Centrifugation is a technique used for the separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. The particles are suspended in a liquid medium and placed in a centrifuge tube. The tube is then placed in a rotor and spun at a define speed.

1. Centrifugation
Md. Saiful Islam
BPharm, MSc
North South University
Fb Group: Pharmacy Universe

2. Centrifugation
 Process by which heterogeneous mixtures undergo sedimentation by
centrifugal force
 Used to separate two immiscible liquids
 A specially-designed equipment know as the centrifuge is used to achieve this

3.  A piece of equipment that puts an object in rotation around a fixed axis
 This applies a potentially strong force perpendicular to the axis of spin
 Works using the sedimentation principle, where the centripetal acceleration
causes denser substances and particles to move outward in the radial
direction
 Objects that are less dense are displaced and move to the centre
 In a laboratory centrifuge that uses sample tubes, the radial acceleration
causes denser particles to settle to the bottom of the tube, while low-density
substances rise to the top
How it WORKS?

4. Centrifugation
 During centrifugation the amount of acceleration to be applied to the sample
is specified, rather than specifying rotational speed
 This is important because two rotors with different diameters running at the
same rotational speed will subject samples to different accelerations
 During circular motion, the acceleration is the product of the radius and the
square of the angular velocity Ω, and the acceleration relative to "g" is named
"relative centrifugal force" (RCF)

5. Centrifugation
 The acceleration is measured in multiples of "g", the standard acceleration
due to gravity at the Earth's surface
 g = earth's gravitational acceleration,
 r = rotational radius
 Ω = angular velocity in radians per unit time
Equation…

6. Types of rotors
Fixed-angle
centrifuges
Swinging
head /
swinging
bucket
centrifuges
Continuous
tubular
centrifuges

7. Fixed-angle centrifuge
 Designed to hold the sample containers at a constant angle relative to the
central axis

8. Swinging-head centrifuge
 Posses a hinge where the sample containers are attached to the central rotor
 This allows all of the samples to swing outwards as the centrifuge is spun

9. Continuous tubular centrifuge
 Do not have individual sample vessels
 Used for high volume applications

10. Classification
 The basic centrifuge consists of two components-
i. an Electric motor with drive shaft to spin the sample, and
Ii. a Rotor to hold tubes or other containers of the sample.
 A wide variety of centrifuges are available ranging from a low speed centrifuge used for routine pelleting of
relatively heavy particles. Mainly there are three types of centrifuges-
1. Low speed centrifuge
2. High speed centrifuge
3. Ultracentrifuge

11. Low speed centrifuge
 A standard low speed centrifuge is used in most laboratories for
routine sedimentation of heavy particles.
 It has a maximum rotor speed of 4000-5000 rpm with RCF value
approx. upto 3000 xg.
 These instruments usually operate at room temperatures with
no means of temperature control.
 Two types of rotors-fixed angle and swinging bucket are used in
low speed centrifuge.
 It is used for sedimentation of coarse precipitates or red blood
cells.
 The samples are centrifuged until the particles are tightly
packed into a pellet and supernatant is separated by
decantation.

12. High speed centrifuge
 High speed centrifuge is used in more sophisticated biochemical
applications.
 It has a maximum speed of 20,000-30,000 rpm with RCF value
approx. 100,000x g.
 The operator of this instrument can carefully control speed and
temperature which is required for sensitive biological samples.
 Three types of rotors-fixed angle,swinging bucket,vertical rotors
are available for high speed centrifugation.
 It is used to sediment-
i. cell debris after cell homogenization.
Ii. Ammonium sulfate precipitate of protein and cellular organelles
such as chloroplasts,mitochondria and nuclei.

13. Ultracentrifuge
 The most sophisticated of the centrifuges are Ultracentrifuge.
 It has a maximum speed of 30,000-70,000 rpm with RCF value approx. up to
10,0000's x g.
 Increase heat is generated in the rotor due to high speed thus the spinning
chamber must be refrigerated and placed under high vacuum to reduce
friction.
 It is mainly used in separation of lipoproteins. Since the separation is a long
process there is generation of heat and thus are provided with internal cooling
system.
 It can be used both for preparative and analytical work.

14. Preparative
VELOCITY
SEDIMENTATION
PREPARATIVE
DIFFERENTIAL
Analytical
DIFFERENTIAL
CENTRIFUGATION
DENSITY
GRADIENT
CENTRIFUGATION
Ultra high
Zonal Centrifugation
Isopynic Centrifugation

15. Preparative
Centrifugation
 Larger sample size can be used
 Generally used to separate
organelles and molecules. Most
centrifugation work done using
preparative ultracentrifuge
 No optical read-out – collect
fractions and analyze them after
the run
 Less pure sample can be used
 Can be used to estimate
sedimentation coefficient and MW
 Uses small sample size (less than 1 ml)
 Built in optical system to analyze
progress of molecules during
centrifugation
 Uses relatively pure sample
 Used to precisely determine
sedimentation coefficient and MW of
molecules
 Beckman Model E is an example of
centrifuge used for these purposes.
Analytical
Centrifugation

16. DENSITY GRADIENT CENTRIFUGATION
DIFFERENTIAL CENTRIFUGATION

17. Rate Zonal
Centrifugation
PRINCIPLE:
 Zonal centrifugation is also known as band or gradient
centrifugation
 In Rate zonal centrifugation the solution have a density gradient. The
sample has a density greater than all the layers in the solution.
 t relies on the concept of sedimentation coefficient (ie movement of
sediment through liquid medium)

18. 1
• a density gradient is created in a test tube
with sucrose and high density at the
bottom.
2
• the sample of protein is placed on the top
of the gradient and then centrifuged.
3
• the proteins sediment according to their
sedimentation coefficient
4
• the fractions are collected by creating a
hole at the bottom of tube.

