A centrifuge is a device for separating particles from a solution according to
their size, shape, density, viscosity of, and rotor speed. In biology, the particles
are usually cells, subcellular organelles, viruses, large molecules such as proteins,
and nucleic acids. To simplify mathematical terminology, we will refer to all
biological material as spherical particles. There are many ways to classify
The single most important advance in the use of centrifugal force to separate
biologically important substances was the combination of mechanics, optics,
and mathematics by T. Svedberg and J.W. Williams in the 1920s. They initiated
the mathematics and advanced the instrumentation.
Nowadays, any technique employing the quantitative application of
centrifugal force is known as ultracentrifugation
Rotors for a centrifuge are either fixed angles, swinging buckets, continuous
flow, or zonal, depending upon whether the sample is held at a given angle
to the rotation plane, allowed to swing out on a pivot and into the plane of
rotation, designed with inlet and outlet ports for separation of large volumes,
or a combination of these.
Fixed angles generally work faster; substances precipitate faster in a given
rotational environment, or they have an increased relative centrifugal force for
a given rotor speed and radius. These rotors are the workhorse elements of a
cell laboratory, and the most common is a rotor holding 8 centrifuge tubes at
an angle of 34°C from the vertical.
Swinging bucket rotors (horizontal rotors) have the advantage that there is
usually a clean meniscus of minimum area. In a fixed-angle rotor, the materials
are forced against the side of the centrifuge tube, and then slide down the wall
of the tube. This action is the primary reason for their apparent faster separation,
but also leads to abrasion of the particles along the wall of the centrifuge tube.
For a swinging bucket, the materials must travel down the entire length of the
centrifuge tube and always through the media within the tube. Since the media
is usually a viscous substance, the swinging bucket appears to have a lower
relative centrifugal force, and it takes longer to precipitate anything contained
within. If, however, the point of centrifugation is to separate molecules or
organelles on the basis of their movements through a viscous field, then the
swinging bucket is the rotor of choice. Most common clinical centrifuges have
Cell biologists employ zonal rotors for the large-scale separation of particles
on density gradients. The rotors are brought up to about 3000 rpm while empty,
and the density media and tissues are added through specialized ports.
In using either a fixed-angle or swinging-bucket rotor, it is necessary to contain
the sample in some type of holder. Continuous and zonal rotors are designed
to be used without external tubes.
For biological work the tubes are divided into functional groups, made of
regular glass, Corex glass, nitrocellulose, or polyallomer. Regular glass centrifuge
tubes can be used at speeds below 3000 rpm, that is, in a standard clinical
centrifuge. Above this speed, the xg forces will shatter the glass.
For work in the higher speed ranges, centrifuge tubes are made of plastic
or nitrocellulose. Preparative centrifuge tubes are made of polypropylene and
can withstand speeds up to 20,000 rpm.
The 2 most common types of centrifugation are analytical and preparative; the
distinction is between the 2 is based on the purpose of centrifugation.
Analytical centrifugation involves measuring the physical properties of the
sedimenting particles, such as sedimentation coefficient or molecular weight.
Optimal methods are used in analytical ultracentrifugation. Molecules are
observed by optical system during centrifugation, to allow observation of
macromolecules in solution as they move in the gravitational field.
The samples are centrifuged in cells with windows that lie parallel to the
plane of rotation of the rotor head. As the rotor turns, the images of the cell
(proteins) are projected by an optical system onto film or a computer. The
concentration of the solution at various points in the cell is determined by
absorption of a light of the appropriate wavelength. This can be accomplished
either by measuring the degree of blackening of a photographic film or by the
deflection of the recorder of the scanning system and fed into a computer.
The other type of centrifugation is called preparative and the objective is
to isolate specific particles that can be reused. There are many type of preparative
centrifugation such as rate zonal, differential, and isopycnic centrifugation.
Ultra centrifugation/Low-Speed Centrifugation
Another system of classification is the rate or speed at which the centrifuge is
turning. Ultracentrifugation is carried out at speed faster than 20,000 rpm.
Super speed ultracentrifugation is at speeds between 10,000 and 20,000 rpm.
Low-speed centrifugation is at speeds below 10,000 rpm.
Moving boundary/Zone Centrifugation
A third method of defining centrifugation is by the way the samples are applied
to the centrifuge tube. In moving boundary (differential) centrifugation, the
entire tube is filled with sample and centrifuged. Through centrifugation, one
obtains a separation of 2 particles, but any particle in the mixture may end up
in the supernatant or the pellet, or it may be distributed in both fractions,depending upon its size, shape, density, and conditions of centrifugation. The
pellet is a mixture of all of the sedimented components, and is contaminated
with whatever unsedimented particles were in the bottom of the tube initially.
The only component that is purified is the slowest-sedimenting one, but its
yield is often very low. The 2 fractions are recovered by decanting the supernatant
solution from the pellet. The supernatant can be recentrifuged at a higher speed
to obtain further purification, with the formation of a new pellet and supernatant.
FIGURE 3 Differential centrifugation.
In rate zonal centrifugation, the sample is applied in a thin zone at the top
of the centrifuge tube on a density gradient. Under centrifugal force, the particles
will begin sedimenting through the gradient in separate zones, according to
their size, shape, and density. The run must be terminated before any of the
separated particles reach the bottom of the tube.
Figure 4 (a)
FIGURE 4(b) Rate zonal centrifugation.
In isopycnic technique, the density gradient column encompasses the whole
range of densities of the sample particles. The sample is uniformLy mixed with
the gradient material. Each particle will sediment only to the position in the
centrifuge tube at which the gradient density is equal to its own density, and
it will remain there. The isopycnic technique, therefore, separates particles into
zone solely on the basis of their density differences, independent of time. In
many density gradient experiments, particles of both the rate zonal and isopycnic
principles may enter into the final separations. For example, the gradient may
be of such a density range that one component sediments to its density in the
tube and remains there, while another component sediments to the bottom of
the tube. The self-generating gradient technique often requires long hours of
centrifugation. Isopycnically banding DNA, for example, takes 36 to 48 hours
in a self-generating cesium chloride gradient. It is important to note that the run
time cannot be shortened by increasing the rotor speed; this only results in
changing the position of the zones in the tube, since the gradient material will
redistribute farther down the tube under greater centrifugal force.
FIGURE 5 Isopycnic separation with a self-generating gradient.
Basic Theory of Sedimentation
Molecules separate according to their size, shape, density, viscosity, and
centrifugal force. The simplest case is a spherical molecule. If the liquid has the
density of do and the molecule has a density of d, and if d > do , then the protein
will sediment. In gravitational field, the motor force (Pg) equals the acceleration
of gravity (g) multiplied by the difference between the mass of the molecule and
the mass of a corresponding volume of medium.
Equation 1. Pg = (m – m0)g
Equation 2. Pg = 4/3 (3.14) r3 dg –4/3 (3.14) r3 do g
Equation 3. Pg = (4/3) r3 (3.14) (d – do )g
Pg = force due to gravity,
g = acceleration of gravity,
do = density of liquid (or gradient)
d = density of molecule,
m = mass of the molecule,
mo = mass of equal volume of medium.
In a centrifugal field, the gravitational acceleration (g) is replaced by the
FIGURE 6 Sedimentation of particles by gravity.