CONICET | Buscador de Institutos y Recursos Humanos

Aqueous two phase systems (ATPSs) formed by a polymer and a lyotropic sal provide a powerful method for separating biomaterials. Those ATPSs containing biodegradable anions such as citrate, tartrate and succinate are particularly interesting due to the lower environmental impact. However, their use in downstream processing has been hampered by the lack of a theory based on fundamental principles capable of explaining and predicting the experimental trends observed. The phase behavior of polymer/salt systems is complicated due to both the size differences between the molecules to the nature of the mutual interactions. Models for the excess Gibbs energy based in Pitzer equation have been successfully employed to describe thermodynamic properties of pure electrolyte solutions. They combine contributions from long and short-range interactions. In this work a modified Pitzer model was applied to solutions that contain both an electrolyte and a neutral polymer in order to calculate partition coefficients of biological molecules in ATPSs. The modifications accounts for i) the dependency of model binary parameters -corresponding to polymer/polymer and polymer/salt short range interactions- on the polymer chain length; ii) the polymer presence like a second solvent that affects density and dielectric constant on the long-range interaction term. ATPS formed by polyethyleneglycols of different MWs (4,000; 6,000 and 8,000) and sodium salts (citrate, tartrate and succinate) were characterized. Polymer and salt concentrations in both phases at the equilibrium condition were measured by independent analytical methods. Partition coefficients (Kp) of seven model proteins: bovine serum albumin, catalase, beta-lactoglobulin, alpha-amylase, lysozyme, pepsin, urease and trypsin in the selected ATPSs were determined spectrophotometrically. The first step in the calculation was to solve phase equilibrium in each ATPS in the absence of partitioned biomolecule. Experimental liquid-liquid equilibrium data were used to estimate the model binary parameters corresponding to polymer/polymer, polymer/salt and cation/anion interactions through the minimization of the deviation between experimental and calculated compositions. Comparison between experimental and calculated tie line data showed mean deviations always lower than 3 % thus indicating a good correlation. In the second step, partitioning of model proteins was analyzed. Pepsine and lisozyme showed to be displaced to the polymer rich.phase (Kp>1) in all the assayed systems while catalase and alpha amylase partitioned unilaterally to the salt-rich phase (Kp <1). Different partitioning trends were observed for the rest of assayed proteins. The model parameters corresponding to binary interaction polymer-protein and salt-protein were then fitted to experimental partition coefficients for each protein. The equilibrium compositions of both phases calculated in absence of biomolecule were assumed to hold in its presence due to the low protein concentration. Predicted and experimental partition coefficients showed a quite successful correlation. Mean deviations showed to be lower than the experimental uncertainty for most of assayed proteins. We conclude that the proposed modified Pitzer model provides a simple thermodynamic framework for correlating and ultimately predicting phase separation and protein partition coefficients. This could contribute to improve the design and optimization of purification processes.The authors gratefully acknowledge financial support from CAPES (Brasil) and MinCyT, PICT508/2006, PIP00196 (Argentina)

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