19. ISOPYCNIC CENTRIFUGATION
PRINCIPLE:
It is also called as density gradient centrifugation.
In this type of centrifugation , the solution contains a greater range
of densities.
Each particle will sediment only to the position in the centrifuge tube
at which the gradient density is equal to its own density.

20. Isopynic or sedimentation
equilibrium centrifugation
Solution of sample and cesium salt is uniformly
distributed in a centrifuge tube and rotated in an
ultra centrifuge.
under the influence of centrifugal force the
cesium salts redistributes to form a density
gradient from top to bottom.
the sample molecules move to the region
where their density equals to the density of
gradient.

21. Isopynic or sedimentation equilibrium
centrifugation
• In Isopycnic centrifugation
separation of particles
occurs into zones on the
basis of their DENSITY
differences, INDEPENDENT
OF TIME.

22.  Differential centrifugation is a common procedure
in microbiology and cytology used to separate certain organelles from
whole cells for further analysis of specific parts of cells.
A tissue sample
is
first homogeniz
ed to break
the cell
membranes and
mix up the cell
content
Homogenate
is then
subjected to
repeated cen
trifugations
Each time
removing the
pellet and
increasing
the
centrifugal
force
Differential
centrifugation

24. Apparatus being used industrially
Perforated basket centrifuge.
Sedimentation type centrifuge
Continuous centrifuge.

25. Perforated basket centrifuge
A vessel about 1m in diameter and its outer wall is perforated. It is mounted on a
vertical shaft by means it can be rotated at a high speed. An outer casing with an
outlet collects the liquid thrown out from the basket. The drive motor may be below
the centrifuge.
Advantage
 It is very compact,occuping very little floor space
 It is capable of handle slurries with high proportions of slides.
 Product generally very low moisture content if compared to a filter cake of a similar
material.
Disadvantages
 Batch process
 It involves a considerable labor cost, making the process expensive .

26. Sedimentation type centrifuge /centrifugal sedimentation .
Tubular bowl centrifuge
 High speed separation & clarification.
 Sharplex Tubular Centrifuge is used to
separate-
- two immiscible liquids with density
difference.
-small quantity of impurities from liquids.
Advantage
 Can be used for both liquid/liquid and
solid/liquid separations
Disadvantage
 Foaming of liquids may occur

27. Conical disc centrifuge
 It is a type of centrifuge that has a series of conical
discs which provides a parallel configuration of
centrifugation spaces.
 The conical plate centrifuge is used to remove
solids (usually impurities) from liquids or to
separate two liquid phases from each other by
means of an enormously high centrifugal force
Advantage
 Can be used for both liquid/liquid and
solid/liquid separations
Disadvantage
 Down time for cleaning lowers efficiency
 Can only be used for feeds with small solid
content

28. Continuous centrifuge
The continuous centrifuge is designed for applications where a continuous process or
very high capacity is required. It is fitted with a conical basket to allow continuous
feeding of a slurry and discharge of the separated solids.
Continuous centrifuge

29. Advantage
 Can be used continuously, eliminating cleaning time.
 These centrifuges can provide excellent washing and low final moisture.
Disadvantage
 Mechanically complex.
 Continuous operation suits for the applications that require high capacity and large
particle size

30.  Preparative Ultracentrifuge: Separation of
• Cells
• Sub-cellular components
• Proteins
• Nucleic acids
 Continuous Centrifuges:
• These centrifuges can provide excellent washing and low final moisture at very high capacity.
 Ultracentrifugation:
• Red cells may be separated from plasma of blood, nuclei from mitochondria in cell homogenates, and one protein
from another in complex mixtures.
 Analytical Centrifugation:
• Involves sedimentation of particles in a medium of homogeneous density or density gradient to measure the
sedimentation coefficient of a particle and the behavior of macromolecules in solution.
= centrifugal force – buoyant force
Application of
Centrifugation

32.  Bioequivalence Studies:
 Plasma separation of blood samples in the bioequivalence study of Propranolol and Norfloxacin.
 Derived analytical data is essential for the evaluation of pharmacokinetic parameters.
 Separation of Secretion Granules:
 Precipitate of protein like Insulin is collected by Cetrifugation.
 Separation of the Mixture of Chiral Compound:
 In both crystallization and supercritical fluid technology drug is separated from mother liquor.
 Production of Bulk Drugs and Vaccines:
 Drug like Aspirin is separated from mother liquor by Centrifugation.
 Vaccines for Diphtheria, Tetanus, Staphylococcus is prepared by Centrifugation by using:

33.  Aluminum Precipitated Toxoids (APT)
 Purified Toxoid Aluminium Phospahte (PTAP)
 Toxoids – Anti Toxin Floccules ( TAF)
Filtrate is collected by Centrifugation.
 Blood Fractionation:
 Separating the blood into its component parts.
This is typically done by Centrifuging the blood.
 Plasma Protein Fractionation:
 The insoluble protein can be collected by
Centrifugation. Figure: Blood components after centrifugation.

34. Other uses:
Separating chalk powder from water.
Removing fat from milk to produce skimmed milk.
Evaluation of suspension and emulsion.
Separating textiles.
Removing water from lettuce after washing it in a salad spinner.
Separating particles from an air-flow using cyclonic separation.
The clarification and stabilization of wine.
Isolation of bacterial cells, fungal and actinomycete mycelium.
Separation of water particles from clothes while spin-drying in washing machines.
Separation of urine components and blood components in forensic and research laboratory.
Used to concentrate samples (preparatory cent).