Dear students I hope this PPT finds you well. This presentation is all about the laboratory instrument centrifuge and its working called centrifugation. A centrifuge is a laboratory device used to separate fluids, gases, or liquids of different densities by spinning them at high speeds. It operates on the principle of centrifugal force, causing heavier particles or substances to move outward and settle at the bottom while lighter ones move toward the top. This separation process is employed in various fields such as medicine, biology, chemistry, and industry for tasks like separating blood components, purifying samples, and separating mixtures based on density variances. Centrifuges come in various types, including microcentrifuges, ultracentrifuges, and refrigerated centrifuges, each designed for specific applications requiring different speeds and capacities.Types of centrifuges and their functions include: Microcentrifuge: Function: Used for small-volume samples (typically ranging from 0.2 mL to 2.0 mL) in molecular biology, biochemistry, and clinical applications. It separates cellular components, proteins, DNA, and RNA. Clinical Centrifuge: Function: Primarily used in medical laboratories for processing blood, urine, and other bodily fluids. It separates components like red blood cells, plasma, and serum for diagnostic purposes. Refrigerated Centrifuge: Function: Similar to standard centrifuges but equipped with cooling systems to maintain low temperatures during separation. Ideal for samples sensitive to heat, like enzymes or biological materials. Ultracentrifuge: Function: Operates at extremely high speeds, separating particles at molecular levels. Used for studying macromolecules, lipoproteins, and subcellular particles. High-Speed Centrifuge: Function: Used in research labs for general separation tasks, capable of higher speeds than standard centrifuges. It's versatile and employed across various scientific disciplines. Differential Centrifuge: Function: Separates particles based on their sedimentation rates and size differences. It's used to isolate specific components from complex mixtures. Preparative Centrifuge: Function: Focuses on large-scale sample separation for purification purposes. It's employed in industrial settings for isolating biomolecules or other substances at higher capacities. Centrifuges find applications in various fields such as molecular biology, biochemistry, medicine, pharmaceuticals, and industrial processes. Their versatility in separating substances based on density variances makes them invaluable tools in scientific research, diagnostics, and manufacturing processes.

1. Centrifugation
Basic Principles, Mathematics &
Theory
(RCF, Sedimentation Coefficient)
- Presented By
Ms. Priti Khadse
Masters In Biotechnology(Semester-I)
Ramaji Pandav Institute Of Computer Science
And Biological Studies

2. • Definition
Centrifugation(Sedimentation)
Centrifugation is a mechanical process which involves the use of the
centrifugal force
to separate particles from a solution according to their size, shape,
density, medium
viscosity and rotor speed. The denser components of the mixture
migrate away from the
axis of the centrifuge, while the less dense components of the mixture
migrate towards the axis.
• Principle
The centrifugation involves the principle of sedimentation, where the
acceleration at centrifugal force causes denser substances to separate
out along the radial direction at the bottom of the tube. In a solution,
particles whose density is higher than that of the solvent sink (sediment),
and particles that are lighter than it float to the top. The greater the
difference in density, faster they move. If there is no difference in
density, the particles stay steady. To take advantage of even tiny
differences in density to separate various particles in solution, gravity

3. Material Density(g/cm3)
microbial cells
mammalian cells
organelles
proteins
DNA
RNA
1.05-1.15
1.04-1.10
1.10-1.60
1.30
1.70
2.00
Table:- Densities of biological materials
Based on data shown in the table one may wrongly assume that
proteins and nucleic acids would settle faster than cells and organelles.
Biological macromolecules in aqueous solution exist in an extensively
hydrated form i.e. in association with a large number of water
molecules. Hence the effective densities of these substances in
solution are only slightly higher than that of water. The substances
listed in table would settle at extremely low velocities under gravity and
hence separation would not be possible. In a centrifugation process,
these settling rates are amplified using an artificially induced
gravitational field.
-The value for a molecule's sedimentation velocity in a centrifugal field

4. Centrifuge
Centrifuge is an equipment which is used to separate
particles or macromolecules.
• Cells- biological components in tissues and cells are
separated by centrifugation and this principle is widely
used in biological laboratory.
• Sub-cellular components- substances like cytoplasmic
fluid, nucleus, mitochondria, Golgi bodies are separated by
this principle.
(a) Proteins- based on density protein in cells and tissues
is separated using high
speed centrifugation
(b) Nucleic acids- DNA, RNA, snRNA etc. are separated
by this method.

