![Differentielle_zentrifugation](https://upload.wikimedia.org/wikipedia/commons/3/36/Differentielle_zentrifugation.png)
In
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
and
cell biology
Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...
, differential centrifugation (also known as differential velocity centrifugation) is a common procedure used to separate
organelle
In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...
s and other sub-cellular particles based on their
sedimentation rate. Although often applied in biological analysis, differential centrifugation is a general technique also suitable for crude purification of non-living suspended particles (e.g.
nanoparticles
A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 1 ...
,
colloidal
A colloid is a mixture in which one substance consisting of microscopically dispersed insoluble particles is suspended throughout another substance. Some definitions specify that the particles must be dispersed in a liquid, while others extend ...
particles,
viruses
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
). In a typical case where differential centrifugation is used to analyze cell-biological phenomena (e.g. organelle distribution), a
tissue sample is first
lysed to break the
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
s and release the organelles and
cytosol
The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells (intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...
. The lysate is then subjected to repeated
centrifugation
Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate ...
s, where particles that sediment sufficiently quickly at a given
centrifugal force
In Newtonian mechanics, the centrifugal force is an inertial force (also called a "fictitious" or "pseudo" force) that appears to act on all objects when viewed in a rotating frame of reference. It is directed away from an axis which is paralle ...
for a given time form a compact "pellet" at the bottom of the centrifugation tube.
After each centrifugation, the ''
supernatant
In an aqueous solution, precipitation is the process of transforming a dissolved substance into an insoluble solid from a super-saturated solution. The solid formed is called the precipitate. In case of an inorganic chemical reaction leading ...
'' (non-pelleted solution) is removed from the tube and re-centrifuged at an increased centrifugal force and/or time. Differential centrifugation is suitable for crude separations on the basis of sedimentation rate, but more fine grained purifications may be done on the basis of density through
equilibrium density-gradient centrifugation.
Thus, the differential centrifugation method is the successive pelleting of particles from the previous supernatant, using increasingly higher centrifugation forces. Cellular organelles separated by differential centrifugation maintain a relatively high degree of normal functioning, as long as they are not subject to denaturing conditions during isolation.
Theory
In a viscous fluid, the ''rate'' of
sedimentation
Sedimentation is the deposition of sediments. It takes place when particles in suspension 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 ...
of a given suspended particle (as long as the particle is denser than the fluid) is largely a function of the following factors:
*
Gravitational force
In physics, gravity () is a fundamental interaction which causes mutual attraction between all things with mass or energy. Gravity is, by far, the weakest of the four fundamental interactions, approximately 1038 times weaker than the strong ...
* Difference in density
* Fluid viscosity
* Particle size and shape
Larger particles sediment more quickly and at lower centrifugal forces. If a particle is less dense than the fluid (e.g., fats in water), the particle will not sediment, but rather will float, regardless of strength of the g-force experienced by the particle.
Centrifugal force
In Newtonian mechanics, the centrifugal force is an inertial force (also called a "fictitious" or "pseudo" force) that appears to act on all objects when viewed in a rotating frame of reference. It is directed away from an axis which is paralle ...
separates components not only on the basis of density, but also of particle size and shape. In contrast, a more specialized
equilibrium density-gradient centrifugation produces a separation profile dependent on particle-density alone, and therefore is suitable for more fine-grained separations.
High g-force makes sedimentation of small particles much faster than
Brownian diffusion
Diffusion is the net movement of anything (for example, atoms, ions, molecules, energy) generally from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in Gibbs free energy or chemical p ...
, even for very small (nanoscale) particles. When a centrifuge is used,
Stokes' law must be modified to account for the variation in g-force with distance from the center of rotation.
:
where
* D is the minimum diameter of the particles expected to sediment (m)
* η (or μ) is the fluid
dynamic viscosity
The viscosity of a fluid is a measure of its resistance to deformation at a given rate. For liquids, it corresponds to the informal concept of "thickness": for example, syrup has a higher viscosity than water.
Viscosity quantifies the inter ...
