2 v = 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.
3 Analytical 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
5 Differential 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]
6 Problems 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®
7 Density Gradient Centrifugation Two Types:1) Rate Zonal p > sseparates primarily by mass2) Isopycnic p < sequilibriumseparates by density
8 Example of Isopycnic Separation Percoll, self-forming gradientlight vs. heavy fraction