4 Centrifugation

Centrifugation
Introduction:
Centrifugation is a separation technique commonly used in clinical and research laboratories. It is

based on the behavior of particles in an applied centrifugal field.
Principle:
Particles which differ in density, size or shape, sediment at different rates in a centrifugal field. The
particles will tend to sediment under the influence of gravity. (The force used to sediment the
particle is gravitational force). (If particles suspended in a liquid are so small or have a density so
close to that of the liquid, then the gravity fails to sediment the particles into a separate layer. So,
the basis of centrifugation techniques is to exert a large force than the gravitational force to
increase the effective sedimentation force for the separating such particles from the liquid).
The rate at which the sedimentation occurs in centrifugation is expressed in terms of sedimentation
coefficient and is expressed in Svedberg units.
Centrifuges:
The equipment used to perform centrifugation is called “centrifuge”. The centrifuge should
essentially have a rotor to keep the sample tube.
The rotor can be three types.
1. Fixed angle rotor

2. Vertical tube rotor
3. Swing bucket rotor
1. Fixed angle rotor
In this, tube will be in a fixed position in a specific slanting position throughout the process.
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2. Vertical tube rotor
In this, tube will be in a fixed position, but its axis will be parallel to the axis of rotation
throughout the process.
3. Swing bucket rotor
In this type of rotor, at rest the tube axis will remain parallel to axis of rotation. But during
centrifugation the tube swings and will have an angle of 90 with the axis of rotation. Aluminium
alloy or titanium alloys have been used as rotor materials as they can withstand the higher stress
force generated during high speed centrifugation. Aluminium alloy rotors, although less expensive,
are far more suspective to corrosion by acids and alkalies. Tritanium alloy rotors can withstand
nearly twice the centrifugal force of rotors made from aluminium alloy.
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Types of centrifuges
Centrifuges may be classified into four major groups:
1. Small bench centrifuges

2. Large capacity refrigerated centrifuges
3. High speed refrigerated centrifuges
4. Ultracentrifuges – Which are of two types,
a. Preparative ultracentrifuges
b. Analytical ultracentrifuges
1. Small bench centrifuges
These are simplest and least expensive and available in many designs (may be driven by mechanical
force or electrical force).These centrifuges generally will have a maximum speed of 4000 – 6000
revolutions per minute (RPM) with maximum relative centrifugal field (RCF) of 3000 to 7000 gm.
They are often used to collect small amounts of material that rapidly sediment (yeast cells,
erythrocytes, coarse precipitates).
2. Large capacity refrigerated centrifuges
These centrifuges have a maximum speed of 6000 RPM and produce a maximum relative
centrifugal field approaching 6500 gm. They have refrigerated rotor chambers and vary only in their
maximum carrying capacity. These are most often used to collect substances that sediment rapidly.
Eg: RBCs, bulky precipitates, yeast cells, nuclei and chloroplasts.
3. High speed refrigerated centrifuges
These are available with maximum rotor speed in the region 25000 RPM (corresponding to a RCF of
60000 gm). These can be used to collect microorganisms, cellular debris and large cellular
organelles. But these cannot generate sufficient centrifugal force to sediment viruses or smaller
organelles such as ribosome.
4. Ultracentrifuges
a. Preparative ultracentrifuges
These are capable of spinning rotors to a maximum speed of 80000 RPM The rotor chamber is
refrigerated, sealed, evacuated to minimize excessive rotor temperatures. These centrifuges are
used to separate, isolate and purify whole cells, subcellular organelles, plasma membranes,
polysomes, ribosomes, chromatin, nucleic acids, lipoproteins and viruses.
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b. Analytical ultracentrifuges
These are capable of operating at a speed of 7000 RPM. These will have a rotor contained in a
protective armored chamber, which is refrigerated and evacuated. These instruments will also have
a special optical system to observe the sedimenting of material and to determine concentration
distributions within the sample at any time during centrifugation. These centrifuges are used to
study pure macromolecules, their sedimentation characteristics and molecular structure.
Applications of Centrifugation
1. Separation of thick precipitates from solution.

2. Separation of serum from clotted blood.
3. Separation of plasma from anticoagulant added blood.
4. Precipitating proteins from serum during colorimetric estimation of serum urea, creatinine.
Trichloroacetic acid is added to serum sample, which denatures proteins and precipitates.
The precipitate is separated by centrifugation. The protein free filtrate is used for further
analysis.
5. Separation of erythrocytes from oxalated (or heparinized) blood.
6. To determine packed cell volume (PCV) or hematocrit values. The oxalated blood is
centrifuged in hematocrit tubes (110 mm long and 3 mm in bore diameter) at 3000 RPM for
30 min. The protective volume of RBCs, settled in the bottom layer below the supernatant
layer of clear plasma in the tube, is read from the graduations of the tube as PCV (normally
it averages 45% in humans).
7. Isolation of subcellular organelles – the cells is subjected to disruption by sonication or by
osmotic shock or by the use of homogenizer. This is usually carried out in an isotonic (0.25M)
sucrose solution. The subcellular particles can be separated by differential centrifugation.
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8. The purity of the subcellular can be checked by the use of marker enzymes. Eg: DNA
polymerase is the marker for nucleus, glutamate dehydrogenase for mitochondrion, glucose
6 phosphatase for ribosome, and hexokinase for cystolic fraction.
9. Determination of mol.wt (in the fields of protein and nucleic acid chemistry) by studying
their sedimentation characteristics.
10. Estimation of purity of macromolecules i.e purity of DNA preparations, viruses and proteins.
11. Detections of conformational changes in macromolecules like DNA and proteins

4 Centrifugation

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Centrifugation

Centrifugation is a separation technique commonly used in clinical and research laboratories. It is

The rotor can be three types.

1. Fixed angle rotor

1. Fixed angle rotor

3. Swing bucket rotor

Centrifuges may be classified into four major groups:

1. Small bench centrifuges

1. Small bench centrifuges

2. Large capacity refrigerated centrifuges

3. High speed refrigerated centrifuges

1. Separation of thick precipitates from solution.

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