Centrifuge, Ultracentrifuge Methods and Theory
Centrifugation of Cell Organelles, Macromolecules and Silt and Clay
Sep 13, 2009
Centrifugation and ultracentrifugation are important research procedures. Molecules, organelles and cells, when suspended in appropriate solutions, may be spun, separated and isolated from one another. Centrifugation is useful, especially when combined with light and electron microscopy, and biochemical studies. Enter this spinning world now.
Centrifuges, What They are, What They Do and Stoke's Law
Centrifuges are devices, or research tools, which enable biologists, chemists and geologists, especially, to spin and separate materials into defined physical layers. Stoke's Law applies to the materials in centrifuge tube solutions or suspensions. Stoke's Law relates the velocity of the rise (or fall) of a particle in water is proportional to the particle's:
- specific gravity minus 1,
- diameter,
- and the temperature of the suspending solution.
As the specific gravity of the particle approaches that of water (usually 1.0), the velocity of the particle approaches 0 and, therefore, the particle moves neither up or down.
Molecular Behavior in Solutions and Suspension
Dense molecules settle fastest, based on gravitational force. Smaller and less dense molecules, organelles, particles, stay suspended longer. The smallest particles may be almost permanently suspended — water molecules constantly bombard and transfer their kinetic energies to other molecules and promote suspension. Characteristically, particles about 0.5 micrometers or smaller stay suspended. These particles display Brownian motion — an erratic, irregular movement caused by random collisions of water molecules with the suspended materials.
Simple and Complex Centrifuge Methods, Differential and Gradient Centrifuges
When suspensions of intact plant, animal or bacterial cells are centrifuged at 200-300 revolutions/minute (RPMs) equal to about 1,000 g ("g" relates to force value as in centrifugation and flight situations) for 5 minutes, the cells sediment as a pellet at the bottom of the centrifuge tube (see plant pellet photo below).
Ultracentrifugation can separate subcellular components of lysed cells. Here, scientists do progressive differential centrifugations at different g forces to spin down the different cell organelles:
- ~1000 g, sediments nuclei, heavy mitochondria, and plasma membrane sheet.
- ~5000 g, sediments light mitochondria, lysosomes, peroxisomes, and intact Golgi.
- ~100,000 g, yields Golgi, endoplasmic reticulum (ER), microsomes, and endosomes.(adapted from Kiri et al., J. Biomol. Tech. 2005 December; 16(4): 371–379)
Gradient densities of sucrose, nycodenz, percoll or Ficoll 400 and long spin times are used for density gradient ultracentrifugations whereupon denser particles (with higher S.G) settle closer to the tube's bottom, and less dense particles float higher in the tube. Spinning occurs at forces of 10,000 g or >, and at the end of 12-20 hr the tube, there are distinct, different layers of cell materials or particles.
A Useful Method and Protocol for Silt and Clay Separations in Geology
In geology, separations of silt and clay are done for X-ray powder diffraction analyses. The entire sample may be recaptured after treatment with acids or oxidizing agents.
A Unisoft C program (based on Stoke's Law) can determine centrifuge run times and speeds for particles of various diameters and densities suspended in media of different temperatures, specific gravities, and viscosities (Poppe et al., U.S. Geological Survey Open-file Report 00-358).
The above program works for any centrifuge and is fast, interactive, and allows the user to select all particle and medium parameters.
The Stoke's Law program provides for:
- initial R1 and R2 distances from axis of rotation ( click photo/figure below).
- viscosity of the medium.
- temperature of medium (affects viscosity).
- angular velocity (RPMs). Non-spherical particles (e.g. phyllosilicates) lack true radii, but may be adjusted to an equivalent radius spherical particle of the same material. Centrifuge operators also must ensure the proper unit measurements and inputs required by the program as well as the initial (R1) and final (R2) distances from the axis of rotation. Because of mechanical differences in the designs of individual centrifuges, operators must measure these distances on the specific centrifuge used.
Sources
U.S. Geological Survey Open File Report 01-041. Separation of the silt and clay fractions for X-ray powder diffraction.
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