CENTRIFUGATION

Centrifugation is a process by which solid particles are sedimented and separated from a
liquid using centrifugal force as a driving force. Centrifugal force is an apparent force that
seems to pull a rotating or spinning object away from a centre. Centrifugal simply means
“fleeing the centre”.

Principle of Centrifugation
Any object moving in a circle at a steady angular velocity is subject to an outward directed
force, F. The magnitude of this force depends on the angular velocity in radians, , and the
radius of rotation, r, in centimetres.

As the rotor spins, centrifugal force is applied to each molecule in the sample.

The centrifugal force, F, is defined by

F = m2r

where

F = intensity of the centrifugal force

m = effective mass of the sedimenting particle
 = angular velocity of rotation in rad/sec
r = distance of the migrating particles from the central axis of rotation in centimetres

The force on a sedimenting particle increases with the velocity of the rotation and the
distance of the particle from the axis of rotation. A more common measurement of F, in terms
of the earth’s gravitational force, g, is relative centrifugal force, RCF, defined by:

This equation relates RCF to revolutions per minute (rpm) of the sample. The RCF value is
reported as “a number times gravity, g.”
It is convenient to determine RCF by use of the nomogram shown below. To use, place a
straight edge connecting the instrumental revolutions (right column) with the distance from
the centre of rotation (left column). The RCF is read where the straight edge crosses the
middle column.

Sedimentation of a molecule is influenced by:

i. properties of the molecules which include size, shape, density;
[Link] of the solvent, or the gradient material which include density, viscosity,
temperature; and
iii. interactions between the solute molecules and the solvent gradient molecule.
Two forces act to counteract the centrifugal force namely, buoyant force (displacement force)
and frictional force. Buoyant force = Mω2rVρ
V=partial specific volume of the solute, ρ=density of the solvent (g/ml) Suspended particles
also generate friction as they migrate through the solution Frictional Force = f(v) = f(dr/dt)

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f = Frictional coefficient unique to the molecules in question, v = dr/dt = velocity of the
sedimenting particle (sedimentation velocity)
For a spherical molecule, f = 6πηrm

η = viscosity of the medium, rm= radius of the molecule

The frictional force increases with the velocity of the particle until a constant velocity is
reached. At this point, the two forces are balanced
Mω2rVρ = fv

A Nomogram

Sedimentation velocity, v = dr/dt = Mω2rVρ/f

It is cumbersome and sometimes impractical to express sedimentation velocity in terms of V,
ρ and f, since these factors are difficult to measure. A new term, sedimentation coefficient, s
(the ratio of sedimentation velocity to centrifugal force) is introduced

Sedimentation coefficient, s = v/ω2r = MVρ/f

s = (dr/dt)/ω2r
The term s is most often defined under standard conditions, 20°C and water as the medium,
and denoted by s20,w. The s value is a physical characteristic used to classify biological
macromolecules and cell organelles. Sedimentation coefficients are in the range of 1 x 10 -13 to
10,000 x 10-13 second. For numerical convenience, sedimentation coefficients are expressed
in Svedberg units, S, where 1 S = 1 x 10 -13 second. There appears to be a direct relationship

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between the S value and the molecular weight or particle size. This, however, is not always
true, as in the case of non-spherical molecules.

CENTRIFUGES
Centrifuges are sedimentation devices in which suspended solids are separated from a liquid
under the action of centrifugal forces generated by spinning the internal bowl of the
centrifuge. Centrifuges can be thought of as sedimentation vessels operating under high
“gravitational” forces. They are devices for separating particles from a solution according to
their size, shape, density, viscosity of the medium and rotor speed.
The basic centrifuge consists of two components, an electric motor with drive shaft to spin
the sample and a rotor to hold tubes or other containers of the sample.

Centrifuge Containers
a. Bottles – take large volumes
b. Screw–top / snap–top tubes
c. Open tubes
d. Flip–Tops (“Eppendorfs”)
These containers may be reusable or disposable, depending on application

Centrifuge Rotors
Three types of rotors are available for high-speed centrifugation: the fixed-angle, the
swinging-bucket, and the vertical rotor (Figure shown below). Fixed-angle rotors are
especially useful for differential pelleting of particles. In swinging-bucket rotors, the sample
tubes move to a position perpendicular to the axis of rotation during centrifugation, as shown
in the diagrammatic representation. These are used most often for density gradient
centrifugation. In the vertical rotor, the sample tubes remain in an upright position (shown in
the diagrammatic representation). These rotors are used often for gradient centrifugation.

Prior to the early 1990s, rotors were constructed from metals such as aluminium and titanium.
Although metal rotors have great strength, they do have several disadvantages: they are
very heavy to handle, they are not corrosion-resistant, and they become fatigued with use.
Rotors are now available that are fabricated from carbon-fibre composite materials. They
have several advantages over heavy metal rotors. These new rotors are 60% lighter than
comparable aluminium and titanium rotors. Because of the lighter weight, acceleration and
deceleration times are reduced; thus, centrifuge run times are shorter. This also results in
lower service and maintenance costs. Instruments are equipped with a brake to slow the rotor
rapidly after centrifugation.