(Pa.s)
* R
f is the final radius of rotation (m)
* R
i is the initial radius of rotation (m)
* ρ
p is particle volumetric mass density (kg/m
3)
* ρ
f is the fluid volumetric mass density (kg/m
3)
* ω is the
angular velocity
In physics, angular velocity or rotational velocity ( or ), also known as angular frequency vector,(UP1) is a pseudovector representation of how fast the angular position or orientation of an object changes with time (i.e. how quickly an objec ...
(radian/s)
* t is the time required to sediment from R
i to R
f (s)
Procedure
Differential centrifugation can be used with intact particles (e.g. biological cells, microparticles, nanoparticles), or used to separate the component parts of a given particle. Using the example of a separation of
eukaryotic
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
organelles from intact cells, the cell must first be lysed and
homogenized
Homogeneity and heterogeneity are concepts often used in the sciences and statistics relating to the uniformity of a substance or organism. A material or image that is homogeneous is uniform in composition or character (i.e. color, shape, siz ...
(ideally by a gentle technique, such as
Dounce homogenization; harsher techniques or over homogenization will lead to a lower proportion of intact organelles). Once the crude organelle extract is obtained, it may be subjected to a varying centrifugation speeds to separate the organelles:
Ultracentrifugation
The lysed sample is now ready for centrifugation in an
ultracentrifuge
An ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as (approx. ). There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge. Both cla ...
. An ultracentrifuge consists of a refrigerated, low-pressure chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed may reach up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima Max-XP or Sorvall MTX150 or himac CS150NX), creating centrifugal speed forces of 800,000g to 1,000,000g. This force causes
sedimentation
Sedimentation is the deposition of sediments. It takes place when particles in suspension 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 ...
of macromolecules, and can even cause non-uniform distributions of small molecules.
Since different fragments of a cell have different sizes and densities, each fragment will settle into a pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers can be done by first centrifuging the original lysate under weak forces, removing the pellet, then exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of different density is sedimented to the bottom of the container and extracted, and repeated application produces a rank of layers which includes different parts of the original sample. Additional steps can be taken to further refine each of the obtained pellets.
Sedimentation depends on mass, shape, and
partial specific volume
The partial specific volume \bar, express the variation of the extensive volume of a mixture in respect to composition of the masses. It is the partial derivative of volume with respect to the mass of the component of interest.
:V=\sum _^n m_i \bar ...
of a macromolecule, as well as solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during the experiment to calculate
molecular weight
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
.
Values of
sedimentation coefficient
The sedimentation coefficient () of a particle characterizes its sedimentation during centrifugation. It is defined as the ratio of a particle's sedimentation velocity to the applied acceleration causing the sedimentation.
: s = \frac
The sedime ...
(S) can be calculated. Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense particle sediments more rapidly. Elongated proteins have larger frictional coefficients, and sediment more slowly to ensure accuracy.
Differences between differential and density gradient centrifugation
The difference between differential and density gradient centrifugation techniques is that the latter method uses solutions of different densities (e.g.
sucrose
Sucrose, a disaccharide, is a sugar composed of glucose and fructose subunits. It is produced naturally in plants and is the main constituent of white sugar. It has the molecular formula .
For human consumption, sucrose is extracted and refined ...
, ficoll) or gels through which the sample passes. This separates the sample into layers by relative density, based on the principle that molecules settle down under a centrifugal force until they reach a medium with the density the same as theirs. The degree of separation or number of layers depends on the solution or gel.
Differential centrifugation, on the other hand, does not utilize a density gradient, and the centrifugation is taken in increasing speeds. The different centrifugation speeds often create separation into not more than two fractions, so the supernatant can be separated further in additional centrifugation steps. For that, each step the centrifugation speed has to be increased until the desired particles are separated. In contrast, the density gradient centrifugation is usually performed with just one centrifugation speed.
See also
*
Buoyant density ultracentrifugation
Buoyant density centrifugation (also isopycnic centrifugation or equilibrium density-gradient centrifugation) uses the concept of buoyancy to separate molecules in solution by their differences in density.
Implementation
Historically a caesium c ...
*
Jerome Vinograd
References
{{DEFAULTSORT:Differential Centrifugation
Cell biology
Centrifugation
Industrial processes
Fractionation
Laboratory techniques