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Types of Centrifuge And Centrifugation (Definition, Principle, Uses)

Types of Centrifuge And Centrifugation Overview

Centrifuge definition

A centrifuge is a device utilised to isolate mixture components depending on their sizes, densities, medium viscosity, and rotor speed.

  • In laboratories, the centrifuge is frequently employed to separate biological components from crude extracts.
  • The sample is held within a rotor which rotates around a fixed point (axis) in a centrifuge, creating a powerful force perpendicular to the axis.
  • The sedimentation concept underlies all of the many types of centrifuges used to separate various molecules.

Centrifugation definition

  • The process of centrifugation involves moving the lower density particles toward the centre while the denser molecules flow toward the periphery due to the centrifugal force or acceleration.
  • Centrifugation is driven by the perpendicular force created while a sample is rotated about a fixed point.
  • The rate of centrifugation is affected by the size as well as the density of the solution’s particles.

Relative Centrifugal Force (RCF)

  • Rotor strength of various types and sizes is measured by relative centrifugal force.
  • This is the force that the spinning of the rotor has on its contents.
  • The sample is subjected to a perpendicular force known as RCF, which is always in relation to the earth’s gravity.
  • For the purpose of comparing rotors, the RCF of several centrifuges may be employed, enabling the choice of the optimal centrifuge for a given function.
  • The relative centrifugal force (RCF) formula is expressed as follows:
  • RCF (g Force)= 1.118 × 10-5 × r × (RPM)2
  • where RPM is the speed of the rotor in rotations per minute and r is the rotor’s radius (in centimetres).

Centrifuge Rotors

The motorised components that house the tubes containing the samples in centrifuges are called rotors. Rotors for centrifuges are made to produce rotational speed that can separate the various components in a sample. A centrifuge uses one of three different types of rotors, and they are as follows:

  1. Fixed angle rotors
  • These rotors hold the sample tubes at a 45° angle with respect to the rotor’s axis.
  • The particles in this kind of rotor impact the other side of the tube, where they eventually slide down and are gathered at the bottom.
  • As the tubes’ pathlengths lengthen, they become quicker than other kinds of rotors.
  • However, some particles may stay at the tubes’ sidewalls since the force’s direction is different from where the tube is located.
  1. Swinging bucket rotors/ Horizontal rotors
  • When the operation is initiated, swinging bucket rotors hold the tubes at a 90° angle as they swing.
  • The tubes of this rotor are suspended in racks that enable them to move far enough to achieve the horizontal position.
  • The force that moves the particles far from the rotor as well as towards the bottom of the tubes is present in this type of rotor along the line or direction of the particles’ presence.
  • The supernatant possessed a flat surface since the tubes were always horizontal, making it feasible to isolate the accumulated particles from the supernatant.
  1. Vertical rotors
  • Of all the rotors, vertical rotors have the shortest pathlength, quickest runtime, and greatest resolution.
  • The tubes in vertical rotors remain vertical while the centrifuge is running.
  • Due to the tube’s location not being in line with the direction of the centrifugal force, the yield of the rotor is not optimal.
  • Particles thus tend to spread towards the outside wall of the tubes rather than settle down.
  • These are frequently employed in density gradient and isopycnic centrifugation.

