Types of centrifugal separations

One way of improving the resolution of particles by size is to minimize the difference in RCF between the top and bottom of the sample. Practically this is achieved by layering the sample as a narrow band over a continuous density gradient. This is called rate-zonal gradient (sedimentation velocity) centrifugation; the conditions are chosen such that ρρ always remains greater than ρl. In a continuous gradient the density increases in a smooth but not necessarily linear fashion and can be generated by a number of methods described later. By allowing the particles to sediment through such a density gradient the resolution and recovery of particles is generally improved. The particles move down through the gradient in the form of discrete zones at a rate that depends principally on their size (see figure below). Resolution between the zones decreases as the radial thickness of the sample increases so the efficacy of rate zonal fractionation is limited by sample size, which is commonly no more than 10% of the total gradient volume. The aim of ratezonal centrifugation is to devise conditions that will allow each zone of particles to move down through the gradient at a more or less constant velocity (isokinetic gradient). In a continuous gradient the particles are decelerated by the increasing viscosity of the medium and the reducing (ρp ρl) factor; ideally the acceleration (RCF) and deceleration forces should balance one another. A discontinuous density gradient (density increases in step-wise fashion) is never used for rate-zonal separations as sudden changes in viscosity and density would lead to irregular separation of the zones of particles as they move from one step to another.