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Centrifugation

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Index
• Introduction



• Problems
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. Problems
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Introduction and Theory
Centrifugation is a method for separating particles from each other in a solutio
n. In biology these particles are usually cells, subcellular organelles or large
molecules. The basic principles of sedimentation originate from Stoke's Law whi
ch 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 = viscosi
ty 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 s
ize.
• 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 medi
um increases.
• The sedimentation velocity increases as the centrifugal force increases.
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Differential Centrifugation
The most common centrifugation method involves fractionation of particles differ
entially between a pellet and supernatant.
The pellet is a mixture of all sedimenting components while the supernatant cont
ains purified components of only the slowest sedimenting components. This works
well only for initial separations of particles with very different sedimention v
elocities. As can be seen at the right, the largest particles will sediment firs
t 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 bou
ndary above which there will not be any molecules of that species. Using this mo
ving boundary methods, each sedimenting species can be monitored with respect to
sedimention velocity and concentration.
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Zone Velocity Centrifugation
A method that results in yields greater than differential centrifugation and all
ows greater resolution of all particles sizes is zone velocity centrifugation. A
sample is layered on top of a shallow density gradient and then centrifuged. Ea
ch 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 th
e particles will resolve. The gradient through which the particles are centrifug
ed is used to stablilze the zones during recovery and helps prevent mixing of re
solved zones. The density of the gradient material should be less than the compo
nents being sedimented.
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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 a
nd a low density at the top. Such gradients are usually made with sucrose or oth
er 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 inc
reasing 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 t
he 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 li
nearly 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 t
hese molecules to be distributed through out the tube. The further these particl
es are from the center of rotation the more concentrated they become, therefore
forming a continuous, linearly increasing density gradient from the top to botto
m 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
Biological Instrumentation

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Course Index
Instructor: Dr. Claire A. Rinehart
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• Centrifugation
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Exam 1 F'97

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