Centrifugation

• Electric motor
• Drive shaft
• Rotor to hold tubes

SEDIMENTATION of suspended and some dissolved particles occurs due to centrifugal force.

Two principal uses

1. Separate out solid matter as a PELLET from dissolved solutes as SUPERNATANT
2. Separate soluble macromolecules of different mass or density

Also sometimes used to provide centrifugal force to drive other processes, eg ultrafiltration.

Centrifuge Rotors

A. Fixed Angle Rotor

Advantage:	Sedimenting particles have only short distance to travel before pelleting. Shorter run time. The most widely used rotor type.

B. Swinging Bucket Rotor

Advantage:	Longer distance of travel may allow better separation eg in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet.

Principles of Centrifugation

Sedimenting force on particle
  = Mass x centrifugal field
  = mw²r

where w = angular velocity of rotor (radians/sec)
      r = radius (ie distance of particle from axis of rotation)

Relative Centrifugal Force (RCF)

RCF value reported as "No. x g" (ie multiples of earth's gravitational (force).

Equation used to calculate NOMOGRAMS (see eg Holme & Peck p 147) for quickly finding RCF at given speed and rotor type (radius).

Interacting Forces in Centrifugation

Sedimenting force, mw²r, is opposed by...

1. Flotation Force (Archimedes)
   = mw²rvr

   where: v = partial specific volume
         (volume displaced by 1g of sedimenting particles)
         r = density of solution

   NET SEDIMENTING FORCE on particle, after allowing for flotation
   

   = mw2r(1- vr)

2. Frictional Resistance
     against particle moving through fluid.
     = f.v
   where: f = frictional coefficient
           v = particle velocity
   
3. Diffusion
     - acting to counter uneven concentration distributions set up when dissolved molecules sediment.

BALANCE between the

SEDIMENTING FORCE and
COUNTERACTING FORCES

leads to various formulae and equations used in

• PREPARATIVE CENTRIFUGATION
  eg to calculate the time required to sediment a particle to the bottom of the tube

  and in

• ANALYTICAL ULTRACENTRIFUGATION
  techniques used to determine SEDIMENTATION COEFFICIENTS and MOLECULAR MASSES of dissolved macromolecules.

10.4 Density Gradient Centrifugation

In absence of a density gradient, separated bands of solute in the centrifuge are gravitationally unstable.

CAN'T OCCUR because layer of concentrated, dense solution overlaying less dense solvent would lead to mixing by convection and nullify the separation.
In absence of stabilising density gradient, can form boundaries (cf electrophoresis 9.3) but not zones. In analytical ultracentrifuge, moving boundaries and concentration distributions observed by optical device.

Create DENSITY GRADIENT in tube
  Use a non-interacting, low M.Wt solute in continuously increasing concentration from meniscus to bottom of tube.

Important technique for purifying proteins and particularly nucleic acids.

Two different types of density gradient centrifugation, for two different purposes are:

• Zonal (or Rate Zonal) Centrifugation
  (Sucrose density gradient centrifugation)
  
• Isopycnic Centrifugation
  (Caesium chloride density gradient centrifugation)

Zonal Centrifugation

Mixture to be separated is layered on top of a
SUCROSE, or FICOLL, GRADIENT
(increasing concentration down the tube)
- provides gravitational stability as different species move down tube at different rates forming separate bands.

Species are separated by differences in SEDIMENTATION COEFFICIENT (S)

S is increased for particle of LARGER MASS
(because sedimenting force a M(1-vr)

S is also increased for MORE COMPACT STRUCTURES of equal particle mass (frictional coefficient is less)

Mild, non-denaturing procedure, useful for protein purification, and for intact cells and organelles.

Isopyncic Centrifugation

Molecules separated on EQUILIBRIUM POSITION, NOT by RATES of sedimentation.
Each molecule floats or sinks to position where density equals density of CsCl solution. Then no net sedimenting force on molecules.
  Isopycnic = Equal density
and separation is on basis of DIFFERENT DENSITIES of the particles.

Very useful for purifying nucleic acid species of different density; also in separating proteoglycans extracted from cartilage.