The process of centrifugation is separation of substances that involves the use to the centrifugal force. It is a method of separating particles from the solution based on their shape, size density, density, viscosity of the medium , and the speed of the rotor.
Definition of Centrifugation
Centrifugation is the method of separation of components. The force or acceleration of the centrifugal force makes the molecules with greater density move toward the outer edges, while the smaller particles are moved towards the center.
Centrifugation is based on the perpendicular force that is generated by a sample being rotated around a fixed location. The speed of centrifugation will depend on dimension and density of particles within the sample.
A centrifuge is a machine designed to separate elements of a mix on the basis of their dimensions, density as well as the viscosity of medium, and also the rotor speed.
The centrifuge is widely employed in labs for getting biological compounds separated from crude extract. In a centrifuge, the specimen is placed in a rotating device that rotates around an axis (axis) that results in an intense force that is perpendicular towards the axis. There are various kinds of centrifuges that are used for the separation of various molecules, however they all operate on the same principle of sedimentation.
What is Relative Centrifugal Force (RCF)?
- Relative centrifugal force is a measurement for the rate of strength in rotors that are of different sizes and types.
- The force is that is exerted on the inside of the rotor by the force of the rotor’s rotation.
- RCF is the force perpendicular to the surface applied to the sample, which is always in relation to the gravity of the earth.
- The RCF of different centrifuges is a good tool for analysis of rotors and allowing to select the best centrifuge to fulfill a specific task.
The formula used to calculate the force of centrifugal relative (RCF) could be written as follows:
RCF (g Force)= 1.118 × 10-5 × r × (RPM)^2
where r is the radius of the rotor (in centimeters), and RPM is the speed of the rotor in rotation per minute.
Objectives of Centrifugation
- To separate the liquids that are immiscible
- Purify the substance by eliminating impurities that are present in the supernatant fluid.
- To separate crystallized drugs from the liquor that is their mother.
- To test the emulsions and suspensions to determine if they are creaming or sedimenting at a speed that is faster.
Centrifugation Principle/how does centrifugation work?
In a solution, the particles that have a density higher than the density from the solvent sink (sediment) or particles that weigh less than the sink floats to the top. The more different density, the more quickly they’ll move. If there’s no distinction between the density of particles (isopycnic conditions) the particles remain constant. To make the most of even small variations in density, to distinguish different particles gravity is replaced by the more effective “centrifugal force” generated by centrifuge.
A centrifuge is an item of equipment that places an object in a rotational motion around an unfixed axis (spins it around a circle) by applying a massive force perpendicularly to the axis of rotation (outward). The centrifuge operates on the principle of sedimentation, in which the centripetal acceleration causes more dense particles and substances to expand away in the radial direction. In the same way objects with lower density are displaced and moved towards the center. In a centrifuge for laboratory use which uses tubes for sample collection this radial acceleration forces the denser particles to sink to the lower part of the tube while those with lower density rise up to the top.
Which factors have an influence on centrifugation :
- Density of both samples as well as the solution
- Displacement of particles’ distance
- The speed of rotation
Examples of centrifugation
Some common examples of centrifugation include:
- The extraction of fat from milk for the production of skimmed milk.
- The removal of water from moist lettuce with the help of a salad spinner.
- The Spin-drying of water in washing machines in order to remove water from the clothing.
- The separation of solid blood and urine materials into forensic and testing laboratories.
- The centrifuge is an container inside which an amalgamation of liquid and solid or two liquids are placed. Then, the container is turned at a very high speed. As the container rotates at high speed, the mixture is separated into its components by the effect of centrifugal force upon their density.
- The liquid or solid with greater specific gravity is thrown into the word with more force.
Sedimentation Principle of Centrifuge
In a solution, the particles with a density that is greater than that of the sink (sediment) and those which are lighter than the sink, float up to the top. The more different density, the more quickly they travel. If there isn’t any distinction between the intensity (isopyknic conditions) the particles remain in a steady state.
To make the most of tiny variations in density, to distinguish different particles within a solution gravity is replaced by the more potent “centrifugal force” that is provided by centrifuge.
The forces works during the centrifugation
Two forces oppose the centrifugal force that acts on suspended particles:
- Buoyant force: force by which particles have to move away from the liquid medium into which they settle.