Types of centrifuges

  1. Benchtop centrifuge
  • Compact centrifuges like the benchtop centrifuge are often employed in clinical and research labs.
  • The tubes are rotated along a fixed axis by an electric motor, producing force perpendicular to the tubes.
  • These are excellent in smaller laboratories with fewer rooms since they are so compact.
  • Benchtop centrifuges come in a variety of designs and may be used for a variety of tasks.
  • A tabletop centrifuge has a rotor containing racks holding the sample tubes and a lid that covers the centrifuge’s operating unit.
  1. Continuous flow centrifuge
  • Large amounts of material may be quickly centrifuged using a continuous flow centrifuge without compromising the rate of sedimentation.
  • This type of centrifuge allows for the isolation of a huge quantity of samples at a high centrifugal force, eliminating the time-consuming procedure of emptying and replenishing the tubes following every cycle.
  • They possess a reduced pathlength, which enables them to control process speed and pelletize the solid fraction of the supernatant more easily.
  • Furthermore, since the sample doesn’t require to be loaded and unloaded frequently, as in conventional centrifuges, that allows for faster.
  • This centrifuge can centrifuge up to 1 litre of samples in no more than 4 hours.
  1. Gas centrifuge
  • A gas centrifuge is a centrifuge that is specifically used to separate gases according to their isotopes.
  • This centrifuge separates molecules based on their masses using the identical centrifugal force principle as all other centrifuges.
  • Uranium-235 and Uranium-238 extraction and separation are the major uses of this centrifuge.
  • In contrast to conventional centrifuges that operate on batch processing, the gas centrifuge is designed to allow for a continuous flow of gas into and out of the device.
  • These centrifuges are set up in a cascade so that the gases are divided into two groups according to their isotopes before being sent to the next centrifuge for additional processing.
  • Due to their ability to produce gases with a greater concentration than earlier technologies, gas centrifuges have taken the place of other gaseous diffusion processes.
  1. Hematocrit centrifuge
  • Specialized centrifuges called hematocrit centrifuges are used to calculate the volume fraction of erythrocytes (RBCs) in a specific blood sample.
  • This centrifuge produces hematocrit readings for testing in biochemistry, immunity, blood tests, and other general clinical procedures.
  • Blood loss, polycythemia (an increase in the erythrocyte count to above-normal levels), anaemia, bone marrow failure, leukaemia, and multiple myeloma can all be diagnosed with hematocrit centrifuges.
  • To spin tube samples, the microhematocrit centrifuge quickly reaches speeds of 11,000 rpm and RCFs of up to 15,000 g.
  • A hematocrit centrifuge has comparable parts to a tabletop centrifuge, but it is designed specifically for using blood samples.
  1. High-speed centrifuge
  • The high-speed centrifuge may be used to spin objects at slightly higher speeds, as the name indicates.
  • The high-speed centrifuge may spin anywhere between 15,000 and 30,000 revolutions per minute.
  • High-speed centrifuging is necessary for the biochemical use of this device, which is frequently used in more advanced laboratories.
  • High-speed centrifuges are outfitted with a system for altering the operation’s speed and temperature for the research of sensitive biological molecules.
  • The high-speed centrifuges come with a number of adapters to accommodate sample tubes of different sizes and shapes.
  • These centrifuges can perform centrifugation using any of the three types of rotors.
  1. Low-speed centrifuge
  • The conventional centrifuges that are frequently employed in labs for the regular separation of particles are called low-speed centrifuges.
  • The highest speed of these centrifuges is 4000-5000 rpm.
  • Since they lack a means for regulating the pace or temperature of the operation, they are typically used at room temperature.
  • These centrifuges can utilise rotors with fixed angles and swinging buckets.
  • These centrifuges are convenient and small, making them excellent for analysing biological materials like blood samples.
  • Similar to other centrifuges, the low-speed centrifuge operates on the same fundamental tenets, but its use is restricted to the separation of less complex solutions.
  1. Microcentrifuge
  • The centrifuges used to separate samples with lower capacities, ranging from 0.5 to 2 l, are called microcentrifuges.
  • The typical speed at which microcentrifuges are run is between 12,000 and 13,000 rpm.
  • This is employed for the molecular separation of DNA and cell organelles like nuclei.
  • The sample tubes used in microcentrifuges, also known as microfuges, are smaller than the conventional test tubes used in larger centrifuges.
  • Some microcentrifuges include adapters that enable the use of both bigger and smaller tubes.
  • For the operation of temperature-sensitive samples, microcentrifuges with temperature controls are offered.
  1. Refrigerated centrifuges
  • Centrifuges that have a temperature control range of -20 °C to 30 °C are known as refrigerated centrifuges.
  • There is a distinct type of centrifuge that features a temperature control system, which is necessary for a number of procedures that call for lower temperatures.
  • Along with the rotors and racks for the sample tubes, centrifuges that are kept chilled also contain a temperature control system.
  • These centrifuges have an RCF of up to 60,000 xg, which is excellent for separating different biological components.
  • These are frequently used to gather materials that separate quickly, such as yeast cells, chloroplasts, and erythrocytes.
  • To suit the requirements of the operations, the chamber of a chilled centrifuge is shut off from the outside.
  1. Ultracentrifuges
  • The separation of considerably smaller molecules like ribosomes, proteins, and viruses is made possible by ultracentrifuges, which spin at extraordinarily high speeds.
  • It is the most advanced kind of centrifuge and can separate molecules that other centrifuges cannot.
  • Such centrifuges have cooling systems that assist in balancing the heat generated by the vigorous spinning.
  • These centrifuges have a maximum speed of 150,000 revolutions per minute.
  • It may be used for both analytical and preparatory tasks.
  • Molecules may be separated using ultracentrifuges in both large batches and a continuous flow system.
  • Ultracentrifuges may be used to separate materials but also to determine the size, shape, and density of macromolecules.
  1. Vacuum centrifuge/ Concentrators
  • Vacuum centrifuges use centrifugal force, vacuum, and heat to hasten the evaporation of substances in the lab.
  • These centrifuges have the capacity to process several samples (up to 148 samples at a time).
  • In chemical and biological laboratories, this kind of centrifuge is employed for the efficient evaporation of sample solvents, which concentrates the samples.
  • These are frequently employed in high-throughput labs for samples that may include a lot of solvents.
  • Solvent bumping is prevented by removing extra solvents with a rotating evaporator.
  • The centrifuge operates by reducing the chamber’s pressure, which also lowers the samples’ boiling points.
  • The liquids evaporate as a result, concentrating the particles for separation.

