Biophysical Tools '02 - Sedimentation 1/3/2019
Biophysical Tools '02 - Sedimentation Principle centrifugal force - buoyancy = frictional force Sedimentation Molecule moves in the centrifuge at constant velocity when: centrifugal force - buoyancy = frictional force w: angular velocity r: distance from the center of spinning m: mass of the molecule n: partial specific volume r: density of solution v: velocity of sedimentation f: frictional coefficient think of this equation when choosing the speed of centrifugation, the solution, or centrifuge tubes separate the experimental parameters from the molecular parameters by defining sedimentation coefficient S the values of sedimentation coefficients are Svedbergs (1S = 10-13 sec) To determine the sedimentation coefficient measure the traveled distance of the sample at various sedimentation times. Partial specific volume for proteins ia 0.725 cm^3 g^-1; nucleic acids 0.5 w: angular velocity r: distance from the center of spinning m: mass of the molecule n: partial specific volume r: density of solution v: velocity of sedimentation f: frictional coefficient Sedimentation coeff. = velocity per unit centrifugal field Biophysical Tools '02 - Sedimentation 1/3/2019
Analytical Centrifuge a sample tube (cell) allowing continuos observation of the sample: detection of sample by Schlieren optics, interferometer or UV absorption. Preparative centrifugation: your common centrifuge does not allow sample observation during sedimentation; Biophysical Tools '02 - Sedimentation 1/3/2019
Boundary sedimentation Velocity is measured as the rate of movement of the sample boundary down the centrifuge tube. velocity = dc/dt thus measure speed with which the boundary moves diffusion spreads the boundary Biophysical Tools '02 - Sedimentation 1/3/2019
Application: Homogeneity equilibria v. heterogenous systems Biophysical Tools '02 - Sedimentation 1/3/2019
Application: Molecular weight determination Sedimentation Equilibrium Sedimentation at low speeds so that the sedimentation is counterbalanced by diffusion. Equilibrium in 16-24 hours Concentration of the molecules in the tube is a function of both the speed and diffusion. Boltzman distribution of the number of moilecules above and below boundary Energy of the centrifugal force (minus buoyancy): Sedimentation Equilibrium Sedimentation at low speeds so that the sedimentation is counterbalanced by diffusion Biophysical Tools '02 - Sedimentation 1/3/2019
Application: Shape determination Stokes 6.2 S 6.7 S 5.4 S Biophysical Tools '02 - Sedimentation 1/3/2019
Application: Purification preformed density gradients Biophysical Tools '02 - Sedimentation 1/3/2019
Zonal - Density Centrifugation In zonal centrifugation density gradient is either preformed (sucrose, Ficoll, Percoll) or self-forming (CsCl). The role of the gradient is to: (a) limit convection of the solution and thus spreading of the boundary (band) or (b) to provide matching buoyancy where sample comes to rest and does not sediment any further (lipoproteins). Biophysical Tools '02 - Sedimentation 1/3/2019
Biophysical Tools '02 - Sedimentation Practical tips K-factor: Pelleting time (t): But if you spin at speed less than the maximum speed (wmax) k is smaller: g force Biophysical Tools '02 - Sedimentation 1/3/2019