- Frictional force: force created by particles when they move throughout the liquid.
Particles are able to move off the plane of motion in the field of a centrifugal force only when the force of centrifugal forces exceeds the buoyant and frictional counteracting forces, resulting in the sedimentation of particles in a steady rate.
Relative Centrifuge Force (RCF) expressed in xg (multiple of earth gravitational force)
RFC = 1,118 x R x (rpm /1000)²
R is radius in cm
rpm : Speed in Revolutions per minute
As a result, for the same speed, a radius 20% bigger gives a number of xg 20% higher.
How to convert between times gravity (×g) and centrifuge rotor speed (RPM)?
Certain procedures require specific centrifugation parameters, which need to be defined using the term the relative centrifugal force (RCF) measured in terms of gravity units (times gravity, or grams). Most microcentrifuges are equipped with the ability to set the speed (revolutions per minute (RPM, also known as RPM) but not the absolute centrifugal force. Therefore, a formula for conversion is needed for ensuring that the correct setting is utilized in the experiment. The relation with RPM as well as RCF is as the following:
g = (1.118 × 10-5) x R x S²
In which g is the ratio of centrifugal force and it is the radius the rotor has measured in centimeters while S represents the speed in rotations per minute. The RCF values in units of the times gravity (x g) for the most common microcentrifuges rotor radius are shown in the table below. In the example below, centrifugation at 5,000 RPM inside microcentrifuge with the rotor has 7 cm radius will produce a centrifugal strength of 1 957 x grams.
Speed and duration of centrifugation typically aren’t the most important factors when it comes to routine procedures for handling samples using the use of a benchtop microcentrifuge. In general the speed and time are adequate to ensure that the cells, debris or resin are effectively pelleted It doesn’t matter whether the speed is greater or the duration is longer than the time needed. For the same reason most protocols do not define the specific centrifugal force that should be used, but rather an overall guideline, such as “centrifuge at a high speed for one minute.”
Types of Centrifuge
1. Low-Speed Centrifuge
- Low-speed centrifuges are the most common centrifuges commonly used in labs to perform the regular sorting of the particles.
- The centrifuges are operated at the maximum speed of 4000-5000 rpm.
- They are typically operated at ambient temperature since they don’t have control mechanisms to regulate the speed or the temperature of the process.
- Fixed angle and swinging bucket types of rotors are used in these centrifuges.
- They are compact and easy to use centrifuges, which are great for the analysis of blood samples as well as different biological sample.
- The centrifuge that operates at low speed follows the same principle that is used in all other centrifuges, however the application is limited to removal between simpler and more efficient solutions.
2. High-Speed Centrifuges
- High-speed centrifuge as the name implies is the type of centrifuge which can operate at speeds that are slightly higher.
- Speed of the centrifuge with high speed may vary between 15,000 and 30,000 rpm.
- The high-speed centrifuge is typically employed in laboratories that are more sophisticated for biochemical applications and requires a fast speed of operation.
- High-speed centrifuges have an instrument for controlling both the temperature and speed of the procedure that is crucial to analyze sensitive biological molecules.
- The high-speed centrifuges are equipped with different adapters to fit samples tubes of different dimensions and volumes.
- The three types of rotors may be used to create the centrifugation process of these centrifuges.
- Ultracentrifuges run at very high speeds which allow separation of smaller molecules like proteins, ribosomes and even viruses.
- This is the highest advanced type of centrifuge which allows to separate molecules which can’t be separated by other centrifuges.
- Refrigeration systems are found in these centrifuges to help control the heat that is generated by the spinning.
- They could be up to 150,000 rpm.
- It can be used to aid in both analytical and preparation work.
- Ultracentrifuges can be used to separate molecules in large batches , and in a continuous flow.
- Apart from separation, ultracentrifuges may also be employed for the analysis of macromolecules’ characteristics, including the shape, size, and density.
- Microcentrifuges can be employed to separate small volumes of samples that range from 0.5 and 2 ul.
- Microcentrifuges usually operate at around 12,000-13,000 rpm.
- This is utilized for to separate molecularly organelles in cells like DNA, nuclei and nuclei as well as for extraction with phenol.