Types of centrifugation

1. Analytical Centrifugation

The particles in a sample are separated using the analytical centrifugation technique based on their density and the centrifugal force they are subjected to. A durable and adaptable technique for analysing macromolecules in solution quantitatively is analytical ultracentrifugation (AUC).

Principle of Analytical Centrifugation

  • The idea behind analytical centrifugation is that denser particles settle down more quickly than less dense ones. Similar to this, bigger molecules spin at a faster rate than smaller ones due to centrifugal force.
  • A sedimentation velocity technique or a sedimentation equilibrium method can be used in analytical ultracentrifugation to determine the relative molecular mass of a macromolecule.
  • The sedimentation coefficients of macromolecules are used to define their hydrodynamic characteristics. The pace at which a concentration boundary of a certain biomolecule travels in the gravitational field may be used to calculate them.
  • The sedimentation coefficient can be used to describe how macromolecules’ sizes and shapes vary when experimental circumstances change.
  • The analytical ultracentrifuge has three optical systems (absorbance, interference, and fluorescence) that allow for accurate and targeted real-time monitoring of sedimentation.

Steps of Analytical Centrifugation

  • In order to be placed inside the ultracentrifuge, analytical cells are used to collect small sample sizes (20–120 mm3).
  • The ultracentrifuge is then set up so that the centrifugal force drives the biomolecules, which are initially dispersed randomly, to migrate radially outward through the solvent.
  • The Schlieren optical system is used to calculate the molecules’ distance from the centre.
  • The molecular mass is calculated using a graph of the solute concentration against the squared radial distance from the centre of rotation.

Uses of Analytical Centrifugation

  • It is possible to employ analytical centrifugation to assess the purity of macromolecules.
  • It may also be used to examine modifications in supramolecular complexes’ molecular masses.
  • Additionally, it makes it possible to estimate the relative molecular weight of solutes in their natural condition.
  • 2. Density gradient centrifugation

When molecules are separated using density gradient centrifugation, the separation is based on the molecules’ densities as they move along a density gradient with a centrifugal force.

