1. Biophysical Tools '02 - Sedimentation
1/3/2019

2. 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 = sec)
To determine the sedimentation coefficient measure the traveled distance of the sample at various sedimentation times.
Partial specific volume for proteins ia 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

3. 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

4. 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

5. Application: Homogeneity
equilibria v. heterogenous systems
Biophysical Tools '02 - Sedimentation
1/3/2019

6. Application: Molecular weight determination
Sedimentation Equilibrium
Sedimentation at low speeds so that the sedimentation is counterbalanced by diffusion.
Equilibrium in 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

7. Application: Shape determination
Stokes
6.2 S
6.7 S
5.4 S
Biophysical Tools '02 - Sedimentation
1/3/2019

8. Application: Purification
preformed density gradients
Biophysical Tools '02 - Sedimentation
1/3/2019

9. 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

10. 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