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Theory module: 11



Centrifugation is widely used to separate and purify biological particles in a liquid medium under applied centrifugal force. Separation of analyte is based on size, shape and density. It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and isolated macromolecules such as proteins or nucleic acids.

Antonin Prandt in the year 1864 developed the first diary centrifuge for separation of cream from milk. Later in the year 1869, Friedrich Miescher first used centrifugation for isolating a cell organelle. In 1879, the first continuous centrifugal separator was started by Gustaf de Lavai, which made commercialization of this system a possible option.

The development of the first analytical ultracentrifuge by Theodor Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades.

Basic principles of sedimentation

Every particle in suspension (such as in tissue homogenate) has tendency to sediment due to earth’s gravitational force. However, the rate of sedimentation of a particle can be accelerated or increased by applying, as during centrifugation, a force greater then the earth’s gravitational force. The rate at which a given particle moves downwards is proportional to the centrifugal force (G), which is directed radially outwards and is represented by following relationship

G=2r (1)

Where G= applied centrifugal force

=angular velocity (in radians/sec)

r= radial distance from the axis of rotation (in cm)

since one revolution of the rotor is equal to the circumference of the circular path, therefore, one revolution is equal to 2π radians. The angular velocity can be expressed in terms of revolution per min (rpm) which is a common way for denoting the centrifugation speeds. So,

= 2π. rpm


2= 2. (rpm)2 (2)


By substituting value of 2 from equation (2) in (1)

G= 2. (rpm)2 .r (3)


The applied centrifugal force (G) is calculated in terms of multiple of earth’s gravitational force (g=981 cm.sec). This is referred to as Relative centrifugal force (RCF) and can be derived from equation (3).

RCF= 4π2. (rpm)2.r (4)

3600 x 981

Equation (4) on simplification becomes

RCF= (1.18x10-5). (rpm)2.r (5)

So the RCF (denoted by xg i.e. times the gravitational force) generated during centrifugation in a given rotor of fixed radius at different speeds (rpm) can be readily computed from equation (5). In fact, nonograms have been prepared and are available (Fig. 1) from which for the desired g values, one can directly read out the required centrifugation speed ( rpm) for the rotor of a given size.

During centrifugation, the rate of sedimentation of a spherical particle suspended in a liquid medium will depend on its mass (volume x density) and the density and viscosity of the medium. The net outward force (F) which is experiences is given by the following equation.

F= Mass of the particle difference in density of the paricle and media.


F= 4 π r3p ( ρpm) 2r (6)



4 π r3p is volume of the spherical particle or radii


ρp= density of the particle

ρm= density of the medium

r= radial distance (in cm)

= angular velocity (radians /sec)

While travelling through the medium, the particle also experiences friction which tends to retard its movement and at its any given velocity, the opposing frictional force will be

F0= f (7)


= velocity or sedimentation rate of the particle

f = frictional coefficient of the particle in the solution

The magnitude of frictional coefficient of the particle is dependent on its size, shape and hydration and also viscosity of the medium. The frictional force for an unhydrated spherical particle is given by the following relationship.

f=6πηrp (8)


f= frictional coefficient

η= viscosity coefficient of the medium

rp= radius of the particle

Substituting the value of f from equation (8) into (7)

F0= 6πηrp.v (9)

An unhydrated spherical particle of fixed volume and density suspended in a medium of uniform density accelerates on application of centrifugal force till the net force of sedimentation equals the frictional force resisting its motion through the medium i.e.