Principle of Density Gradient Centrifugation

  • Density gradient centrifugation is based on the idea that molecules would settle down while being pulled toward a liquid that has the same density as them.
  • In this situation, a medium having a gradient in density is used, and the density must either drop or increase.
  • A centrifugal force is produced as molecules in a sample travel through the medium while it is spun.
  • As molecules pass over the gradient in density, they start to migrate towards the bottom.
  • When the particle density reaches the surrounding medium, the molecules start to float in midair.
  • This separates molecules with varied densities at distinct levels, where they may subsequently be retrieved using a variety of techniques.

Steps of Density Gradient Centrifugation

  • In a centrifuge tube, a density gradient of a medium is made by carefully smearing the lower concentration over the higher concentrations.
  • The tubes are then inserted into an ultracentrifuge with the sample deposited over the gradient.
  • The particles move across the gradient until they arrive at a location where their density is the same as that of the medium around them.
  • In order to extract the particles as isolated units, the fractions are taken out and separated.

Uses of Density Gradient Centrifugation

  • It is possible to use density gradient centrifugation to purify huge quantities of biomolecules.
  • It may even be used to purify certain viruses, facilitating future research on such viruses.
  • This method may be used to separate particles as well as to figure out what the different particles’ densities are.

Examples of Density Gradient Centrifugation

  • This technique was applied in the well-known experiment that demonstrated DNA’s semi-conservative nature by employing various nitrogen isotopes.
  • Another example is the use of this approach to separate membrane vesicles with different densities after isolating the microsomal fraction from muscle homogenates.
  • 3..Differential centrifugation

Differential centrifugation is a type of centrifugation technique in which substances are separated and brought to the bottom of a centrifuge tube by successively applying greater centrifugal forces.

Principle of Differential Centrifugation

  • Based on changes in the rates at which biological particles of different sizes and densities settle, differential centrifugation is used.
  • The bigger molecules begin to first settle when the centrifugal force is increased.
  • Depending on the density and relative sizes of the particles, as well as the rate and duration of each centrifugation stage, more particles settle down.
  • Smaller-sized structures are left in the supernatant after the biggest class of particles forms a pellet at the bottom of the centrifuge tube.
  • Consequently, bigger molecules sediment more rapidly and with lower centrifugal forces than smaller molecules, which require more time and stronger pressures to do so.
  • Less dense particles will float in the absence of settling as compared to the medium.

Steps of Differential Centrifugation

  • The buffer-containing medium homogenises the sample solution.
  • The sample is then placed in the centrifuge tube and rotated for the predetermined period of time at the predetermined temperature using a predetermined centrifugal force.
  • At the end of this procedure, a pellet will finally form at the bottom of the tube, which is then isolated from the supernatant.
  • The supernatant is transferred to a fresh centrifuge tube and centrifuged at a different speed for a certain period of time at a predetermined temperature.
  • Once more, the pellets are separated from the supernatant.
  • All particles are separated from one another by repeating these stages.
  • Testing for signs that are particular to the individual particles will then enable the identification of the particles.

Uses of Differential centrifugation

  • The separation of cell organelles and membranes often involves differential centrifugation.
  • It may also be used to separate the nucleus with limited resolution.
  • This method may be used to purify extracts that contain larger-sized contaminants since it separates particles depending on their sizes.
  • 4. Isopycnic centrifugation

Isopycnic centrifugation is a form of centrifugation in which the sample is subjected to centrifugal force while the particles are separated according to their densities.

Principle of Isopycnic centrifugation

  • Since particles are separated based only on their densities and not their sizes, isopycnic centrifugation is often referred to as equilibrium centrifugation.
  • Depending on the size of the particles, the particles migrate in a downward direction. And when the particle’s density reaches parity with that of the medium, it stops flowing. 
  • As we descend the tube toward the bottom, the gradient’s density rises. As a result, the less dense particles form bands above the denser particles, which are followed by the denser particles that fall to the bottom.
  • Since this is directly dependent on the buoyant densities and not the particle sizes, it is regarded as a real equilibrium.

