, but little has been published about the complex problem of how the guanidine hydrochloride effects on the phase diagram behavior and determining partition coefficient the guanidine hydrochloride in these systems. Aqueous two- phase systems, based on polyethylene glycol and sodium sulfate, have earlier been successfully used in the presence of urea for the recovery of active insulin like growth factor (IGF- 1) from inclusion bodies [7]. Recovery of active protein in phase systems containing PEG and a chaotropic salt such as Manuscript received June 15, 2007. This work was supported in part by the research vice departments of Razi University. Farshad Rahimpour, Assistant Professor, Biotechnology research lab, Chemical Engineering Department, Faculty of Engineering, Razi University, Kermanshah 67149-67346, Iran (phone: +98-831-4274530; fax: +98-831- 4274542; e-mail: f_rahimpour@razi.ac.ir). Mohsen Pirdashti, MSc, Biotechnology research lab, Chemical Engineering Dept. Faculty of Engineering, Razi Univ. Kermanshah 67149- 67346, Iran (e-mail: Pirdashti@razi.ac.ir). guanidine hydrochloride has also been shown to be possible [8]. Novel bioseparation research based on aqueous two-phase systems needs to focus on determining phase diagrams, partition coefficients and other thermodynamic data for the design of industrial-scale process [1]. A comprehensive review of the early experimental liquid–liquid equilibria (LLE) of the aqueous two-phase systems containing two different kinds of polymers or a polymer and a salt have been reported by Albertsson [2] and Walter et al. [3], but the knowledge of phase systems containing chaotropic compounds is very limited [9], Rämsch et al. [9],[10], determined phase diagrams of poly(ethylene glycol)(PEG)/ sodium sulfate/ urea/ water and PEG/ dextran T-500(DEX)/ phosphate buffer/water at different concentration of urea and different PEG molecular weights. Guanidine hydrochloride is preferred due to the problem that urea solutions may contain and spontaneously produce cyanate [11], which can carbamylate the amino groups of the protein [12]. In addition, inclusion body solubilization by urea is pH dependent and optimum pH conditions must be determined for each protein [12]. At the present study, the effect the guanidine hydrochloride on phase behavior of PEG4000/ phosphate/ guanidine hydrochloride/ water at different guanidine hydrochloride concentrations and pH was investigated. Furthermore the partition coefficient of guanidine hydrochloride and parameters that affect it, e.g., pH and PEG/Salt (%w/w) ratio was studied. These data would be useful to increasing the knowledge of aqueous two-phase separation process and improving the yield of protein refolding. II. EXPERIMENTAL SECTION A. Materials Polyethylene glycol, with a mass average 4000, di- potassium hydrogen phosphate and sodium di-hydrogen phosphate were of analytical grade (Merck) and were used without further purification. Guanidine hydrochloride was purchased from Sigma-Aldrich. Distilled water was used in all experiments. B. Preparation of the Aqueous Two-Phase Systems Biphasic systems were prepared by a mixture of PEG 4000 and phosphate salt solution at required pH. The pH of the salt solution was adjusted by mixing appropriate ratio of sodium di hydrogen phosphate and di potassium hydrogen phosphate. In M World Academy of Science, Engineering and Technology 29 2007 124