The document discusses centrifugation techniques used in biochemical research. It defines centrifugal force and relative centrifugal force (RCF). It states that to centrifuge a sample at 100,000g with an average radius of 4cm would require setting the centrifuge to over 25,000rpm. Different types of centrifuges are used for separating particles of various sizes. Centrifugation is used for preparative and analytical techniques like sedimentation analysis to determine properties of particles like sedimentation coefficients.

1. Centrifugation in biochemical research
The centrifugal force is defined as
F = m 2r
F = intensity of the centrifugal force
m = effective mass of the sedimenting
particle
 = angular velocity of rotation in rad/sec
r = distance of the migrating particles from
the central axis of rotation

2. Relative centrifugal force (in g units):
RCF = (1.119 x 105)(rpm)2(r)

4. Problem
You wish to centrifuge a biological sample so that
it experiences an RCF of 100,000 g. At what rpm
must you set the centrifuge assuming an average
r value of 4.

5. Instrumentation
Low speed centrifuges (maximum speed in the
range of 4,000 – 6,000 rpm or RCF up to 6,000 g).
-Rapid sedimentation of coarse precipitates or cells
High-speed centrifuges (speed 1,000 – 25,000 rpm
or 50,000 g)
-Pelleting of cells, nuclei, organelles
Ultracentrifuges (20 – 80,000 rpm or 600,000 g)
-Pelleting of ribosomes and macromolecules

6. Applications of centrifugation
Preparative techniques:
Velocity sedimentation centrifugation separates
particles such as coarse precipitates from the bulk
of the solution
Differential centrifugation consists of successive
centrifugations at increasing rotor speeds

7. Typical sedimentation
rates of subcellular
components

9. Applications of centrifugation
Analytical centrifugation involves sedimentation of
particles in a medium of homogeneous density or
density gradient to measure the sedimentation
coefficient of a particle
= centrifugal force – buoyant force
m is the mass of the particle,  is the angular velocity, Vp is the
particle volume, and  is the density of the solution.
motion of the particle through the solution is opposed
by the frictional force: Ffriction = vf, where v = dr/dt and
f is the frictional coefficient.

10. Under the influence of the centrifugal force the particle
accelerates until the forces on it balance:
The particle mass m is equal to the molar mass (g/mol)
divided by Avagadro’s number (NA):

11. where is the partial specific volume, which is the
is the volume change when 1 g (dry weight) of particles
is dissolved in an infinite volume of the solute. It has
units of cm3/g and for most proteins the value is
0.73 cm3/g.
define:
The sedimentation coefficient is usually expressed in
units of 1013 s, which are known as Svedbergs (S).

13. Prior to 1970 most molecular mass determinations
of macromolecules were made via analytical
ultracentrifugation techniques. This method of
estimating molar mass has largely been replaced
by simpler methods such as?
The technique still enjoys application to help
researchers answer questions about macromolecular
association and for purification of nucleic acids in CsCl
gradients.

14. Use of density gradients greatly enhances resolving
power, which leads to two approaches:
1) zonal centrifugation – macromolecular solution is
layered on top of a preformed density gradient.
2) isopycnic or equilibrium density – spinning of
sample in a concentrated solution of a dense, fast-diffusing
substance (e.g., CsCl) causes the substance
to form a steep gradient such that sample molecules
sediment to positions where their density equals that
of the surrounding portion of the solution.
-famous example: The Meselson-Stahl Expt.

17. The Meselson-Stahl
Experiment a.k.a. “The
Most Beautiful Experiment”