Centrifugation – Principle, Types, Rotor & Applications (Complete Easy Notes for Biology Medical Students)
Introduction
* Centrifugation is one of the most important and widely applied research cellular techniques in bio-chemistry and molecular biology, pharmacy and in medicine
* Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge
* It is carried out by spinning a biological sample at high rate of speed
Categories
Preparative Centrifugation
Separation & purification of Biological samples
( cells, organelles, macromolecules)
Analytical Centrifugation
Physical characteristics of biological sample such as size, shape and density
PRINCIPLE
* Utilizes density difference between the particles and the medium in which these are dispersed
* Dispersed systems are subjected to artificially induced gravitational fields
* The centrifugal force causes the sedimentation of heavier solid particles
Continued
* A particle is subjected to centrifugal force when it is rotated at high rate of speed
* The centrifugal force, F is defined by the equation
F = mω²r
Where,
F = intensity of the centrifugal force
m = effective mass of the sedimenting particle
ω = angular velocity of rotation
r = distance of the migrating particles from the central axis of rotation
Continued
A more common measurement of F in terms of the earths gravitational force, g, is relative centrifugal force, RCF its defined by
RCF = (1.119 × 10⁻⁵) (rpm)² (r)
This equation relates RCF to revolutions per minute of the sample . Equation dictates that the RCF on a sample will vary with r, the distance of the sedimenting particles from the axis of rotation . The RCF value is reported as “ a number times gravity g .”
Instrumentation
Consist of two components
Electric motor
( Spin sample)
Rotor
( hold tubes and containers of sample )
Types of Centrifuge
* Low speed centrifuge
* High speed centrifuge
* Ultra centrifuge
Low Speed Centrifuge
* Most laboratories have a standard low-speed centrifuge used for routine sedimentation of heavy particles.
* The low speed centrifuge has a maximum speed of 4000-5000rpm.
* These instruments usually operate at room temperatures with no means of temperature control.
* Two types of rotors are used in it, fixed angle and swinging bucket.
* It is used for sedimentation of red blood cells until the particles are tightly packed into a pellet and supernatant is separated by decantation.
High Speed Centrifuge
* High speed centrifuges are used in more sophisticated biochemical applications, higher speeds and temperature control of the rotor chamber are essential
* The operator of this instrument can carefully control speed and temperature which is required for sensitive biological samples
* Three types of rotors are available for high speed centrifugation- fixed angle, swinging bucket, vertical rotors
Ultra centrifuge
* It is the most sophisticated instrument
* Intense heat is generated due to high speed thus the spinning chambers must be refrigerated and kept at high vacuum
* It is used for both preparative work and analytical work
Types of rotors
A centrifuge rotor is the rotating unit of the centrifuge, which has fixed holes drilled at an angle. Test tubes are placed inside these holes and the rotor spins to aid in the separation of the materials
Types
Swing-bucket Rotor
Fixed-angle Rotor
Vertical rotor
Swing-bucket Rotor
* A swing-bucket rotor usually supports samples ranging in volume from 36 mL to 2.2 mL
* Swing-buckets can support two types of separations: rate-zonal and Isopycnic
* Swing-buckets are preferred for rate-zonal separations, because the distance between the outside of the meniscus and the outside of the bottom of the tube is long enough for separation to occur
Fixed-angle rotors
* Fixed-angle rotors are usually used for pelleting applications to either pellet particles from a suspension and remove the excess debris, or to collect the pellet
* Rotor cavities range from 0.2 mL to 1 mL
* The most important aspect in deciding to use a fixed-angle rotor is the K factor
* The K factor indicates how efficient the rotor can pellet at maximum speed
* The lower the K factor, the higher the pelleting efficiency
Vertical rotors
* Vertical rotors are highly specialized
* They are typically used to band DNA in cesium chloride
* Vertical rotors have very low K factors, which is useful if the particle must only move a short distance until it pellets
* Run time on vertical rotors is short
Types of Centrifugation Techniques
* Density Gradient Centrifugation
* Differential Centrifugation
* Ultra Centrifugation
Density gradient centrifugation
* It is the preferred method to purify subcellular organelles and macromolecules.
* Density gradient can be generated by placing layer after layer of gradient media such as sucrose in tube, with heaviest layer at the bottom and lightest at the top in either.
* Classified into two categories:
Rate-zonal (size) separation
Isopycnic (density) separation
Rate zonal centrifugation
* In Rate zonal centrifugation the solution have a density gradient. The sample has a density i.e. greater than all the layers in 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 sedimenting through the gradient.
Continued
The particles will begin sedimenting in separate zones according to their size shape and density
Isopycnic or sedimentation equilibrium centrifugation
* In this type of centrifugation , the solution contains a greater range of densities
* The density gradient contains the whole range of densities of the particles in the sample
* Each particle will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density
Continued
* In Isopycnic centrifugation separation of particles occurs into zones on the basis of their density differences, independent of time
Differential Centrifugation
* 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
* In the process, a tissue sample is first homogenized to break the cell membranes and mix up the cell contents
* The homogenate is then subjected to repeated centrifugations, each time removing the pellet and increasing the centrifugal force
Ultra Centrifugation
* An important tool in biochemical research is the centrifuge, which through rapid spinning imposes high centrifugal forces on suspended particles, or even molecules in solution, and causes separations of such matter on the basis ULTRACENTRIFUGATION of differences in weight
* Example: Red cells may be separated from plasma of blood, nuclei from mitochondria in cell homogenates, and one protein from another in complex mixtures
Applications
Pharmaceutical Industry
➤ Production of bulk drug
➤ Biopharmaceutical analysis of drugs
➤ Determination of molecular weight of colloids
➤ Evaluation of Suspension and Emulsions
➤ Production of biological products
Advantages
* Centrifuges have a clean appearance and have little to no odor problems
* Not only is the device easy to install and fast at starting up and shutting down, but also only requires a small area for operation
* They can be selected for different applications
* The device is simple to operate
* Centrifuge has more process flexibility and higher levels of performance
Disadvantages
* The machine can be very noisy and can cause vibration
* The device has a high-energy consumption due to high G-forces
* High initial capital costs
Precautions
* A centrifuge user should strictly observe the following precautions
* Manufacturer’s manual should be strictly followed
* Rotor should be stored in proper containers
* Attention should be given to imbalance detectors
* Rotor speed should not exceed the assigned speed
* Lid of the rotor chamber should remain locked during operation
* To avoid the rotor failure, manufactures instructions regarding rotor care and use should always be followed
