SUBJECT FORENSIC SCIENCE

Paper No. and Title PAPER No. 4: Instrumental Methods and Analysis

Module No. and Title MODULE No. 31: Centrifugation

Module Tag FSC_P4_M31

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 TABLE OF CONTENTS
1. Learning Outcomes
2. Introduction
3. Principle of Centrifugation
4. Methodology of Centrifugation
5. Types of Centrifugation
6. Types of Centrifuges
7. Application of Centrifuges
8. Summary

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 1. Learning Outcomes
After studying this module, you shall be able to know about-

 The significance of Centrifuges

 The basic principle and methodology of Centrifugation
 The types and application of Centrifuges

2. Introduction
Centrifugal force is a combination of two Latin words centrum, meaning ‘center’, and fugere,
means ‘to flee’. It is the ostensible force that draws a gyratory body away from the centre of
rotation and is generated by the inertia of the body as the body’s path is constantly redirected.
The centrifuge is a significant instrument in cellular and molecular biology research
laboratory. Basically, all centrifuge instruments consist of a motor which spins a rotor
holding the experimental sample. It is predominantly used to segregate particles or macro-
molecules like cells, sub-cellular components like nucleic acids and proteins on the basis of
their sedimentation rate. The sample gets separated due to variation in their sedimentation
rate because of their different shape, size, viscosity of the medium, density, and the rotational
speed of the rotor to separate

In 1925, Theodor Svedberg designed the first Analytical Ultracentrifuge. The Vacuum
Ultracentrifuge was developed by Edward Greydon Pickels. Vacuum systems similarly
facilitated the upkeep of constant temperature. In 1946, Pickels cofounded Spinco
(Specialized Instruments Corporation) and marketed an ultracentrifuge based on his design.
In 1949, Spinco presented first Preparative Ultracentrifuge to attain a maximum speed of
40,000 rpm. In an Analytical Ultracentrifuge, a sample being whirled can be examined in real
time through an Optical Detection System, by means of Ultraviolet Light Absorption and/or
Interference Optical Refractive Index Sensitive system. The types of information that can be
achieved from an analytical ultracentrifuge consist of the gross shape of macromolecules, the
conformational changes in macromolecules, and size allocations of macromolecular testers.

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 3. Principle of Centrifugation
The centrifuge encompasses principle of sedimentation, where the acceleration at centripetal
force reasons denser constituents to isolate out beside the radiating direction at the bottom
most of the tube. With the similar theory lighter substances will be likely to move to the top
of the tube. Particle with more density than the solvent residue and the particle with lesser
density than, it drift the solution to the top. The greater the difference in density, the faster
they move. In Isopycnic conditions, i.e., where there is no difference in density, the particles
remains stable. To take benefit of even minute differences in density to separate different
particles in a solution, gravity can be substituted with the much more potent “centrifugal
force” delivered by a separator.

According to the Sedimentation Theory, sedimentation is the affinity of particles in

suspension to settle down in the fluid where they are pulled and come to rest against a barrier.
This is on account of their motion through the fluid in response to the forces acting on them.
These forces are due to of gravity, centrifugal speeding up or electromagnetism. The value
for a molecular sedimentation velocity in a centrifugal field is called as its Svedberg constant
or S.

Calculation of S:

Where,

M = Molecular weight (m × NAV)

S = Svedberg coefficient

ῡρ = Partial specific volume of the molecule

N = Avogadro’s number

f = Frictional coefficient

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 4. Methodology
Centrifuge makes use of density difference among the particles or macromolecules and the
medium where they are spread. An artificially induced gravitational field is exposed for the
dispersed systems. A centrifuge is generally propelled by an electric motor, which sets an
object in rotation around a stationary axis, applying a force vertical to the axis. Procedures for
centrifugation characteristically specify the amount of acceleration to be applied to the
sample, relatively than postulating a rotational speed such as revolutions per minute. This
peculiarity is significant since two rotors with diverse diameters running at the similar
rotational acceleration will subject samples to different accelerations. Throughout circular
motion the acceleration is the product of the radius and the square of the angular velocity and
it is conventionally known as the Relative Centrifugal Force (RCF). The acceleration is
measured in multiples of g which is the standard acceleration due to gravity at the Earth's
surface.

