Centrifugation
is a process used to separate or concentrate materials suspended
in a liquid medium. The theoretical basis of this technique is
the effect of gravity on particles (including macromolecules)
in suspension. Two particles of different masses will settle
in a tube at different rates in response to gravity. Centrifugal
force (measured as xg, gravity) is used to increase this settling
rate in an instrument called a centrifuge. Two common examples
of the use of centrifugal force are: (1) When you do the "around
the world" trick with a yo-yo, it is centrifugal force that
makes the yo-yo body stay at the end of the string as you rotate
it; and (2) When you wash clothes in a washing machine, it is
centrifugal force generated in the "spin" cycle that
forces water out of the fabric to facilitate faster drying.
Centrifuges are devices used in a variety
of scientific and technical applications which spin carrier vessels
(centrifuge tubes) at high rotation speeds and very high centrifugal
force. The centrifugal force (expressed as # gravities
or, # xg) generated is proportional to the rotation rate
of the rotor (in rpm) and the distance between the rotor center
and the centrifuge tube. Therefore, a given centrifuge may use
multiple rotor sizes to give flexibilty in choosing centrifugation
conditions. Each centrifuge has a special graph, a nomograph,
or a table which relates rotation rate (rpm) to centrifugal force
(xg) for each size of rotor it accepts.
Typically, the material to be "spun"
is placed in a centrifuge tube which is then placed in a rotor.
The rotor is generally a dense metal which dissipates heat quickly,
and is of sufficient mass that it generates momentum, i.e., once
its spinning it requires little energy to keep it going. Centrifuges
generally work under vacuum and are refrigerated to reduce heating
caused by frictional forces as the rotor spins. Rotors are usually
stored in refrigeration units to keep them at or near the operating
temperature.
Centrifuges come in all shapes and sizes,
and the rotors vary, therefore, the universal and transferable
unit of centrifugation is centrifugal force in gravities (xg).
Different makes of centrifuges use different rotors and each
model comes with a table or a graph that relates centrifugal
force to rotational speed (rpm) for each rotor (or swing buckets)
it can use. In lab write-ups you should ALWAYS report the centrifugal
force used (#gravities) and duration of time at that force because
centrifugal force is the only transferable unit between different
centrifuges.
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Differential Centrifugation
A commonly used technique for cell fractionation,
called differential centrifugation, is used to separate
particles from a liquid medium or to separate particles of different
masses into separate fractions of the supernatant. We will use
this technique in a several ways in this course.
1. In the Bio 242 Amylase lab we will
use centrifugation to pellet the cellular debris and excess starch
during the enzyme extract preparation. The enzyme, which is soluble,
will remain in the supernatant. During the actual experiment,
we will use centrifugation to separate the enzyme (soluble) from
its substrate (insoluble amylose-azure) to stop the reaction.
2. In the molecular labs we will use centrifugation
to promote a chemical reaction by forcing small quantities of
reactants together in the bottom of microcentrifuge tubes. We
will also use centrifugation to prepare bacterial cells for transformation
by alternately pelleting them and then resuspending them with
different chemical solutions.
3. In the Hill Reaction lab we will use
a multi-step differential centrifugation (Fig. 9-3) to isolate
cell organelles (chloroplasts) from crude cellular homogenate.
Because the organelles have much less mass than the cell wall
components, the first pellet that forms at low centrifugal force
is primarily cellular debris. The organelle fraction is then
pelleted at higher centrifugal force.
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Centrifuge Cautions:
These cautions presume you have had proper
instruction in the use of the centrifuge AND have read the instructions
for using the instrument thoroughly.
1. Make sure the correct rotor is being
used and that it is installed properly on the spindle. Make sure
the rotor is secured before starting a run. On the prep centrifuges
the rotor cap screws onto the spindle.
2. Balance the load in the rotor -
every tube must have a balance tube in the opposite slot
with the same volume of fluid. Imbalanced rotors can damage or
destroy the machine, and, in some instances kill people.
3. Make sure you are using the appropriate
centrifuge tube for the job - they can rupture at too high a
speed. You may need special, high density tubes for high force
centrifugation.
4. Pre-cool the centrifuge and the rotor
before use. Rotors should be stored in a refrigerator when possible.
5. DO NOT attempt to override any safety
features of the centrifuge.
6. NEVER leave the centrifuge unattended
until it reaches maximum speed and is going smoothly.
7. When in doubt, ASK FOR HELP.
