is a process that involves the use of the centrifugal force
Centrifugal force can generally be any force directed outward relative to some origin. More particularly, in classical mechanics, the centrifugal force is an outward force which arises when describing the motion of objects in a rotating reference frame...
for the sedimentation of mixture
In chemistry and chemical engineering, a separation process, or simply a separation, is any mass transfer process used to convert a mixture of substances into two or more distinct product mixtures, at least one of which is enriched in one or more of the mixture's constituents. In some cases, a...
s with a centrifuge
A centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...
, 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 gravitation
Gravitation, or gravity, is a natural phenomenon by which physical bodies attract with a force proportional to their mass. Gravitation is most familiar as the agent that gives weight to objects with mass and causes them to fall to the ground when dropped...
al 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
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
is properly called the "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decanted
Decantation is a process for the separation of mixtures. This is achieved by carefully pouring a solution from a container in order to leave the precipitate in the bottom of the original container...
from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette
Pasteur pipettes, also known as droppers or eye droppers, are used to transfer small quantities of liquids. They are usually glass tubes tapered to a narrow point, and fitted with a rubber bulb at the top. The combination of the Pasteur pipette and rubber bulb has also been referred to as a teat...
The rate of centrifugation is specified by the angular velocity
In physics, the angular velocity is a vector quantity which specifies the angular speed of an object and the axis about which the object is rotating. The SI unit of angular velocity is radians per second, although it may be measured in other units such as degrees per second, revolutions per...
measured in revolutions per minute
Revolutions per minute is a measure of the frequency of a rotation. It annotates the number of full rotations completed in one minute around a fixed axis...
(RPM), or acceleration expressed as g
The g-force associated with an object is its acceleration relative to free-fall. This acceleration experienced by an object is due to the vector sum of non-gravitational forces acting on an object free to move. The accelerations that are not produced by gravity are termed proper accelerations, and...
. The conversion factor between RPM and g
depends on the radius
In classical geometry, a radius of a circle or sphere is any line segment from its center to its perimeter. By extension, the radius of a circle or sphere is the length of any such segment, which is half the diameter. If the object does not have an obvious center, the term may refer to its...
of the sample in the centrifuge rotor
Rotor may refer to:*A rotating part of a mechanical device, for example in an electric motor, generator, alternator or pump.In engineering:...
. The particles' settling velocity
Settling is the process by which particulates settle to the bottom of a liquid and form a sediment. Particles that experience a force, either due to gravity or due to centrifugal motion will tend to move in a uniform manner in the direction exerted by that force...
in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
difference between the particle and the liquid, and the viscosity
Viscosity is a measure of the resistance of a fluid which is being deformed by either shear or tensile stress. In everyday terms , viscosity is "thickness" or "internal friction". Thus, water is "thin", having a lower viscosity, while honey is "thick", having a higher viscosity...
In the chemical and food industries, special centrifuges can process a continuous stream of particle-laden liquid.
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
Uranium hexafluoride , referred to as "hex" in the nuclear industry, is a compound used in the uranium enrichment process that produces fuel for nuclear reactors and nuclear weapons. It forms solid grey crystals at standard temperature and pressure , is highly toxic, reacts violently with water...
Microcentrifuges are used to process small volumes of biological molecules, cells
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
, or nuclei
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
. Microcentrifuge tubes generally hold 0.5 - 2 mL of liquid, and are spun at maximum angular speeds of 12000–13000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a refrigeration
Refrigeration is a process in which work is done to move heat from one location to another. This work is traditionally done by mechanical work, but can also be done by magnetism, laser or other means...
This is the important process.
High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30000 rpm). The rotors may come with different adapters to hold various sizes of test tubes, bottles, or microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
Method of fractionation procedure:
Cell sample is stored in a suspension which is:
- Buffered - neutral Ph, preventing damage to the structure of proteins including enzymes (which could affect ionic bonds)
- Isotonic (of equal water potential) - this prevents water gain or loss by the organelles
- Cool - reducing the overall activity of enzyme released later in the procedure
- Cells are homogenised in a blender and filtered to remove debris
- The homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet
- The supernatant (suspension containing remaining organelles) is spun at a higher speed - chloroplasts settle out
- The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out
- The supernatant is spun at an even higher speed - ribosomes, membranes settle out
The ribosomes, membranes and Golgi complexes can be separated by another technique called density gradient centrifugation.