Let us assume an element of mass m in a centrifuge tube filled with a liquid. The particle is
acted on by three forces:

1) Fc : Centrifugal Force or Relative Centrifugal Force

2) Fb : Buoyant Force
3) Ff : Frictional Force between the particle and the liquid

The particle in a centrifugal field will experience a Centrifugal Force defined by:

Fc = m r ω2
Where,

m = Mass of the particle

ω = Angular Velocity = 2 π revolutions/ 60 minutes

r = Distance from the axis

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 5. Types of Centrifugation

5.1 Differential Centrifugation

This technique is occasionally speak of differential sedimentation, and is fundamentally a
method of successive centrifugation with increasing centrifugal force. Separation is mainly
dependent on particle’s mass and size, where heavier particles or cells settle first at lower g
values. However, in various cases, differential centrifugation is used to separate out
intracellular matter, and consequently this method is important for purported subcellular
fractionation. But, to obtain a pure organelle fraction from differential centrifugation is
practically impossible.

5.2 Moving Boundary or Zone Centrifugation

In this, the whole tube is occupied with sample and centrifuged. With centrifugation parting
of two units can be achieved but any unit in the combination may result in the supernatant or
in the pellet or it may be distributed in both fractions. However, it depends upon the shape,
size, density, and conditions of centrifugation. The mixture of all of the sedimented
components is known as “pellet”, and it is mixed with unsedimented particles of any kind in
the bottom of the tube formerly. The only constituent which is refined is the slowest
sedimenting one, but its yield is habitually very little. The two portions are recuperated by
transferring the supernatant solution from the pellet. The supernatant can be re- centrifuged at
greater speed to obtain extra purification, with the formation of a new pellet and supernatant.

5.3 Rate Zonal Centrifugation

In this, we keep the sample on the topmost position of the tube in a very thin zone of density
gradient in the influence of centrifugal force. The particles will start sedimenting over the
gradient in distinct zones as per their size shape and density. The run must be terminated
before any of the separated particles reach the bottom of the tube. Particles of the same size
but different shapes will distinct, the particle having more frictional coefficient (f) will
transfer slower (rod shaped moves slower than globular). This technique is called velocity
gradient centrifugation

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 5.4 Isopycnic or density gradient centrifugation
In isopycnic technique, the density gradient column encompasses the whole range of
thicknesses of the sample material. The matter is consistently agitated with the gradient
material. Each particle would residue at the point, where the gradient density becomes equal
to its specific density in the centrifuge tube, and remains constant. Isopycnic technique
separates particles into separate zones exclusively on the source of their density differences.
For example, the gradient may possibly be of such a density range that one component
sediments to its density in the tube and remains there, while other component sediments to
the bottom of the tube. The self- generating gradient technique over and over again entails
long hours of centrifugation.

6. Types of Centrifuges
Broadly, Centrifuges are classified into two categories:

6.1 Laboratory Centrifugation

Laboratory centrifuges are used for small-scale separation and clarification. Characteristic
liquid capacities controlled by such devices are in the range of 1 - 5000 ml. The solid to be
centrifuged is dispersed into suitable numbers of centrifuge tubes (that appears as test tubes)
which is joined in a symmetric method to a revolving block known as the rotor. There are two
kinds of rotors i.e., fixed angle rotors and swing out rotors.

The fixed angle rotor grips the centrifuge in an immovable way at specific angle to the axis of
rotation whereas swing out rotors clamp the tubes analogous to the axis of rotation though the
rotor is immobile but when the rotor comes in motion, the tubes blow out in a way that they
are aligned perpendicular to the axis of rotation.

6.2 Preparative Centrifugation

Preparative centrifuges can handle considerably more liquid capacities as compare to the
laboratory centrifuges, which characteristically fluctuating from unit litre to some thousand
litres. Its various types are discussed further.

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 6.2.1 Tubular Bowl Centrifugation

The suspension to be separated is put into these devices from one end and the supernatant and
precipitate are taken out from the other end of the device in a continuous or semi-continuous
manner. At any point the motion of a particle within a tubular centrifuge in the radial
direction is governed by the following equation:

Force exerted on a particle = Frictional force suffered by the particle

The particle will settle down because to centrifugal acceleration, till the two forces are
balanced.

6.2.2 Disk Stack Centrifugation

The disc stack centrifuge is a different type of preparative centrifuge that is compressed in
design and provides better solid-liquid parting in comparison to the standard tubular bowl
centrifuge. The particles are thrown outward and these come into contact with the angled disc
stack. Once this take place the disc is slide down, and the product is collected at the edge of
the bowl and discharged from the device in the form of slurry. The liquid flows up the device
along the central regions and is discharged from the top.