Steps of Isopycnic centrifugation

  • A gradient is created with a gradient of increasing density at the tube’s bottom. The usage of a pre-created gradient is also possible.
  • The centrifuge tube is filled with a homogenous mixture of salt and biological material, and the centrifuge is then turned on.
  • A salt density gradient is created in the tube after the centrifuge has been running.
  • When the particles reach the area with their different densities, they travel down the tube and settle down.
  • Following that, the particles are sorted and identified using several additional procedures.

Uses of Isopycnic centrifugation

  • It is possible to use isopycnic centrifugation to purify huge quantities of biomolecules.
  • This method may be applied to figure out the densities of different types of particles.
  • 5. Rate-zonal density gradient centrifugation/Moving Zone Centrifugation

The process of rate-zonal density gradient centrifugation uses the same principles as density gradient centrifugation but operates differently to separate particles based on their shape rather than their size. Another name for it is moving zone centrifugation.

Principle of Rate-zonal density gradient centrifugation

  • Particles are separated by rates of zonal centrifugation according to size and form.
  • The process involves pouring a pre-poured density gradient over which a sample is to be layered in a confined area. After that, the density gradient is centrifuged.
  • Since the density gradient only contains densities significantly lower than the densities of the particles being centrifuged, all particles migrate into it.
  • The primary factors used to divide the particles are size and shape. A particle sediments more quickly the bigger it is.
  • A particle deposits more quickly the more spherically symmetrical it is.
  • The sedimentation coefficient of the particles determines how quickly they move along the gradient.
  • In rate-zonal centrifugation, as opposed to differential centrifugation, where the sample is dispersed throughout the medium, the sample initially only exists as a narrow band on top of the gradient.

Steps of rate-zonal density gradient centrifugation

  • A density gradient is created in a centrifuge tube before the sample is applied.
  • The same is then overlaid in the shape of a band on top of the gradient.
  • Because faster-moving, bigger, more spherical particles migrate ahead of slower ones during centrifugation, different particles are separated into distinct bands on varied gradients.
  • The particles are collected from the bottom of the tube through a puncture and are sorted based on their sedimentation coefficients. 

Uses of rate-zonal density gradient centrifugation

  • As each virus comprises components that are distinct in size and density, rate-zonal differential centrifugation has been utilised to separate the various viruses.
  • RNA on sucrose gradients has been fractionated using this technique.
  • Additionally, rate-zonal differential centrifugation has been used to separate, purify, and divide DNA molecules from bacteria and viruses.
  • One of the first uses of this technique was to fractionate polysomes and ribosomal subunits.
  • 6. Differential velocity (Moving Boundary) centrifugation

Differential velocity centrifugation is a type of centrifugation in which materials are isolated into distinct settlings down the length of a centrifuge tube with the use of a series of escalating speeds.

Principle of Differential velocity (Moving Boundary) centrifugation

  • Differential centrifugation is focused on differences in the settling rates of biological particles of varying sizes and densities.
  • The first stage of the bigger molecules’ sedimentation occurs as the rotors’ speed increases.
  • Depending on the density and relative sizes of the particles, as well as the rate and duration of each centrifugation stage, more particles settle down.
  • Smaller-sized structures are left in the supernatant after the biggest class of particles forms a pellet at the bottom of the centrifuge tube.
  • After removing the pellet, the supernatant is further centrifuged to separate the larger particles from the supernatant.
  • As a result, bigger molecules sediment more rapidly and at lower speeds than smaller molecules, which take longer and move at greater speeds.
  • Less dense particles will float in the absence of settling as compared to the medium.