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 A: Fixed-angle Rotor ; B: Swinging-bucket Rotor ; C: Vertical Rotor.

Types of Centrifuges
Two types of rotors, fixed angle and swinging bucket, may be used in the instrument.
Centrifuge tubes or bottles that contain 12 or 50 mL of sample are commonly used. They can
take approximately (up to) 100 tubes, depending on diametre. Low-speed centrifuges are
especially useful for preparative work like rapid sedimentation of coarse precipitates or red
blood cells. The sample is centrifuged until the particles are tightly packed into a pellet at the
bottom of the tube. (This technique is sometimes called pelleting.) The upper liquid portion,
the supernatant, is then separated by decantation. Examples include: Small Bench-top or
Desktop centrifuges.

A Benchtop Centrifuge

2. Microcentrifuges (“microfuge”, “Eppendorf”): Widely used in the category of medium-

speed centrifuges is the microfuge. These instruments, which are designed for the bench-top,
are used for rapid pelleting of small samples (preparative work). It is very common in
biochemistry/molecular biology/ biological laboratories and operated with or without
refrigeration. Fixed-angle rotors are able to hold up to eighteen 1.5 or 0.5 mL tubes. The
maximum speed of most commercial microfuges is between 12,000 and 15,000 rpm, which
deliver a force of 11,000-12,000 x g.

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3. High-Speed Centrifuges: For more sophisticated biochemical applications, higher speeds
and temperature control of the rotor chamber are essential. A typical high-speed centrifuge
has these features. The operator of this instrument can carefully control speed and
temperature, which is especially important for carrying out reproducible centrifugations of
temperature sensitive biological samples. It can reach a speed of 20,000 – 25,000 rpm. Rotor
chambers in most instruments are maintained at or near 4°C. The preparation of biological
samples almost always requires the use of a high-speed centrifuge. High-speed centrifuges
find applications in research for preparative work and may be used to sediment:
(a) cell debris after cell homogenization;
(b) ammonium sulphate precipitates of proteins;
(c) microorganisms; and
(d) cellular organelles such as chloroplasts, mitochondria, and nuclei.

4. Ultracentrifuges: These are the most sophisticated of the centrifuges. The spin chamber
must be refrigerated and placed under a high vacuum to reduce friction because intense
heat is generated in the rotor due to the high speeds (65, 000 – 150,000 rpm) attainable.
These require special rotors, are expensive, have limited lifetime. Although it is rare, metal
rotors sometimes break into fragments when placed under high stress. The rotor chamber on
all ultracentrifuges is covered with a protective steel armour plate. The drive shaft of the
ultracentrifuge is constructed of a flexible material to accommodate any “wobble” of the
rotor due to imbalance of the samples. It is still important to counterbalance samples as
carefully as possible before inserting them in the rotor.

Ultracentrifuges can be used both for preparative work and for analytical measurements.
Thus, two types of ultracentrifuges are available: preparative models, primarily used for
separation and purification of samples for further analysis, and analytical models, which are
designed for performing physical measurements on the sample during sedimentation.
Analytical ultracentrifuges have the same basic design as preparative models, except that they
are equipped with optical systems to monitor directly the sedimentation of the sample during
centrifugation.
These find applications in research like nucleic acid and protein analyses.

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APPLICATIONS OF CENTRIFUGATION
A) Preparative Centrifugation: This technique is quite straightforward, consisting of
placing the sample in a tube or similar container, inserting the tube in the rotor, and spinning
the sample for a fixed period. (Filled centrifuge tubes or bottles must be weighed and
balanced before centrifugation. Tubes across from each other in the rotor should have
approximately the same weight.) The sample is removed and the two phases, pellet and
supernatant (which should be readily apparent in the tube), may be separated by careful
decantation. It is used to separate organelles and molecules, can handle larger liquid volumes
and requires no optical read-out.

B) Analytical Centrifugation: This uses small size samples. It has built-in optical system
and uses relatively pure sample. A variety of analytical measurements can be made on
biological samples during and after a centrifuge run. Most often, the measurements are taken
to determine molecular weight, density, and purity of biological samples. All analytical
techniques require the use of an ultracentrifuge

Centrifugation Techniques
1. Differential Centrifugation – pelleting.
- It is based on the size of the particles
- Used for simple pelleting, for the separation of sub-cellular organelles and macromolecules
- Sedimentation depends on mass, shape and partial specific volume of a macromolecule, as
well as solvent density, rotor size, rate of rotation.
- Usually uses a fixed angle rotor.

