Technical Considerations for Ultracentrifugation

Initially developed in the 1920’s, ultracentrifuges have the ability to generate forces thousands or millions of times stronger than the force of gravity. Further development of this class of devices permitted the fractionation of subcellular components which were previously visible only through the use of an electron microscope. As a result, modern ultracentrifugation can be used to determine the shape, size and weight of macromolecular complexes.

Extremely high speeds can be obtained through the combination of specialised rotors, tubes and bottles. When spun in micro-volumes, particles can be separated at 150,000 rpm or in excess of 1,000,000 x g. This high-speed separation capability has made ultracentrifugation an ideal technique for applications such as cell biology (sub-cellular fractionation), proteomics (protein and lipoprotein purification and fractionation), genomics (RNA and DNA purification), microbiology (pelleting, virus purification/concentration), and nanotechnology (purification and separation of nanoparticles). The size and density of the material to be separated forms

the basis behind the theory of ultracentrifugation. Depending upon the application in question, differential or density gradient centrifugation should be used. Differential centrifugation enables the successive pelleting of particles, which decrease in sedimentation velocities. As a result, denser components pellet at the bottom of the tube and less dense components will remain in suspension. Density gradient centrifugation causes components to come to rest at points in the tube at which they are in density equilibrium with the surrounding solvent, and can be subdivided into: rate-zonal (separation based on molecular weight) and isopycnic (an equilibrium separation).

TUBE VARIATIONS

Due to the large number of variables associated with ultracentrifugation, each selection directly impacts on the next. As such, when choosing tubes and bottles, it is important to consider rotor and chemical compatibility.

Size and volume

The sample volume to be processed will naturally impact on the tube size required and in order to maximise efficiency, the tube must be compatible with the rotor type.

Tube volumes are generally referred to as either nominal or fill: the nominal volume is the maximum amount of liquid that may fit into the rotor cavity and the fill volume is the volume that manufacturers recommend the tube or bottle should hold. If not filled correctly, the centrifugal forces can adversely affect the tube material, causing it to warp and bend, resulting in a loss of volume.