Steps of Differential Velocity (Moving Boundary) Centrifugation

  • The buffer-containing medium homogenises the sample solution.
  • The sample is then put into the centrifuge tube, which is run for a certain amount of time at a predetermined temperature.
  • A pellet will eventually form at the bottom of the tube, which is then separated from the supernatant at the conclusion of this process.
  • The supernatant is transferred to a fresh centrifuge tube and centrifuged at a different speed for a certain period of time at a predetermined temperature.
  • Once more, the pellets are separated from the supernatant.
  • All particles are separated from one another by repeating these stages.
  • Testing for signs that are particular to the individual particles will then enable the identification of the particles.

Uses of Differential Velocity (Moving Boundary) Centrifugation

  • The separation of cell organelles and membranes often involves differential centrifugation.
  • It may also be used to separate the nucleus with limited resolution.
  • This method may be used to identify and compare particles of various sizes since it separates particles according to their sizes.
  • 7. Equilibrium density gradient centrifugation.

A specialised and modified kind of density gradient centrifugation is equilibrium density gradient centrifugation.

Principle of Equilibrium density gradient centrifugation

  • The theory behind equilibrium density gradient centrifugation is that particles in a solution are separated according to their densities.
  • In this instance, the particles travel along the gradient in densities until coming to a standstill when the medium’s density is equal to that of the particles.
  • At this moment, the buoyant force pushing the particles upward is equivalent to the centrifugal force exerted on the particles. The particles stop moving as a result, and they may then be divided into several layers.
  • As we descend the tube toward the bottom, the gradient’s density rises. As a result, the less dense particles form bands above the denser particles, which are followed by the denser particles that fall to the bottom.

Steps of Equilibrium density gradient centrifugation

  • A gradient is created with a gradient of increasing density at the tube’s bottom. The usage of a pre-created gradient is also possible.
  • The centrifuge tube is filled with a homogenous mixture of salt and biological material, and the centrifuge is then turned on.
  • A salt density gradient is created in the tube after the centrifuge has been running.
  • When the particles reach the area with their different densities, they travel down the tube and settle down.
  • Following that, the particles are sorted and identified using several additional procedures.

Uses of Equilibrium density gradient centrifugation

  • The purification of significant amounts of biomolecules can be accomplished via equilibrium density gradient centrifugation.
  • This method may be applied to figure out the densities of different types of particles.

Examples of Equilibrium density gradient centrifugation

  • Meelson and Stahl used this in their studies to calculate the densities of various DNA molecules based on where they reached along the density gradient.
  • 8. Sucrose gradient centrifugation

A particular sort of density gradient centrifugation called sucrose gradient centrifugation changes the sucrose concentration to create a density gradient.

Principle of Sucrose Gradient Centrifugation

  • Sucrose gradient centrifugation is based on the idea that molecules would settle down while being pulled apart by a centrifugal force until they come into contact with a medium that has the same density as them.
  • In this instance, a medium with a sucrose gradient—one with a lower density at the top and a larger density at the bottom—is used.
  • A centrifugal force is produced as molecules in a sample travel through the medium while it is spun.
  • As molecules pass over the gradient in density, they start to migrate towards the bottom.
  • When the particle density reaches the surrounding medium, the molecules start to float in midair.
  • This separates molecules with varied densities at distinct levels, where they may subsequently be retrieved using a variety of techniques.

Steps of Sucrose gradient centrifugation

  • The lower concentration of sucrose is carefully layered over the higher concentrations in a centrifuge tube to produce a sucrose density gradient.
  • The tubes are then inserted into an ultracentrifuge with the sample deposited over the gradient.
  • The particles move across the gradient until they arrive at a location where their density is the same as that of the medium around them.
  • The fractions are eliminated and separated, resulting in the separation of the particles.

Uses of Sucrose gradient centrifugation

  • Centrifugation using a gradient of sucrose is a potent method for separating macromolecules like DNA and RNA.
  • Additionally, it has been applied to analyse protein complexes and figure out the size and density of different other macromolecules.

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  • <1% – https://en.wikipedia.org/wiki/Differential_centrifugation
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