Putting the values of F from equation (6) and F0 from equation (9)

4 π r3p ( ρpm) 2r = 6πηrp.v


This balancing of opposing forces is attained quickly and soon the particle sediment at a constant velocity because the frictional resistance increase at higher sedimentation velocity of the particle and under these condition the net force experienced by particle is zero. The particle does not accelerate any more but achieves a maximum velocity and hence sediment at a constant rate. The rate of sedimentation of particle then is defined by the following equation.

v =dr = 2 r2p ( ρpm). 2rp (11)

dt 9η

From equation 11 it may be observed that sedimentation rate of a particle is proportional to its size, difference in densities of the particle and the medium and the applied centrifugal field. When ρpm, then ρpm =0 and under such a situation, its sedimentation velocity also becomes zero or in other words it does not move any further. It may also be observed that its sedimentation rate decreases its more viscous media and increases at higher centrifugal force. Equation (11) contains square of particle radius (r2p) as one of the terms and it becomes immediately apparent that size of the particle will have very profound effect on its sedimentation rate. Particle which have almost identical density but differ slightly in their size sediment at significantly different rates.

The following equation can be derived by integrating equation (11)

t= 9 η ln rb (12)

22 rp2pm) rt
t= sedimentation time in sec

rt = radial distance from the axis of rotation in liquid minicus

rb= radial distance from the axis of rotation to bottom of the tube
This equation relates time for sedimentation of a particle in a centrifuge tube with different variable such as centrifugal force, viscosity of media, particle size, difference in densities of the particle can be separated on the basis of their densities and/or their size by centrifugation of the sample for time required for complete sedimentation of the particle after a fixed time. Equation (11) and (12) are of paramount importance and form the basis of separation of sub cellular organelles by centrifugation.

The sedimentation rate or velocity of a biological particle can also be expressed as

its sedimentation coefficient (s), whereby:

s= v (13)


When designing a centrifugation protocol, following factors could play important role.

  • The more dense a biological structure is, the faster it sediments in a centrifugal field;

  • The more massive a biological particle is, the faster it moves in a centrifugal field

  • The denser the biological buffer system is, the slower the particle will move in a centrifugal field;

  • The greater the frictional coefficient is, the slower a particle will move;

  • The greater the centrifugal force is, the faster the particle sediments;

  • The sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal.

Many different types of centrifuges are commercially available including:

  • large-capacity low-speed preparative centrifuges;

  • refrigerated high-speed preparative centrifuges;

  • analytical ultracentrifuges;

  • preparative ultracentrifuges;

  • large-scale clinical centrifuges; and

  • Small-scale laboratory microfuges.

Microfuges (so called because they centrifuge, small volume samples in Eppendorf tubes), large-capacity preparative centrifuges, high speed refrigerated centrifuges and ultracentrifuges are most widely used centrifuge techniques by undergraduates students.

Simple bench-top centrifuges are mainly used to collect separate biological material, such as blood cells, bacterial cells, clinical samples ect. Refrigerated centrifuges are widely used for heat labile substances such as protein samples.

Modern refrigerated microfuges are equipped with rotor and adapters to hold standardised plastic tubes to centrifuge small volume up to 0.5 to 1.5 ml and at speed of approximately 10 000 g. Microfuges can also be used to concentrate protein samples in ultra filters by applying low speed.

microfugerefri centrifuge avantij20xp
Fig.2. Different types of centrifuges (a) refrigerated microfuges, (b) refrigerated centrifuge and (c) preparative centrifuge

In larger preparative bench-top centrifuges, sedimentation can be achieved upto 3000 to 7000 g adaptable with different types of containers. Depending on the range of available adapters, considerable quantities of 5 to 250 ml plastic tubes or 96-well ELISA plates can be accommodated. It is useful tool in many high-throughput biochemical assays where the quick and efficient separation of coarse precipitates or whole cells is critical.

High-speed refrigerated centrifuges are absolutely essential for the sedimentation of protein precipitates, large intact organelles, cellular debris derived from tissue homogenisation and microorganisms. They operate at maximum centrifugal fields of approximately 100 000 g. However, this centrifugal force is not sufficient to sediment smaller microsomal vesicles or ribosomes, but can be employed to differentially separate nuclei, mitochondria or chloroplasts or protein aggregates. In order to harvest yeast cells or bacteria from large volumes of culture media, high-speed centrifugation may also be used in a continuous flow mode with zonal rotors. This approach does not therefore use centrifuge tubes but a continuous flow of medium. As the medium enters the moving rotor, biological particles are sedimented against the rotor periphery and excess liquid removed through a special outlet port.

