1. Biotechniques 410
Centrifugation

2. 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

3. 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

4. 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

6. Gravity Gravitational forces are predicated on mass
Greater mass the grater the attractive force of gravity

7. Gravitational Force Force = mass (m) * acceleration (a)
Gravitational force (g)
g = m/s2 on earth
g = 16 psi pressure
Acceleration agaisnt gravity requires forces greater than g

8. 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

10. Centrifugation NASA’s Human Centrifuge
For Training pilots and astronauts to prepare for flight with high angular momentum

11. 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

12. 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

13. Parts of the Cell Cells Organelles vary in size/density
Use centrifugation for identification
Isolation and individual observation

14. Cell Fractionation

15. Cell Fractionation
The larger the organelle form pellets at slower speed centrifugation

16. 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

17. Differential Centrifugation

18. 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

19. 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

20. Density Gradient
Sample will settle amongst he gradient, based on density of molecules
Can be used in conjunction with differential fractionation

21. Blood centrifugation Common method for blood prep
Transfusion of specific blood parts
Clinical diagnostics
Research on blood-born disease and medicine

23. 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

24. Parts of the Centrifuge

25. Rotor Styles
Bucket (swinging)
Fixed Angle

26. 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

27. Loading Samples Samples must be loaded properly
Unequal weight will cause the rotor to spin lopsided
Damage the centrifuge
Create a dangerous situation

28. 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

30. 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