- Microcentrifuges also referred to as microfuge, utilize test tubes which are smaller as compared to the traditional test tubes that are utilized in larger centrifuges.
- Certain microcentrifuges have adapters that allow making use of bigger tubes with smaller ones.
- Microcentrifuges with temperature controls are available for the operation of temperature-sensitive samples.
5. Benchtop centrifuge
- Benchtop centrifuges are compact centrifuge, which is widely used in research and clinical laboratories.
- It is powered through an electric motor. the tubes rotate around an undetermined axis, which results in force perpendicularly towards the tubes.
- Because they are small, they can be useful for smaller labs that have smaller space.
- A variety of centrifuges for benchtop use are available on the market for a variety of uses.
- A benchtop centrifuge is equipped with an rotor that has racks for the tubes that are used to test and a lid that covers the unit that is used to operate the centrifuge.
6. Continuous flow centrifuge
- Continuous flow centrifuge can be described as a centrifuge that permits the centrifugation of huge volumes of samples, without impacting the rate of sedimentation.
- This kind of centrifuge permits the separation of an enormous amount of samples with high centrifugal force, thereby getting rid of the laborious task of emptying and filling tubes after each cycle.
- They have a shorter length of path that makes it easier for getting the solid portion from the supernatant thereby allowing for a faster pace that the operation can be carried out.
- They also have bigger capacities, which can speed up the process as the sample does not have to be loaded and unloaded repeatedly again , as in conventional centrifuges.
- One liter of sample can be removed using this centrifuge in a that lasts 4-hours or less.
7. Gas centrifuge
- A gas centrifuge can be described as a device specifically designed for the separation of gases that are based on isotopes.
- The centrifuge works on the principle of centrifugal force the same as other centrifuges in which it is possible to separate the molecular on basis of their mass.
- The centrifuge is used principally to extract and separate of uranium-238 and uranium-235.
- The gas centrifuge is based according to the plan of constant flow of gases into and out of the centrifuge unlike other centrifuges that work with batch processing.
- They are placed in cascades in order they can be split into two parts based on their isotopes. They are then transferred to the next centrifuge to continue processing.
- Gas centrifuges are replacing other gaseous diffusion techniques because they produce more gas concentration than previous methods.
8. Hematocrit centrifuge
- Hematocrit centrifuges are specially designed centrifuges that are used to determine the amount of the volume fraction of the erythrocytes (RBCs) within a specific blood sample.
- The centrifuge can provide hematocrit levels which can be used for tests in biochemistry, immune as well as blood tests, among other tests in general clinical use.
- Hematocrit centrifuges can be used to determine the cause of bleeding as well as polycythemia (an an elevation of the number of erythrocytes to higher than normal levels) anemia bone marrow dysfunction, leukemia, and myeloma.
- Microhematocrit’s centrifuge is able to reach rates of around 11,000 rpm, and RCFs up to 15,000 g for spin tubes with samples.
- A hematocrit centrifuge’s parts centrifuge for hematocrit are similar to those of the benchtop centrifuge, however this particular centrifuge is designed specifically to work with blood samples.
9. Refrigerated centrifuges
- Centrifuges that are refrigerated that come with temperature controls that span from -20degC up to 30degC.
- A different type of centrifuge is available with the control of temperature that is crucial for different processes that require lower temperatures.
- Refrigerated centrifuges are equipped with an instrument for controlling temperature as well as racks and rotors for the test tubes.
- These centrifuges offer an RCF that can reach 60,000 xg which is perfect for the separation of different biological molecules.
- They are commonly employed to collect substances which are rapidly separated, like the yeast cell, chloroplasts and erythrocytes.
- The refrigerated centrifuge’s chamber is shut by a barrier to ensure that it is suitable for the operation.
10. Vacuum centrifuge/ Concentrators
- Vacuum centrifuge makes use of centrifugal force and vacuum heat to speed up lab evaporation of the samples.
- These centrifuges can be used for processing large numbers of sample (up to up to 148 samples at once).
- This type of centrifuge can be employed in both biological and chemical laboratories to facilitate the efficient extraction of solvents from samples, thereby concentration of the samples.
- These are often employed in high-throughput labs for samples that may contain an abundance of solvents.
- A rotary evaporator can be used to eliminate solvents that are not needed and reduce bumping in the solvent.