The ultracentrifuge is a  centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 2 000 000 g. Theodor Svedberg invented the analytical ultracentrifuge in 1925 and won the noble prize in Chemistry in 1926 for his valuable contribution in this field.

There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge, a valuable tool and has decisively advanced the detailed biochemical analysis of subcellular structures and isolated biomolecules.

Preparative ultracentrifugation can be operated at RCF up to 900 000 g. In order to minimise excessive rotor temperatures generated by frictional resistance between the spinning rotor and air, the rotor chamber is sealed, evacuated and refrigerated. To avoid delays during biochemical procedures involving ultracentrifugation, the cooling and evacuation system of older centrifuge models should be switched on at least an hour prior to the centrifugation run. On the other hand, modern ultracentrifuges can be started even without a fully established vacuum and will proceed in the evacuation of the rotor chamber during the initial acceleration process.

On the other hand, modern ultracentrifuges can be started even without a fully established vacuum and will proceed in the evacuation of the rotor chamber during the initial acceleration process.

In an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical detection system, using ultraviolet light absorption and/or interference optical refractive index sensitive system. This allows the operator to observe the evolution of the sample concentration versus the axis of rotation profile as a result of the applied centrifugal field. With modern instrumentation, these observations are electronically digitized and stored for further mathematical analysis.
Two kinds of experiments are commonly performed on these instruments: sedimentation velocity experiments and sedimentation equilibrium experiments.

Sedimentation velocity experiments aim to interpret the entire time-course of sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as their size-distribution. The size resolution of this method scales approximately with the square of the particle radii, and by adjusting the rotor speed of the experiment size-ranges from 100 Da to 10 GDa can be covered. Sedimentation velocity experiments can also be used to study reversible chemical equilibria between macromolecular species, by either monitoring the number and molar mass of macromolecular complexes, by gaining information about the complex composition from multi-signal analysis exploiting differences in each components spectroscopic signal, or by following the composition dependence of the sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins theory.

Sedimentation equilibrium experiments are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time-independent concentration profile.

This method is simple, more reliable, more precise, is a common method of measuring absolute molecular weight. The disadvantage is balanced centrifugal too long, the conventional sedimentation equilibrium method requires dozens of hours of experimental time, the present method can be reduced to use short-course ten hours.
Zones of equal density centrifugation

This technique is used to separate molecule with same size of particles (M) but different shapes (e.g., linear versus globular). In this the particle with the greater frictional coefficient (f) will move slower (rod shaped moves slower than globular). Alternately this technique is called velocity gradient centrifugation (a gradient of sucrose is used to linearize the motion of the particles). In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient (Fig. 3). Under centrifugal force, the particles will begin sediment 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.


Fig. 3. Rate zonal centrifugation

In isopycnic centrifugation, 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 at the position in the centrifuge where the gradient density is equal to its own density (Fig.5). The isopycnic technique, therefore, separate particles into zone solely on the basis of their density differences, independent of time. 


Fig. 5. Isopycnic centrifugation

Types of rotors

Rotors can be broadly classified into three common categories.

