Centrifugal Separations in Molecular and Cell Biology by G. D. Birnie, D. Rickwood

By G. D. Birnie, D. Rickwood

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Two separate peaks form if the reaction rates are slow. 57) Skerrett (1975) showed that the apparent molecular weight {M^^^) defined by can be where Mq is the local weight-average weight and Β = (c^ - d)lr the linear relationsnip between concentration and radius with α = intercept. where β is the weighted diffusion coefficient, (afi^), and α,· is the weight fraction of Ρ in PAi, = M(M + iMa )"^. 60). 62) throughout by η gives Σλ:{ and dividing by η gives the means x'^. 65) Hence, οοΣΛ:' + Λ ι Σ χ χ ' + α 2 Σ χ ν + .

Density is the factor which determines the stability of the gradient; the density gradient must be steep enough to prevent convection and to maintain the stability of sedimenting zones throughout centri­ fugation. A gradient which needs to be steep at the top to support the sample zone need not be so steep further down, where the zones have separated from each other and have spread by diffusion and because of particle heterogeneity. For this reason, so-called convex density gradients are often advocated, that is, gradients which are steep at the top and shallower towards the bottom.

However, a sharp increase in viscosity may concentrate a zone enough to cause instability. There are advantages and disadvantages in the use of the various types of gradient shape, which will be dealt with briefly as step, constant slope, concave and convex, constant velocity, and complex. The mathematical derivations for defining the shape of gradients have been given elsewhere (Noll, 1969a). Step Gradients Gradients formed from two or more layers of differing densities are useful in isopycnic centrifugation and in many pelleting techniques, but are of limited use in rate-zonal centrifugation.

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