Spotlight
Technical Considerations for Ultracentrifugation Centrifuges, Stirrers & Shakers
“Matching the correct rotor to each application type can be confusing, but in order to maximise experimental efficiency, it is important that this is performed as accurately as possible”
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 sub- cellular 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.
Format
As shown in Table 1, multiple tube formats are available to meet the complete range of applications conducted within various laboratories.
Table 1. Tube format and rotor compatibility Rotor type Tube format Thin wall open top Thick wall open top
Author Details: Nadia Boujtita PhD,
Thermo Fisher Scientific & Stephanie R. Noles, PhD, Thermo Fisher Scientific
Thin wall sealed Oak ridge style
Fixed angle
No Yes Yes Yes
Swinging bucket
Yes Yes
Some tube formats
No Vertical No No Yes No Banding or pelleting
Pelleting small volumes when high g-forces are required
For use with delicate samples
Popular choice as suitable for a wide range of applications
Materials
Various samples will have different properties and as such, the effect of the tube material must be taken into consideration. Tubes are commonly available in polyethylene (PE), polypropylene (PP), polyallomer (PA) thin wall or thick wall, and polycarbonate (PC). These materials need to be chemically resistant, transparent, thin, and flexible. However, a balance needs to be obtained between being thin and tolerant, since some of the tubes will need to be piercable for sample extraction, as well as resistant to high pressures and temperatures to prevent the occurrence of deformation. Various tube materials and their properties are detailed in Table 2 below.
Table 2: Centrifuge tube materials and properties Property Tube material PE PP
PA thin wall
PA thick wall
PC Clarity
Transparent/ translucent
Transparent Transparent Transparent Transparent Piercable Yes No Yes No No Sliceable No No Yes
No (yes for tubes Ø 5-13 mm)
No (yes for tubes Ø 5-13 mm)
ROTOR COMPATIBILITY
After tube or bottle selection, an appropriate rotor needs to be chosen and each rotor type has different operational characteristics at rest and at speed (Figure 1). Of the three standard classes of rotor (swinging bucket, fixed angle and vertical/near vertical) fixed angle and swinging bucket styles are predominantly used in ultracentrifugation applications. Rotors have traditionally been made of steel, titanium or aluminum, but more recently Thermo Fisher Scientific has introduced Fiberlite carbon rotors, providing a lightweight and versatile performance with a robust, corrosion-free design.
Autoclavable No Yes Yes Yes Yes
Chemical resistance
Good Good Good Good Good
Figure 1. Rotor Characteristics. At rest, tubes are held parallel or at a fixed angle relative to the axis of rotation. At speed, tubes may move positioning depending upon rotor type.
Application
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