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) K2HPO4 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. (C) 2008 Elsevier B.V. All rights reserved.