Biotechniques 410 Centrifugation
Centrifugation The use of centrifugal forces Inertial force Angular momentum Reaction to centripetal force Rotation around a central axis creates higher force away from axis
Terms Mass – the amount of matter (atoms) an object has Volume – the amount of space an object takes up Density – the ration of mass to volume How much mass in a set amount of space Weight – the force of gravity on an object Greater mass = greater weight
Stone on a string The further from the axis the greater the force on the object Objects with equal mass have a greater weight the further they rotate from the axis
Gravity Gravitational forces are predicated on mass Greater mass the grater the attractive force of gravity
Gravitational Force Force = mass (m) * acceleration (a) Gravitational force (g) g = 9.80665 m/s2 on earth g = 16 psi pressure Acceleration agaisnt gravity requires forces greater than g
Centrifugation Using gravity we can observe the effects of angular momentum on an object Heavier objects Those with more mass per given volume Experience greater force
Centrifugation NASA’s Human Centrifuge For Training pilots and astronauts to prepare for flight with high angular momentum
Centrifugation We can use angular momentum, and density to separate material Density dependent Gravitation forces (G) Proportion of earths gravitation force, exerted on an object
History 1923 Theodore Svedberg developed the first high speed centrifuge to separate large grain sols (sediment particles) To separate out trace amounts of gold efficiently Centrifuges quickly became a laboratory tool Cellular organelle identification in 1929 Used to identify subunits of Hemoglobin in 1930’s
Parts of the Cell Cells Organelles vary in size/density Use centrifugation for identification Isolation and individual observation
Cell Fractionation
Cell Fractionation The larger the organelle form pellets at slower speed centrifugation
Two methods for centrifugation Differential Multiple steps Sample run at increasingly higher speeds Pellet or supernatant removed at each step Density gradient Sucrose of other solution provided in a gradient Sample passed through the gradient Separate based on relative densoity
Differential Centrifugation
Differential Centrifugation After each spin in the centrifuge the pellet is removed Removing the densest material So sample must be run at higher speed and for longer to isolate remaining materials
Density Gradient Suspension gradient Solution poured with different gradient of dissolved materials (e.g. sucrose) Sample is then spun in the centrifuge Gradient does not mix, because of density of solute
Density Gradient Sample will settle amongst he gradient, based on density of molecules Can be used in conjunction with differential fractionation
Blood centrifugation Common method for blood prep Transfusion of specific blood parts Clinical diagnostics Research on blood-born disease and medicine
Centrifuges High-speed centrifuge Ultracentrifuge Minicentrifuge Benchtop or Free-standing Max speed: ~10,000 RPM Ultracentrifuge Free-standing Max speed: >30,000 RPM Minicentrifuge Table top
Parts of the Centrifuge
Rotor Styles Bucket (swinging) Fixed Angle
Measuring Force Relative centrifugal force (RCF) RPM (Revolutions per minute) can be used to determine the amount of force on a sample. Relative centrifugal force (RCF) RCF = g R = distance from axis to end of the sample tube
Loading Samples Samples must be loaded properly Unequal weight will cause the rotor to spin lopsided Damage the centrifuge Create a dangerous situation
Loading Samples E.g. 10g * 10,000 rpm 1667 g/s2 force Samples of equal mass must be loaded on opposite sides of the rotor If an uneaven number of samples use a blank opposite the odd sample
Today’s Lab Lyse spinach cells Using differential centrifugation Use sand to help homogenize cells Using differential centrifugation 3 spins, 3 pellets We will examine the contents of each under the microscope Use the table top centrifuge to re-pellet part of a sample Use vital stains to observe samples