Ultracentrifugation makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges 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 velocities in excess of 70000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
, caesium chloride
Caesium chloride is the inorganic compound with the formula CsCl. This colorless solid is an important source of caesium ions in a variety of applications. Its crystal structure forms a major structural type where each caesium ion is coordinated by 8 chlorine ions...
, or iodixanol
Iodixanol is a contrast agent, sold under the trade name Visipaque. Visipaque is commonly used as a contrast agent during coronary angiography. It is the only iso-osmolar contrast agent, with an osmolality of 290 mOsm/kg H2O, the same as blood. It is sold in 2 main concentrations 270 mgI/ml and...
. The high-density solution may be at a uniform concentration throughout the test tube ("cushion") or a varying concentration ("gradient
In vector calculus, the gradient of a scalar field is a vector field that points in the direction of the greatest rate of increase of the scalar field, and whose magnitude is the greatest rate of change....
"). Molecular properties can be modeled through sedimentation
Sedimentation is the tendency for particles in suspension to settle out of the fluid in which they are entrained, and come to rest against a barrier. This is due to their motion through the fluid in response to the forces acting on them: these forces can be due to gravity, centrifugal acceleration...
velocity analysis or sedimentation equilibrium analysis. During the run, the particles or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution, and eventually form a pellet at the bottom of the tube, or bands at various heights.
Particle dispersion and sedimentation can be analyzed using the Lamm equation
The Lamm equation describes the sedimentation and diffusion of a solute under ultracentrifugation in traditional sector-shaped cells. It was named after Ole Lamm, later professor of physical chemistry at the Royal Institute of Technology, who derived it during his Ph.D...
. 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 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:
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.
- Separating textile
A textile or cloth is a flexible woven material consisting of a network of natural or artificial fibres often referred to as thread or yarn. Yarn is produced by spinning raw fibres of wool, flax, cotton, or other material to produce long strands...
- Removing water from lettuce after washing it in a salad spinner
- Separating particles from an air-flow using cyclonic separation
Cyclonic separation is a method of removing particulates from an air, gas or liquid stream, without the use of filters, through vortex separation. Rotational effects and gravity are used to separate mixtures of solids and fluids...
- The clarification and stabilization of wine
The clarification and stabilization of wine in winemaking involves removing insoluble and suspended materials. The insoluble material causes a wine to be cloudy. These processes may include fining, filtration, centrifugation, flotation, refrigeration, barrel maturation, pasteurization and racking...
- Harrison, Roger G., Todd, Paul, Rudge, Scott R., Petrides D.P. Bioseparations Science and Engineering. Oxford University Press, 2003.
- Dishon, M., Weiss, G.H., Yphantis, D.A. Numerical Solutions of the Lamm Equation. I. Numerical Procedure. Biopolymers, Vol. 4, 1966. pp. 449–455.
- Cao, W., Demeler B. Modeling Analytical Ultracentrifugation Experiments with an Adaptive Space-Time Finite Element Solution for Multicomponent Reacting Systems. Biophysical Journal, Vol. 95, 2008. pp. 54–65.
- Cole, J.L., Hansen, J.C. Analytical Ultracentrifugation as a Contemporary Biomolecular Research Tool. Methods and Reviews, 1999/2000.
- Howlett, G.J., Minton, A.P., Rivas, G. Analytical Ultracentrifugation for the Study of Protein Association and Assembly. Current Opinion in Chemical Biology, Vol. 10, 2006. pp. 430–436.
- Dam, J., Velikovsky, C.A., Mariuzza R.A., et al. Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s). Biophysical Journal, Vol. 89, 2005. pp. 619–634.
- Berkowitz, S.A., Philo, J.S. Monitoring the Homogeneity of Adenovirus Preparations (a Gene Therapy Delivery System) Using Analytical Ultracentrifugation. Analytical Biochemistry, Vol. 362, 2007. pp. 16–37.