Differential Centrifugation in bio separation

The force of gravity will cause sedimentation of particles from a heterogeneous mixture; larger and
denser particles sedimentfaster than the smaller and less dense particles. This phenomenon is useful for
separating heterogeneous solutions into independent components, and for the isolation and enrichment
of target molecules, cells, and cell organelles. Differential centrifugation accelerates the separation
process by introducing centripetal forces many times greater than gravity. The precipitated particles
form a pellet at the bottom of the tube during centrifugation. The rate of sedimentation is dependent on
the size and density of the particles, so centrifugation can be used to isolate target particles simply by
controlling centrifugal force or the rate of centrifugation. The rate of centrifugation is reported as
angular velocity by revolutions per minute (rpm) or as acceleration(g). RPM is dependent on the radius
of the rotor in the centrifuge.

Differential centrifugation (also differential velocity centrifugation) is a common procedure in

biochemistry and cell biology used to separate organelles and other sub-cellular particles on the basis of
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,
colloidal particles, viruses). 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
membranes and release the organelles and cytosol. The lysate is then subjected to repeated
centrifugations, where particles that sediment sufficiently quickly at a given centrifugation force for a
given time form a compact "pellet" at the bottom of the centrifugation tube. After each centrifugation,
the supernatant (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
sedimintation rate, but more fine grained purifications may be done on the basis of density through
equilibrium density-gradient centrifugation.

Procedure Edit

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 organelles from intact cells, the cell must first be lysed and homogenized
(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:

Centrifugation is a technique of separating substances which involves the application of centrifugal

force.

The particles are separated from a solution according to their size, shape, density, the viscosity of the
medium and rotor speed.

In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that
are lighter than it floats to the top.
The greater the difference in density, the faster they move. If there is no difference in density (isopycnic
conditions), the particles stay steady.

To take advantage of even tiny differences in density to separate various particles in a solution, gravity
can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.

A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a
circle), applying a potentially strong force perpendicular to the axis of spin (outward).

The centrifuge works using the sedimentation principle, where the centripetal acceleration causes
denser substances and particles to move outward in the radial direction.

At the same time, objects that are less dense are displaced and move to the center.

In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to
settle to the bottom of the tube, while low- density substances rise to the top.

Differential Pelleting (differential centrifugation)

It is the most common type of centrifugation employed.

Tissue such as the liver is homogenized at 32 degrees in a sucrose solution that contains buffer.

The homogenate is then placed in a centrifuge and spun at constant centrifugal force at a constant
temperature.

After some time a sediment forms at the bottom of a centrifuge called pellet and an overlying solution
called supernatant.

The overlying solution is then placed in another centrifuge tube which is then rotated at higher speeds
in progressing steps.

Density Gradient Centrifugation

This type of centrifugation is mainly used to purify viruses, ribosomes, membranes, etc.

A sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher
concentrations in centrifuge tubes

The particles of interest are placed on top of the gradient and centrifuge in ultracentrifuges.

The particles travel through the gradient until they reach a point at which their density matches the
density of surrounding sucrose.

The fraction is removed and analyzed.

Most animal and plant tissues contain a mixture of cell types. However, an investigator often wishes to
study a pure population of one type of cell. In some cases, cells differ in some physical property that
allows different cell types to be separated. White blood cells (leukocytes) and red blood cells
(erythrocytes), for instance, have very different densities because erythrocytes have no nucleus; thus
these cells can be separated on the basis of density. Since most cell types cannot be differentiated so
easily, other cell-separation techniques have had to be developed. Similarly, it is essential to isolate
quantities of each of the major subcellular organelles to study their structures and metabolic functions
in detail.

we discussed the principles of centrifugation and the uses of centrifugation techniques for separating
proteins and nucleic acids. Similar approaches are used for separating and purifying the various
organelles, which differ in both size and density.

Most fractionation procedures begin with differential centrifugation at increasingly higher speeds
(Figure 5-23), also called differential-velocity centrifugation. The different sedimentation rates of various
cellular components make it possible to separate them partially by centrifugation. Nuclei and viral
particles can sometimes be purified completely by such a procedure. After centrifugation at each speed
for an appropriate time, the supernatant is poured off and centrifuged at higher speed. Each pelleted
fraction can be resuspended and further separated by equilibrium densitygradient centrifugation
(discussed next).

Figure 5-23. Cell fractionation by differential centrifugation.

Figure 5-23

Cell fractionation by differential centrifugation. Generally, the cellular homogenate is first filtered or
centrifuged at relatively low speeds to remove unbroken cells. Then centrifugation of the homogenate
at a slightly faster speed or for a longer (more...)

Differential centrifugation does not yield totally pure organelle fractions. One method for further
purifying fractions is equilibrium density-gradient centrifugation, which separates cellular components
according to their density. The impure organelle fraction is layered on top of a solution that contains a
gradient of a dense nonionic substance, such as sucrose or glycerol. The tube is centrifuged at a high
speed (about 40,000 rpm) for several hours, allowing each particle to migrate to an equilibrium position
where the density of the surrounding liquid is equal to the density of the particle. In typical preparations
from animal cells, the rough endoplasmic reticulum (density = 1.20 g/cm3) separates well from the Golgi
vesicles (density = 1.14 g/cm3) and from the plasma membrane (density = 1.12 g/cm3). (The higher
density of the rough endoplasmic reticulum is due largely to the ribosomes bound to it.) This method
also works well for resolving lysosomes, mitochondria, and peroxisomes in the initial mixed fraction
obtained by differential centrifugation (Figure 5-24).
SUMMARY

 Flow cytometry can identify different cells based on the light they scatter or the fluorescence they emit.
The fluorescence-activated cell sorter (FACS) is particularly useful in separating different types of white
blood cells (see Figure 5-21). A cell’s DNA and RNA content also can be measured with a FACS.

 Disruption of cells by vigorous homogenization, sonication, or other techniques releases their

organelles. When placed in a hypotonic solution, cells swell, thereby weakening the plasma membrane
and making it easier to rupture.

 Sequential differential-velocity centrifugation of a cell homogenate yields partially purified organelles

that differ in mass (see Figure 5-23).

 Equilibrium density-gradient centrifugation, which separates cellular components according to their

density, can further purify cell fractions obtained by differential centrifugation.

 Because the membrane surrounding each type of organelle contains organelle-specific proteins,
immunological techniques are very useful in purifying organelles and vesicles, particularly those that
have a similar size and density.

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.

Ultracentrifuges are laboratory centrifuges with rotors that spin at very high speeds, usually ranging
from 60,000 RPM and 200,000 x g to 150,000 RPM and 1,000,000 x g. Ultracentrifuges are segmented
into two main classes, preparative and analytical. Preparative ultracentrifuges isolate or pellet biological
particles, viruses, organelles, membranes and biomolecules such as DNA, RNA and lipoproteins.