5. Basis of separation-
• Size-size of the particle matters a lot while application of this
principle. It has the basis that as much lesser the size will be,
more the particle will be towards the base.
• Shape- the shape of particle is important. The circular particles
will settle down easily as compared to polygonal shape
particles.
• Density- this component is the main aspect of centrifugation
principle, denser the object, lower the settling
Mechanism:
The acceleration achieved by centrifugation is expressed as a
multiple of the earth's gravitational force (g-9.81ms). Bench-top
centrifuges can reach acceleration values of up to 15000 g (where
g is centrifugal force), while high speed refrigerated centrifuge can
reach 50000 g and ultra centrifuges, which operate with
refrigeration and in a vacuum, can reach 500000 g. Two types of
rotor are available in high powered centrifuges: fixed angle rotors
and swing-out rotors that have movable bucket containers. The
tubes or buckets used for centrifugation are made of plastic and

6. What is Relative Centrifugal Force?
The relative centrifugal force (RCF) or the g force is the radial
force generated by
the spinning rotor as expressed relative to the earth's gravitational
force. The g force
acting on particles is exponential to the speed of rotation defined
as revolutions per
minute (RPM).
Relative centrifugal force- M r ω²
M: mass of particle
r. radius of rotation (cm) (i.e. distance of particle from axis of
rotation)
ω: Average angular velocity (radians/sec),
ω= 2π revolutions/ 60minutes.
-The velocity (v) of particle sedimentation during centrifugation
depends on the angular velocity (c) of the rotor, its radius (the
distance from the axis of rotation), and the particle's sedimentation
properties.

7. Centrifuge: General view, Centrifugation Procedure and
Components Separation

8. Types of Centrifugation Techniques
1. Density gradient centrifugation
A procedure for separating particles (such as viruses or ribosomes
or molecules such as DNA in which the sample is placed on a
preformed gradient such as sucrose or cesium chloride. Upon
centrifugation either by rate zonal or equilibrium procedures, the
macromolecules are 'banded' in the gradient and can be collected
as a pure fraction.
There are 2 forms of density gradient centrifugation one is rate zonal
centrifugation and the second is isopycnic or sedimentation
equilibrium centrifugation.

9. A) Rate zonal centrifugation
In rate zonal centrifugation the solution has a density gradient.
The sample has a
density therefore greater than all the layers the solution. 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 segmenting through the gradient. The
particles will begin segmenting in separate zones according to
their size, shape and density.

10. B) Isopycnic or sedimentation equilibrium centrifugation – In
isopycnic separation, also called buoyant or equilibrium
separation, particles are separated singly on the basis of
their density. Particle size only affects the rate at which
particles move until their density is the same as the
surrounding gradient medium. In sedimentation
centrifugation separation of particles occur into the zone
based on their density difference, independent of time.

11. 2) Differential Centrifugation
Differential centrifugation is a common procedure in microbiology
and cytology useful to separate certain organelles for further
analysis of specific parts of cells. In the process, a tissue sample
is first homogenized generalized to break the cell membranes and
mix up the cell contents. The homogenate is then subjected to
repeated centrifugation, each time remove in the palate and
increasing the centrifugal force.
3) Ultracentrifugation
an ultracentrifuge is a special type of centrifuge in which rotor
rotates at much higher speed than a standard centrifuge. typical
rotation speeds in ultracentrifuges ranges from 30000 to 50000
rpm. the high rotating speeds used in ultracentrifuges can
generate considerable amount of heat. therefore cooling
arrangements are required in these devices. it is usually used for
separating macromolecules from solvents or for fractioning
mixtures of macromolecules.

12. Applications
Clinical laboratory applications
1. Remove cellular elements from blood to provide cell free plasma or
serum
for analysis.
1. Remove chemically precipitated protein from an analytical
specimen.
2. Separation of the subcellular organelle, DNA, RNA.
3. Extract solutes in biological fluids from aqueous to organic
solvents.
4. Separate lipid components.
Industrial applications
1. Production of bulk drugs.
2. Production of biological products.
3. Evaluation of suspensions and emulsion.
4. Separating chalk powder from water.
5. Removing fat from milk to produce skimmed milk.
6. The clarification and stabilization of the wine.
7. Biopharmaceutical analysis of drugs.

13. THANKING
YOU.