  1. swinging-bucket

  2. fixed-angle

  3. vertical

Swinging Bucket rotors: In this type of rotors, the sample tubes are loaded into individual buckets that hang vertically while the rotor is at rest. When the rotor begins to rotate the buckets swing out to a horizontal position (Fig. 6) It is useful when samples are to be resolved in density gradients. The longer path length permits better separation of individual particle types from a mixture. However, this rotor is relatively inefficient for pelleting. Also, care must be taken to avoid “point loads” caused by spinning CsCl or other dense gradient materials that can precipitate.

swing bucket rotor

Fig. 6. Design of the swinging bucket rotor

Fixed-angle rotors: In this type of rotors, the sample tubes are held fixed at the angle of the rotor cavity. When the rotor begins to rotate, the solution in the tubes reorients (Fig. 7).It is widely used for pelleting applications for example include pelleting bacteria, yeast, and other mammalian cells. It is also useful for isopycnic separations of macromolecules such as nucleic acids.

fixed angle rotor flow digaram

Fig. 7. Design of the fixed –angle rotor

Vertical rotors: Sample tubes are held in vertical position during rotation (Fig. 8 ). This type of rotor is not suitable for pelleting applications but is most efficient for isopycnic (density) separations due to the short path length. It is widely used in plasmid DNA, RNA, and lipoprotein isolations.


Fig. 8. Design of vertical rotor

Cell fractionation is the separation of homogeneous sets, usually organelles, from a population of cells. Cellular and subcellular fractionation techniques are indispensable methods used in biochemical research. Although the proper separation of many subcellular structures is absolutely dependent on preparative ultracentrifugation, the isolation of large cellular structures, the nuclear fraction, mitochondria, chloroplasts or large protein precipitates can be achieved by conventional high-speed refrigerated centrifugation.

Cell fractionation can be performed at two levels

Differential centrifugation: Differential centrifugation is based upon the differences in the sedimentation rate of biological particles of different size and density. Crude tissue homogenates containing organelles, membrane vesicles and other structural fragments are divided into different fractions by the stepwise increase of the applied centrifugal field. Following the initial sedimentation of the largest particles of a homogenate (such as cellular debris) by centrifugation, various biological structures or aggregates are separated into pellet and supernatant fractions, depending upon the speed and time of individual centrifugation steps and the density and relative size of the particles. To increase the yield of membrane structures and protein aggregates released, cellular debris pellets are often rehomogenised several times and then recentrifuged. This is especially important in the case of rigid biological structures such as muscular or connective tissues, or in the case of small tissue samples as is the case with human biopsy material or primary cell cultures.

The differential sedimentation of a particulate suspension in a centrifugal field is diagrammatically shown in Fig. 3.4a. Initially all particles of a homogenate are evenly distributed throughout the centrifuge tube and then move down the tube at their

Since the sedimentation rate per unit centrifugal field can be determined at different temperatures and with various media, experimental values of the sedimentation coefficient are corrected to a sedimentation constant theoretically obtainable in water at 20ºC, yielding the S20,W value. The sedimentation coefficients of biological macromolecules are relatively small, and are usually expressed (see Section 3.5), as Svedberg units, S. One Svedberg unit equals 10–13 s.
Types of centrifugation

Centrifugation techniques take a central position in modern biochemical, cellular and molecular biological studies. Depending on the particular application, centrifuges differ in their overall design and size. However, a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated. Particles of biochemical interest are usually suspended in a liquid buffer system contained in specific tubes or separation chambers that are located in specialised rotors. The biological medium is chosen for the specific centrifugal application and may differ considerably between preparative and analytical approaches. As outlined below, the optimum pH value, salt concentration, stabilising cofactors and protective ingredients such as protease inhibitors have to be carefully evaluated in order to preserve biological function. The most obvious differences between centrifuges are:

  • The maximum speed at which biological specimens are subjected to increased sedimentation;

  • The presence or absence of a vacuum;

  • The potential for refrigeration or general manipulation of the temperature during a centrifugation run; and

  • The maximum volume of samples and capacity for individual centrifugation tubes.

Many different types of centrifuges are commercially available including:

  • large-capacity low-speed preparative centrifuges;

  • refrigerated high-speed preparative centrifuges;

  • analytical ultracentrifuges;

  • preparative ultracentrifuges;

  • large-scale clinical centrifuges; and

  • Small-scale laboratory microfuges.



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