centrifugation lecture

Centrifugation in biochemical research

The centrifugal force is defined as F = mω2rF = intensity of the centrifugal forcem = effective mass of the sedimenting particleω = angular velocity of rotation in rad/secr = distance of the migrating particles from the central axis of rotation

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

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.

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

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

Applications of centrifugation

Typical sedimentation rates of subcellular components

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

motion of the particle through the solution is opposed by the frictional force: Ffriction = vf, where v = dr/dt andf is the frictional coefficient.

m is the mass of the particle, ω is the angular velocity, Vp is theparticle volume, and ρ is the density of the solution.

Under the influence of the centrifugal force the particleaccelerates until the forces on it balance:

The particle mass m is equal to the molar mass (g/mol)divided by Avagadro’s number (NA):

where is the partial specific volume, which is theis the volume change when 1 g (dry weight) of particlesis dissolved in an infinite volume of the solute. It has units of cm3/g and for most proteins the value is0.73 cm3/g.

define:

The sedimentation coefficient is usually expressed inunits of 10−13 s, which are known as Svedbergs (S).

Prior to 1970 most molecular mass determinationsof 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 This method of estimating molar mass has largely been replaced by simpler methods such as?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 macromolecularassociation and for purification of nucleic acids in CsClgradients.

Use of density gradients greatly enhances resolving power, which leads to two approaches:

1) zonal centrifugation – macromolecular solution islayered 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 substanceto form a steep gradient such that sample moleculessediment to positions where their density equals that of the surrounding portion of the solution.

-famous example: The Meselson-Stahl Expt.

The Meselson-StahlExperiment a.k.a. “TheMost Beautiful Experiment”