Ultracentrifugation

Ultracentrifugation is a specialized technique used to spin samples at exceptionally high speeds. Ultracentrifuges: Operate at speed of 75,000rpm, providing the centrifugal force of 500,000g. Rotor chamber is sealed and evacuated by pump to attain vacuum. Refrigeration system (temp 0-4 degree C). Rotor chamber is always enclosed in a heavy armor plate. Centrifugation for isolation and purification of components is known as preparatory centrifugation, while that carried out with a desire for characterization is known as analytical centrifugation. The denser a biological structure is, the faster it sediments The more massive a biological particle is, the faster it moves The denser the biological buffer system is, the slower the particle moves The greater the frictional coefficient the slower a particle moves The greater the centrifugal force is, the faster the particle sediments The sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium is equal. Analytical Ultracentrifuge Uses small sample size (less than 1 ml). Built in optical system to analyze progress of molecules during centrifugation. Uses relatively pure sample. Used to precisely determine sedimentation coefficient and MW of molecules. Beckman Model E is an example of centrifuge used for these purposes. Preparative Ultracentrifuge Larger sample size can be used. No optical read-out collect fractions and analyze them after the run. Less pure sample can be used. Can be used to estimate sedimentation coefficient and MW, used to separate organelles and molecules. Commonly used Types of Ultracentrifugation A. Analytical B. Preparative Applications In clinical laboratory, centrifuge is used to; Remove cellular elements from blood to provide plasma or serum analysis. Remove chemically precipitated protein from an analytical specimen. Separate protein bound from free ligand in immunochemical assays etc.. Separation of the sub-cellular organelle, DNA, RNA. Extract solutes in biological fluids from aqueous to organic solvents. Separate lipid components.