6.2.3 Chamber Bowl Centrifugation

The chamber bowl with intermittent discharge has no disk stack. The chamber bowl is like a
tubular centrifuge but with insignificant L/D (< 1) ratio. It is appropriate for moving viscous
feed or more concentrated solid feeds. As discussed, the clarification capacity is less than that
with the disk-stack centrifuge due to a reduced settling area. Though, in comparison to the
Disk- Stack centrifuge the chamber bowl can take higher feed solids. The feed rate usually is
smaller for a chamber bowl compared to that of disk-stack centrifuge for the same bowl size.

6.2.4 Ultracentrifuge

An ultracentrifuge is an extraordinary type of centrifuge where the rotor revolves at a much

higher speed than a standard centrifuge. Typical rotation speeds in ultracentrifuges range
from 30,000 rpm – 50,000 rpm. An ultracentrifuge is usually used for separating
macromolecules from solvents or for fractionating mixtures of macromolecules.
Ultracentrifuges are developed for analytical as well as for preparative applications. An
Analytical Ultracentrifuge (AUC) is largely used for examining the properties of
macromolecules besides for analysing complex mixtures of macromolecules.

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 Preparative ultracentrifuges are employed to purify macromolecules such as proteins and
nucleic acids based on their physical properties such as size, molecular weight, density and
mobility. The high rotating speeds used in ultracentrifuges can generate substantial amount of
heat requiring cooling measures to be attached with these devices.

7. Applications of Centrifuges

7.1 Separation

Centrifugation is employed for solid-liquid isolation in a condition that the solids are denser
as compared to liquid. It can be applied to isolate a heavy phase, and two lighter liquid
phases, in which one will be lighter phases being lighter with respect to other. As stated,
solids can be lighter than liquid and separation is by flotation of the dispersed solid phase.

7.2 Interpretation

Centrifuge is used to segregate the discharged separated lighter liquid phase. The purpose is
to reduce the discrete suspended solids in the light continuous phase. Usually, only fine
submicron bio-solids are left uncaptured by centrifugation and they leak with the discharged
light phase.

7.3 De-gritting

De-gritting is like classification where surplus particles, larger or denser, are rejected in the
sediment, with smaller or less dense product, overflowing in the lighter liquid phase. Another
condition is where smaller surplus particles are excluded in the light liquid phase, and
valuable heavier solids are settled with the heavier phase.

7.4 Thickening or concentration

In order to concentrate the solid phase by sedimentation and compaction, removing the
excess liquid phase in the overflow or centrate centrifuge devices are regularly used. This
decreases the volume of the product in subsequential processing.

7.5 Separation and re-pulping

With a concentrated suspension containing impurities such as salts and ions, it is diluted and
washed so that the impurities are dissolved in the wash liquid. Afterwards, the suspension is
directed for centrifugation to remove the expended wash liquid with dissolved contaminants
or finely suspended solids. Subsequently, the product can be further concentrated by
centrifugation.

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation
 8. Summary

 Centrifugal force, word from Latin centrum, meaning “center”, and fugere, means “to
flee”, is the ostensible force that draws a gyratory body away from the centre of rotation.
It is produced by the inertia of the body as the body’s path is constantly redirected. The
centrifuge is an important instrument in cellular and molecular biology research.

 The centrifuge encompasses principle of sedimentation, where the acceleration at

centripetal force reasons denser constituents to isolate out beside the radiating direction
at the bottommost of the tube. With the similar theory lighter substances will be likely to
move to the top of the tube.

 According to the Sedimentation Theory, sedimentation is the affinity of a particle in

suspension to settle down in the fluid where they are pulled over and come to rest against
a barrier. This is due to their motion through the fluid in response to the forces acting on
them. These forces are due to of gravity, centrifugal speeding up or electromagnetism.

 Centrifugal evaporators are designed to evaporate multiple samples down using a

combination of centrifuge / rotor, vacuum pump and solvent trap (refrigerated cold trap
or recirculating suction water trap). The centrifuge chamber can have the option of
heating (to speed sample evaporation) also.

 A refrigerated laboratory centrifuge applies an in-built cooling system in order to retain

the centrifuge chamber and thus, the samples being separated is already set under
ambient temperature. Centrifugation in an unrefrigerated centrifuge generally makes
samples to warm up to certain degree and temperature delicate samples could be
affected, thus the use of a refrigerated centrifuge.

FORENSIC SCIENCE PAPER No.4: Instrumental Methods and Analysis

MODULE No.31: Centrifugation