Flow cell separation in fluctuating g-field

Abstract

Field flow fractionation of particles in rotating coiled column has been investigated in recent year. In contrast to the classical mode of field flow fractionation in narrow channels, the use of rotating coiled columns offers the possibility of large sample loading. In this thesis, the potential for new cell separation methods based on the use of flow fractionation in fluctuating g-fields generated in rotating coil columns is examined. The effects of operational conditions (flow rate and rotational speed – Chapter 3 and Chapter 5); cell properties (cell flexibility – Chapter 4); and column shapes (different inner diameters and coil geometries – Chapter 6) on the flow behaviour of a model system of red blood cells (RBCs) from different species, which differ markedly in size, shape & density, flowing in a single phase of buffered saline have been characterised. Operational Conditions: For a particular rotational speed, there was a minimum flow rate which caused all the cells to be retained in the column and a maximum flow rate at which all cells were eluted. Both the minimum and maximum flow rate were increased when a higher rotational speed was applied. Differences in the behaviour of sheep & hen RBCs have been used to develop a separation method using a continuously increasing flow gradient. This separation could be speeded up by using a step flow gradient. The effects of cell load and rotational direction on the behaviour of RBCs in the column was also studied in this thesis. Cell Properties: The minimum flow rate was found to correlate with cell diameter/cell volume of the RBCs as expected for a sedimentation related process and was partially described by a theoretic equation developed for particles by Fedotov and colleagues (Fedotov et al. 2005). However cell dependent departures from this equation were found which appear to indicate that cell specific surface properties may also be involved for cells (Chapter 3). By contrast the maximum flow rate showed no correlation with cell diameter/cell volume. An effect of cell deformability on the flow separation behaviour of the cells has been demonstrated. Chemical fixation of sheep RBCs with glutaraldehyde rendered the normally deformable RBCs rigid and non-deformable and resulted in the fixed sheep RBCs eluting significantly earlier than unfixed sheep RBCs. This difference was great enough that a mixture of deformable (unfixed) and non-deformable (fixed) sheep RBCs could be separated. Fixed cells tended to show cell aggregation, which could be reduced by the addition of surfactant. Column Geometry: An effect of column shapes on the flow separation behaviour of cells has been demonstrated showing that the optimisation of column design is an important feature of this mode of cell separation. For columns with the same cross sectional area, a “horizontal” rectangular column provided better separation than a circular column and a “vertical” rectangular column gave the least efficient separation. A possible explanation for this behaviour is suggested the thinner sedimentation layer and less secondary flow. Differences in the behaviour of various species of RBCs in the “horizontal” rectangular column have been used to study the efficiency of separation of a mixture of sheep and hen RBCs, and a mixture of rabbit and hen RBCs. This work shows similarities and differences with other reports on cell/particle separations in rotating coiled columns in single phases and also in aqueous two phases systems (ATPS) and these are discussed. Fedotov P.S., Kronrod V.A. & Kasatonova O.N. (2005). Simulation of the motion of solid particles in the carries liquid flow in a rotating coiled column. J. Anal. Chem., 60, 4, 310-316.