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 (xg). In lab write-ups you should report the centrifugation force used (#gravities) because it is the transferable unit between different centrifuges.
http://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html
The centrifuge is one of the most powerful tools available to biochemists and biologists. It can be used for separation, purification and fractionation of materials, and for the study and physical characterization of macromolecules. Centrifugation of particles in a suspending medium causes the particles to sediment rapidly in the direction outward from the center of rotation. The centrifugal force generated by centrifugation is proportional to the speed of rotation and the radius of the rotor. At a fixed centrifugal force and medium viscosity, the sedimentation rate of the particle is proportional to the molecular weight of the particle and the difference between its density and the density of the medium. There are two types of centrifugation procedures: one is "preparative" which is used to isolate specific particles; the other is called "analytical" which is used to measure the physical properties of sedimentating particles.
DIFFERENTIAL CENTRIFUGATION
Differential centrifugation is a common separation procedure. In this method, a centrifuge tube filled with a uniform mixture of a sample is centrifuged at an appropriate speed and time. After centrifugation two fractions are obtained: the pellet containing sedimented materials and the supernatant containing unsedimented materials. These two fractions can then be recovered simply by decanting the supernatant from the pellet. The supernatant can then be recentrifuged at a higher centrifugal force to obtain further purification, with the formation of a new pellet and supernatant. The pellet can also be recentrifuged after suspension in a small volume of a suitable solvent. Purity of fractions can be increased further by repeating the process of differential centrifugation.
DENSITY GRADIENT CENTRIFUGATION
Density gradient centrifugation is a popular method for fractionation of nucleic acids, virus particles and proteins. This is done by centrifugation of a mixture of particles or components in a density gradient column. Particles or components with different densities will be separated at different positions in the density gradient column. Basically, there are two types of density gradient centrifugation, termed rate zonal and isopycnic.
Preparation of density gradients. In either zonal or isopycnic density gradient centrifugation, a density gradient has to be prepared prior to centrifugation by either a hand-layering process or by employing a density gradient former. A number of materials such as sucrose, Ficoll, or salts such as NaCl, NaBr, or CsCl, can be used for preparation of the density gradient. A sucrose density gradient can be prepared by pipetting into a centrifuge tube layers of progressively lower concentrations of sucrose on top of higher concentrations. Density gradient columns can also be prepared by the use of a syringe with a piece of tubing attached to the syringe needle (20-22 gauge). To prepare a 5-20% sucrose density gradient in a 15 ml tube, start by placing 3 ml of 5% sucrose in the tube and then carefully inject the 3 ml of 10% sucrose into the tube by keeping the tip of the syringe tubing at the bottom of the centrifuge tube. Repeat the process with 3 ml of 15% and 3 ml of 20% sucrose. When the preparation is completed, remove the syringe tubing carefully by holding the tip of tubing against the wall of the centrifuge tube. Ficoll and cesium chloride density gradients can be prepared in a similar manner. Density gradients thus prepared can either be used immediately as a step gradient or made into a linear gradient by allowing it to diffuse in a refrigerator overnight.
Rate Zonal Density Gradient Centrifugation. In rate zonal density gradient centrifugation, a sample solution containing particles to be fractionated is layered on top of the density gradient column. Under centrifugation the particles will start to sediment through the density gradient into separate zones. Each zone consists of particles with the same sedimentation rate. In the rate zonal centrifugation, centrifugation must be terminated before any of the separated zones reach the bottom of the tube, since the density of some zones may be higher than the highest density area in the density gradient.
Isopycnic (="same density") density gradient centrifugation. In isopycnic density gradient centrifugation, the density gradient column encompasses the whole range of densities of sample particles. Each particle will sediment only to the position in the gradient where the density in the gradient column equals its own density, and the particle will remain at this position. In the isopycnic method, it is not always convenient to form a gradient artificially and layer the sample on top of the gradient column. It is sometimes necessary to start with a uniformly-mixed solution of gradient material and sample. During centrifugation, gradient material redistributes in the tube and forms a linear density gradient. At the same time, sample particles which are initially distributed throughout the tube either sediment or float to their isopycnic positions. This type of procedure is termed the self-generating gradient technique.
Historically, self-generating isopycnic density gradient centrifugation have generally required long hours of centrifugation. For example, isopycnic "banding" of DNA can take 36 - 48 hours in a self-generating cesium chloride density gradient using standard swinging-bucket or fixed-angle ultracentrifuge rotors. The running time can not be shortened by increasing the rotor speed, since this only results in changing the positions of zones in the tube due to the redistribution of gradient material further down the tube. Run times can be decreased by shortening the distance over which the gradient forms, however. A recent innovation to decrease running times for DNA preparation (down to 3-4 hours) has been the use of "vertical" or "near-vertical" rotors, in which the gradient forms across the diameter, rather than the length of the tube.
Separated zones ("bands") from both rate zonal and isopycnic density gradient centrifugation can be removed by: (i) puncturing a hole on the bottom of the tube and collecting the fractions or drops either manually or using a fraction collector, (ii) removing successive zones from the top of the unpunctured tube, or (iii) pucturing the tube through the side to recover a band as a single fraction.
Application of Density Gradient Centrifugation. Density gradient centrifugation has been used extensively in separation and purification of a wide variety of biological materials. It is particularly well suited for the study of viruses and nucleic acids. Cells and subcellular components such as bacteria, nucleoids, ribosomes, membranes, etc. have been isolated and purified with this technique. Separation and sedimentation coefficients of macromolecules can also be measured through density gradient centrifugation by comparison with another similar macromolecule of known sedimentation coefficient. Several proteins and nucleic acids are available commercially for this purpose. Both known and unknown samples are sedimented in the same centrifuge tube and the sedimentation coefficient is calculated as follows:
http://microvet.arizona.edu/Courses/MIC328/Waynes World/AppendixE_centrif.html