Steps
- Sample must be homogenised first
- Material initially uniformly distributed in the solution
- During spin, particles move with varying velocities down tube
- After spin, pellet contains larger to smaller particles (usually mixture)
- Supernatant = liquid + most slowly sedimenting component
- Pellet can be washed and re-spun and this results in reduced yield
If material is not cleanly pelleted (smeared; fixed angle rotor), then it means acceleration is
too rapid and/or sample too concentrated.

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Diagrammatic Representation of Differential Centrifugation

Further characterization or analysis is usually carried out on the individual phases. This
technique, called velocity sedimentation centrifugation, separates particles ranging in size
from coarse precipitates to cellular organelles. Relatively heavy precipitates are sedimented
in low-speed centrifuges, whereas lighter organelles such as ribosomes require the high
centrifugal forces of an ultracentrifuge. Much of our current understanding of cell structure
and function depends on separation of subcellular components by centrifugation. The specific
method of separation, called fractional centrifugation, consists of successive centrifugations
at increasing rotor speeds.

Fractional centrifugation of a cell homogenate

2. Density Gradient Centrifugation: This method is used to purify subcellular organelles
and macromolecules. Density
gradients are generated by placing layer after layer of gradient media Density gradient
centrifugation classified into two:

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a. Rate-Zonal Separation (size)
Tube is filled with inert liquid/solvent of continuous density gradient such as sucrose which
gives a pre-formed gradient within tube. The density of the solution increases towards the
bottom of the tube. Sample is layered on the top and molecules of the sample form discrete
bands after centrifugation. The gradient of the solvent is unchanged by centrifugation.
Separation is based on relative velocities of the molecules which is a function of their
sizes. With centrifugation, faster-sedimenting particles in sample move ahead of slower ones.
Swinging-bucket rotors are preferred.

b. Isopycnic Density-Gradient Centrifugation

Sample is loaded into tube with gradient-forming solution (on top of or below pre-formed
gradient, or mixed in with selfforming gradient). Particles move to point where their bouyant
density equals that part of gradient and form bands. The “true” equilibrium procedure
depends on buoyant densities, not velocities. Example of gradient materials for
macromolecules and nucleotides are CsCl, NaI. These are “self-forming” gradients under
centrifugal force. It uses swinging-bucket or fixed-angle rotor. Density barrier centrifugation
occurs when a single-step gradient is used to block the path of a particular particle in mixture.

Rate-zonal centrifugation Isopycnic centrifugation

Choice of Material for Forming Gradients

1. Stable, soluble.
2. Minimum osmotic effect, minimum change to ionic strength and pH.
3. Totally inert toward biological materials (i.e. has no effect on physical or chemical
compositions)
4. Sterilizable.
5. Not hydrated in aqueous solutions.
6. No interference to assay.
7. Readily and completely separable from fractionated particles
8. Readily, obtainable, inexpensive or recoverable
9. Chemical, physical and thermodynamic properties known (especially in quantitative work)
Note: No one material meets all criteria, so you need to choose carefully.

Examples of Gradient Materials

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i. Simple sugars – sucrose, sorbitol, glycerol
ii. Polysaccharides – Ficoll, dextran, glycogen
iii. Protein- bovine serum albumin
iv. Deuterium oxide
v. Inorganic salts – CsCl, Cs2SO4
vi. Iodinated organic compounds – Metrizamide, Renographin
vii. Colloidal silica – e.g. Percoll (15nm silica coated with polyvinylpyrrolidone)
viii. Solvents other than water – DMSO, formamide
ix. Mixtures of the above

Care of Centrifuge and Rotor

1. Carefully read the operating manual or receive proper instructions before you use any
centrifuge.
2. Check the rotor chamber for cleanliness and for damage. Clean with soap and warm water,
rinse with distilled water, and dry.
3. Select the proper operating conditions on the instrument. If refrigeration is necessary, set
the temperature to the appropriate level and allow 1 to 2 hours for temperature equilibration.
4. Select the proper rotor. Many sizes and types are available. Follow guidelines already
stated in this topic or consult your instructor.
5. Be sure the rotor is clean and undamaged. Observe any licks, scratches, or other damage
that may cause imbalance. If dirty, the rotor should be cleaned with warm water and a mild,
non-basic detergent. A soft brush can be used inside the cavities. Rinse well with distilled
water and dry. Scratches should not be made on the surface coating, as corrosion may result.
6. Filled centrifuge tubes or bottles should be weighed carefully and balanced before
centrifugation.
7. Rotor manufacturers provide a maximum allowable speed limit for each rotor. Do not
exceed that limit.
8. Keep an accurate record of centrifuge and rotor use. Centrifuge maintenance is usually
determined by hours of use and total revolutions of the rotor. It is also essential to maintain a
record of the use for each rotor. Metal rotors weaken with use and the maximum allowable
speed limit decreases. Rotor manufacturers usually provide guidelines for decreasing the
allowable speed for a rotor (de-rating a rotor).
9. If an unusual noise or vibration develops during centrifugation, immediately turn the
centrifuge off.
10. Carefully clean the rotor chamber and rotor after each centrifugation.