2v = 2rm(p - s)/fThe greater the centrifugal force (2r), the faster the particle sediments.The more massive a particle, the faster it moves in a centrifugal field.The denser a particle, the faster in moves in a centrifugal field.The denser the solvent, the slower the particle will move in a centrifugal field.The particle velocity is 0 when the solvent density is greater than the particle density.The greater the frictional coefficient (factors such as solvent viscosity, particle shape, etc.), the slower the particle will move.
3Analytical vs Preparative Centrifugation Sedimentation Coefficient (s)is the velocity per Fc, ors = v/2runits are the Svedberg (S), where 1 S = sec= m(p - s)/fAnalytical vs Preparative Centrifugation
5Differential Centrifugation prepare cell lysatesubject to centrifugationcentrifugal forcetime (g ·min)tube size and shaperotor angleseparate supernatant and pelletre-centrifuge supernatantRelative Centrifugal Forceexpressed as ‘x gravity’RCF = Fc/Fg = 2r/980[ = (rpm)/30]RCF = x 105 (rpm)2r[radius in cm]
6Problems Density Gradients contamination resolution large particles contaminated with smaller particlesresolutionparticles of similar sizes not separatedvibrations and convection currentsDensity Gradientscentrifugation in a dense mediumincreases stabilityprovides greater resolutioncommon media: sucrose, CsCl, Ficoll®, Hypaque®, Percoll®
7Density Gradient Centrifugation Two Types:1) Rate Zonal p > sseparates primarily by mass2) Isopycnic p < sequilibriumseparates by density
8Example of Isopycnic Separation Percoll, self-forming gradientlight vs. heavy fraction