Centrifugation

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Index

• Introduction
• Problems

Introduction and Theory

Centrifugation is a method for separating particles from each other in a solution. In biology these particles are usually cells, subcellular organelles or large molecules. The basic principles of sedimentation originate from Stoke's Law which describes the settling of a sphere in a gravitational field.

                  d^2 (pd - ld)          v = -------------  x g                  18(lv)where v = sedimentation rate or velocity of the sphere      d = diameter of the sphere     pd = particle density     ld = liquid density     lv = viscosity of liquid medium      g = gravitational force

From this arrangement of the Stoke's Law equation, it can be seen that:

• The sedimentation velocity is proportional to the square of the sphere size.
• The sedimentation velocity is proportional to the difference between the particle and liquid density and is zero when these two densities are equal.
• The sedimentation velocity decreases as the viscosity of the liquid medium increases.
• The sedimentation velocity increases as the centrifugal force increases.

Differential Centrifugation

The most common centrifugation method involves fractionation of particles differentially between a pellet and supernatant.

The pellet is a mixture of all sedimenting components while the supernatant contains purified components of only the slowest sedimenting components. This works well only for initial separations of particles with very different sedimention velocities. As can be seen at the right, the largest particles will sediment first leaving a mixture of the slower sedimenting species. Notice that yield of the lighter species is not very high since many of these particles are being trapped in the pellet.

An analytical application of this methods looks at the zone of clearing for each sedimenting species. For example, as each species sediments there will be a boundary above which there will not be any molecules of that species. Using this moving boundary methods, each sedimenting species can be monitored with respect to sedimention velocity and concentration.

Zone Velocity Centrifugation

A method that results in yields greater than differential centrifugation and allows greater resolution of all particles sizes is zone velocity centrifugation. A sample is layered on top of a shallow density gradient and then centrifuged. Each particle size will migrate as a zone or band at a characteristic velocity. If the velocities of the particles are sufficiently different then the zones of the particles will resolve. The gradient through which the particles are centrifuged is used to stablilze the zones during recovery and helps prevent mixing of resolved zones. The density of the gradient material should be less than the components being sedimented.

Gradients

There are two types of gradients used in centrifugation, the preformed and self-forming gradients.

The preformed gradient has a high density at the bottom of the centrifuge tube and a low density at the top. Such gradients are usually made with sucrose or other inert substrate. Samples are usually layered on top of these gradients.
The shapes of preformed gradients can be of several types as shown at the right. Linear gradients of sucrose have not only an increasing density but also an increasing viscosity (stickiness) from the top to bottom of the tube. To compensate for the increased viscosity, the shape of the gradient can be made similar to the upper right panel so that at the bottom of the tube the change in density of the gradient decreases as the viscosity increases. This is called an isokinetic gradient because particle sedimentation responds as if it were encountering a linearly increasing gradient. The other shapes have special purposes.

The self-forming gradient is generated during the centrifugation process and is usually composed of a high molecular weight salt such as CSCl. These salts have sufficient mass to be concentrated toward the bottom of the centrifuge tube but are also small enough such that diffusion against the centrifugal force causes these molecules to be distributed through out the tube. The further these particles are from the center of rotation the more concentrated they become, therefore forming a continuous, linearly increasing density gradient from the top to bottom of the tube.

Problems

Unknown A was found to have a minimal molecular weight of 100 amu.

IR spectroscopy gave the following scan:

The C13 NMR gave the following scan:

The proton NMR gave the following scan:

After you have determined the structure, send the answer to C.A. Rinehart

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Last Modified: Oct. 18 1995
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