Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.
The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or g. The particles' settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity.
In the chemical and food industries, special centrifuges can process a continuous stream of particle-laden liquid.
It is worth noting that centrifugation is the most common method used for uranium enrichment, relying on the slight mass difference between atoms of U238 and U235 in uranium hexafluoride gas.
Centrifugation in Biotechnology
Microcentrifuges and Superspeed Centrifuges
In microcentrifugation, centrifuges are run in batch to isolate small volumes of biological molecules or cells (prokaryotic and eukaryotic). Nuclei is also often purified via microcentrifugation. Microcentrifuge tubes generally hold 1.5-2 mL of liquid, and are spun at maximum angular speeds of 12000-13000 rpms. Microcentrifuges are small and have rotors that can quickly change speeds. Superspeed centrifuges work similarly to microcentrifuges, but are conducted via larger scale processes. Superspeed centrifuges are also used for purifying cells and nuclei, but in larger quantities. These centrifuges are used to purify 25-30 mL of solution within a tube. Additionally, larger centrifuges also reach higher angular velocities (around 30000 rpm) and also use a larger rotor.
Ultracentrifugation makes use of high centrifugal force for studying properties of biological particles. While microcentrifugation and superspeed centrifugation are used strictly to purify cells and nuclei, ultracentrifugation can isolate much smaller particles, including ribosomes, proteins, and viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocites in excess of 70000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batch, ultracentrifuges can separate molecules in batch and continuous flow systems.
In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of macromolecular properties, including the amino acid composition of a protein, the protein's current conformation, or properties of that conformation. In analytical ultracentrifuges, concentration of solute is measured using optical calibrations. For low concentrations, the Beer-Lambert law can be used to measure the concentration. Analytical ultracentrifuges can be used to simulate physiological conditions (correct pH and temperature).
In analytical ultracentrifuges, molecular properties can be modeled through sedimentation velocity analysis or sedimentation equilibrium analysis. In sedimentation velocity analysis, concentrations and solute properties are modeled continuously over time. Sedimentation velocity analysis can be used to determine the macromolecule's shape, mass, composition, and conformational properties. During sedimentation equilibrium analysis, centrifugation has stopped and particle movement is based on diffusion. This allows for modeling of the mass of the particle as well as the chemical equilibrium properties of interacting solutes.
Centrifugation Analysis
Particle dispersion and sedimentation can be analyzed using the Lamm equation. The calculation of the sedimentation coefficient and diffusion coefficient is useful for determining the physical properties of the molecule, including shape and conformational changes. However, the Lamm Equation is most ideal for modeling concentrations of ideal, non-interacting solutes. Chemical reactions are unaccounted for by this equation. Additionally, for large molecular weight particles, sedimentation is not always smooth. This may lead to the overestimation of the diffusion coefficient, or oscillation effects at the bottom of a solution cell.
Sigma Analysis
Sigma Analysis is a useful tool determining centrifuge properties. It is similar to the continuity equation that relates volumetric flow rate Q, fluid velocity u, and flow path cross-sectional Area A:
Q = uA
In the case of sigma analysis, u is replaced by vg,the settling velocity at centripetal acceleration of g (9.81 m/s2), Σ replaces area, and is a property of the type of centrifuge, and Q is the input fluid flow rate. Σ has the same units as area.
Q = vgΣ
Other applications
Sources
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See also
