The importance of column design for protein separation using aqueous two-phase systems on J-type countercurrent chromatography

• Published in: Separation and purification technology Vol. 65; no. 1; pp. 79 - 85
• Main Authors: Guan, Y.H., Bourton, E.C., Hewitson, P., Sutherland, I.A., Fisher, D.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 02.02.2009
• Subjects: Aqueous two-phase systems; Centrifugal partition chromatography; Countercurrent chromatography; Multilayer column; Protein separation; Spiral column; Toroidal column; Protein separation; Toroidal column; Countercurrent chromatography; Multilayer column; Aqueous two-phase systems; Centrifugal partition chromatography; Spiral column
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2008.07.016

The separation of lysozyme and myoglobin has been used as a model system with which to study protein separation in aqueous two-phase systems (ATPS) containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K 2HPO 4 in Brunel multilayer J-type countercurrent chromatography (CCC) centrifuges, where wave mixing pertains. Poor separations of the proteins with this type of CCC columns were observed in ATPS and the proteins did not elute as predicted from their equilibrium partition coefficients. As mass transfer of the proteins appeared to be a limiting factor, methods of improving mixing of the phases in the J-type CCC were explored. This has led to the design, construction and application of a toroidal column fitted to the Brunel Dynamic Extraction Centrifuge, providing pseudo-hydrostatic CCC in which much improved mixing for the stationary and mobile phases was obtained. In this toroidal column the protein mixture was separated with the locations of the protein peaks being consistent with those predicted by the partition coefficients. This toroidal coil system will form the basis for optimisation studies and scaling up separations.