Centrifugation Flashcards
Uses of centrifugation
Preparative - to separate
Analytical
Analytical applications of centrifugation
o Needs pure material
o Mr of proteins
o Density
o Shape
o Association/Disassociation
Movement of particles depends on…
- Applied force
- Density difference with solvent
- Size of particles
- Shape of particles
- Viscosity
The rate at which particles sediment in a centrifuge
o Centrifugal force
o Buoyancy
o Resultant force
o Velocity produced
Centripetal force
Force keeping an object moving in a circle.
Acts towards the centre
Centrifugal force
Centripetal force → tube & liquid in circle
Centrifugation means that over time particles move _
down the tube
What is the Archimedes principle?
Upthrust force = weight of solvent displaced
V0 (solvent volume displaced) = Vp (particle volume)
What is the resultant force
Resultant force = Centrifugal – Upthrust
What is the diffusion coefficient?
How long a particle takes to diffuse across a volume
What is the sedimentation velocity?
Speed at which particle moves away from rotation axis
What is the sedimentation coefficient s?
Speed of sedimentation / speed of centrifuge
If you know size of particle (M) and speed of sedimentation required, you can determine _
w
Preparative centrifugation
Analytical centrifugation
Set w, determine speed of sedimentation (dx/dt) and D => M
Can calculate mass of proteins accurately
Fractionation
Precipitation with increasing salt ie: (NH4)2SO4
To find approximately where precipitation occurs
Stages of preparative centrifugation
Precipitation
Cell fractionation
Sedimentation pattern in analytical centrifugation
o Where sediment is and when
o End with clear solvent with some back diffusion
Fitting absorbance curves to Svedburg equation gives s and D and hence _
M
Summary of analytical centrifugation
- Method uses sedimentation velocity.
- Absolute method for native Mr
- Sedimentation pattern shows some back diffusion
What is the sedimentation equilibrium?
the point at which there is an equilibrium between sediment and solution.
Can use the concentration to give Mr
Sedimentation velocity vs sedimentation equilibrium
Sedimentation is fast and good for multiple non-interacting species
Sedimentation equilibrium is slower and gives M of structure, good for single or interacting species
True or false:
Both sedimentation velocity and equilibrium give absolute values
True
True or false:
Both sedimentation velocity and equilibrium have constant solvent density
True
What is density gradient centrifugation?
Sedimentation (Velocity or Equilibrium) in uniform solvent
Separation by size (M) with some shape (D)
Separation by density
Two types of Density Gradient Centrifugation
- Pre-formed gradient: rate zonal centrifugation
- Gradient formed during experiment: equilibrium (isopycnic)
So particle will sediment until the _ of the protein reaches the _ of the solvent
Density
Rate zonal centrifugation
Equivalent to sedimentation velocity
Material layered on top
Sediment on basis of size
Slows as rp approaches “ρ0”
Fractionate at end of run to remove
Equilibrium or isopycnic gradient centrifugation
Solvent usually salt solution e.g. CsCl
Gradient forms on centrifugation
Simpler experimental setup
Molecules move up or down until point where it has the same density as the solvent.
Opposed by diffusion, therefore finite band width
Rate zonal vs isopycnic
Rate zonal: shallow gradient, time dependent and size separation
Isopycnic: steep gradient, sufficient time and density separation
The centrifuge apparatus needs to be _
Balanced
Swing out rotor
Low speeds, low rmax used in challenginf separations
Fixed angle centrifuge
High speed, also micro centrifuges
rmax short so more effective pelleting
Vertical centrifuge
Isopycnic, high/ultra speeds
No pellets formed, no harvesting
Relative centrifugal force (RCF)
Multiple of acceleration due to gravity (g-value)
Depends on speed, n (revs per minute)
And radius of rotation r, (mm)
There isn’t a constant RCF throughout the tube, so we use an average.
