Centrifugation Theory
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 a 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.
Because centrifuges come in all shapes and sizes, and the rotors vary, the universal and transferable unit of centrifugation is centrifugal force in gravities (xg). In lab write-ups you should report the centrifugation force used (#gravities) because it is the only transferable unit between different centrifuges.
Differential Centrifugation
The most common technique, 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.
Centrifuge Cautions:
These cautions presume you have had proper instruction in the use of the centrifuge OR have read the instruction manual 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.
2. Balance the load in the rotor - every tube must have a balance tube in the opposite slot with the same volume of fluid.
3. Make sure you are using the appropriate centrifuge tube for the job - they can rupture at too high a speed. You will need special 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.
Modified 9-30-08 gja
Department of Biology, Bates College, Lewiston, ME 04240