Ultracentrifugation

Essentially, centrifuges used in laboratory settings or in the industry are devices for separating particles from a heterogeneous mixture. Fundamental to separation is the increase in the effective gravitational force by spinning the sample in the single cavity or the tubes of a rotor as the ‘mobile part’ of a centrifuge. Subjected to the respective centrifugal force thus applied, the individual constituents of a sample will differentially sediment according to their physical properties as well as the density and viscosity of the sample solution.

Ultracentrifuges are optimised for spinning a rotor at very high speeds generating centrifugal forces as high as 1 000 000g. There are two kinds of ultracentrifuges, preparative and analytical ultracentrifuges. Preparative ultracentrifuges are used in biology for the fractionation of fine particulate samples such as tissue homogenates aiming to isolate subcellular organelles, macromolecules, bacteria or viruses. By means of analytical centrifuges, the physico-chemical properties of a sedimenting particle or molecular interactions of macromolecules and their possible subunits, respectively, can be unravelled.

Key Concepts:

  • Centrifugation, in particular high-speed (ultra)centrifugation, is a widely used technique to elucidate fundamental processes such as adenosine triphosphate (ATP) synthesis in mitochondria, the synthesis of proteins by ribosomes or the interactions of intracellular multiprotein complexes.
  • Ultracentrifugation is basically carried out in two ways: preparative and analytical centrifugation.
  • The former aims to isolate and purify, for example, subcellular organelles or multiprotein complexes; the latter allows to analyse interactions between macromolecules and to unravel physico-chemical properties like mass and size of such particles.
  • Preparative centrifugation performed as batch-type (conventional) centrifugation is mostly used to separate and enrich organelles out of complex biological mixtures.
  • The alternative, continuous-flow centrifugation, is particularly useful for the large-scale collection of particles out of a diluted solution as it combines high centrifugal force with high throughput.
  • Theoretically, two kinds of preparative centrifugation have to be distinguished: differential centrifugation and density gradient centrifugation.
  • Differential centrifugation fractionates organelles of a tissue homogenate according to their size and shape, yet leads only to an enriched rather than a highly purified preparation of a particular organelle.
  • To get a preparation genuinely purified, common contaminants have to be subsequently removed by density gradient centrifugation.
  • Rate zonal and isopycnic density gradient centrifugations differ in their basic concepts and the types of density gradients used.

Keywords: cell fractionation; ultracentrifuge; rotor; density gradient; preparative and analytical centrifugation; conventional and continuous-flow centrifugation

Figure 1. Differential pelleting.
Figure 2. Flow chart summarising the sequential steps in the subfractionation of a homogenate by differential centrifugation.
Figure 3. The density of a gradient may be increased continuously or discontinuously from top to bottom of the centrifuge tube.
Figure 4. Modes of density gradient centrifugation: (upper) rate zonal centrifugation and (lower) isopycnic centrifugation.
Figure 5. Photograph of an actual tissue separation: subfractionation of a rat hepatic light mitochondrial fraction by rate zonal centrifugation using a vertical rotor.
Figure 6. Buoyancy (B), friction (F) and centrifugal (C) forces acting on a particle during centrifugation.
Figure 7. A vertical tube rotor.
Figure 8. A fixed-angle rotor.
Figure 9. A swing-out rotor.
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 References
    Anderson NG (1966) Zonal centrifuges and other separation systems. Science (New York) 154: 103–112.
    book Birnie GD (ed.) (1972) Subcellular Components: Preparation and Fractionation, 2nd edn. London: Butterworths.
    Claude A (1946) Fractionation of mammalian liver cells by differential centrifugation. 1: Problems, methods, and preparation of extract. Journal of Experimental Medicine 84: 51–59.
    De Duve C (1971) Tissue fractionation. Past and present. Journal of Cell Biology 50: 20D–55D.
    De Duve C (1975) Exploring cells with a centrifuge. Science (Washington, DC) 189: 186–194.
    Drahos KL, Tran HC, Kiri AN et al. (2005) Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles. Journal of Biomolecular Techniques 16: 347–355.
    book Hinton RH, Mullock BM and Gilhuus-Moe CC (1974) In: Reid E (ed.) Methodological Developments in Biochemistry, vol. 4, p. 103. London: Longmans.
    Kiri AN, Tran HC, Drahos KL et al. (2005) Proteomic changes in bovine heart mitochondria with age: using a novel technique for separation and enrichment. Journal of Biomolecular Techniques 16: 371–379.
    Miescher F (1971) Über die Zusammensetzung der Eiterzellen. Medizinisch-chemische Untersuchungen 4: 441–460.
    Sanchez-Lopez V, Fernandez-Romero JM and Gomez-Hens A (2009) Evaluation of liposome populations using a sucrose density gradient centrifugation approach coupled to a continuous flow system. Analytica Chimica Acta 645: 79–85.
    Schumaker VN (1967) Zone centrifugation. Advances in Biological and Medical Physics 11: 245–339.
    book Svedberg T and Pedersen KO (1940) The Ultracentrifuge. Oxford: Clarendon Press.
 Further Reading
    book Birnie GD and Rickwood D (eds) (1978) Centrifugal Separations in Molecular and Cell Biology. London: Butterworths.
    ePath Dorin M and Cummings J (2004) Principles of Continuous Flow Centrifugation. Technical Information, Beckman Coulter. www.Beckman.com/literature/Bioresearch/T-1780B
    book Price CA (ed.) (1982) Centrifugation in Density Gradients. New York: Academic Press.
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How to Cite close
Völkl, Alfred(Jun 2010) Ultracentrifugation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002969.pub2]