- The centrifuge functions by lowering the chamber’s pressure and also reducing temperature of sample.
- The solvents will evaporate, causing the particles and causing them to separate.
Types of Centrifugation
1. Analytical Centrifugation
Analytical centrifugation is an approach to separation which is where the particles within the sample are separated based on their density as well as the force generated by centrifugal forces. The process of analytical ultracentrifugation (AUC) is a flexible and durable method to perform qualitative analysis of macromolecules in solution.
Principle of Analytical Centrifugation
Analytical centrifugation works on the idea that particles with greater density than other particles settle down more quickly. The larger molecules are more able to move when they exert centrifugal force than smaller ones. Analytical ultracentrifugation to determine of the molecular weight of a macromolecule could be carried out using a sedimentation speed method or a sedimentation equilibrium technique. The properties of macromolecules’ hydrodynamics are outlined in terms of their coefficients of sedimentation. They are determined by the speed at which the boundary of concentration for specific biomolecule moves in the field of gravitational force.
The sedimentation coefficient may be used to identify the changes in structure and size of macromolecules due to changes in the experimental conditions. There are three optical methods accessible for the ultracentrifuge for analytical purposes (absorbance as well as interference and fluorescence) which permit exact and precise monitoring of sedimentation in real time.
The steps of Analytical Centrifugation
- Small samples (20-120 millimeters) are collected in analytical cells and placed in the ultracentrifuge.
- The ultracentrifuge is operated to create a centrifugal force that creates a movement of biomolecules distributed randomly throughout the solvent, radiating away from the center of rotation.
- The distance of particles from their center are calculated using the Schlieren optical system.
- The graph can be drawn using the concentration of solutes against the squared radial separation from the centre of rotation upon the molecular mass measured.
Uses of Analytical Centrifugation
- Analytical centrifugation may be employed to determine the quality of macromolecules.
- It is also used to study modifications in the molecular mass and supramolecular structures.
- Additionally, it permits the calculation of the molecular mass for solutes in their original state.
2. Density gradient centrifugation
Density gradient centrifugation refers to an approach to separation between molecules in which the separation is determined by the concentration of molecules as they move through a density gradient under the influence of a centrifugal force.
Principle of Density gradient centrifugation
Density gradient centrifugation relies on the idea that molecules settle under the force of centrifugal forces until they find the same density in a medium similar to their own. In this instance the medium has the density gradient is utilized that needs to reduce density or increase density. Molecules within a sample travel through the medium while the sample rotates producing an axial force.
The molecules that are more dense start to shift towards the bottom of the gradient as they travel through the densities gradient. The molecules are then suspended at a level at which the density of particles is greater than that of the medium. This way molecules of different density are separated into various layers. They can be recovered through various methods.
Steps of Density gradient centrifugation
- A density gradient in the medium is produced by gently spreading the concentrations with lower levels over more concentrated ones in the centrifuge tube.
- The sample is placed over the gradient after which the tubes will be then placed inside an ultracentrifuge.
- The particles move along their gradients until they arrive at a point where their density is equal to with the denseness of medium around them.
- The fragments are then removed and separated, giving particles that are isolated.
Uses of Density gradient centrifugation
- Density gradient centrifugation may be used to purify huge quantities of biomolecules.
- It could even be utilized to purify various viruses, which can aid in their future research.
- This method can be utilized to separate particles as well as a method to determine the densities of different particles.
- Examples of centrifugation using a density gradient
- This technique was utilized for the long-running experiment which demonstrated that DNA was semi-conservative employing different nitrogen isotopes.
- Another instance is the use of this method for the removal of the microsomal fraction in muscle homogenates and then the segregation of membrane vesicles that have different density.
3. Differential centrifugation
Differential centrifugation is one type of centrifugation procedure where components are separated and placed into the centrifuge tube using an increasing centrifugal forces.
Principle of Differential centrifugation
Differential centrifugation is based on the variations in the rate of sedimentation of biological particles that vary in dimensions and densities. When the greater force of centrifugal forces is applied, the initial sedimentation of the bigger molecules begins. The particles continue to settle, based on the speed and length of the individual centrifugation processes as well as the density and relative size of the particles.
The biggest category of particles form in a small pellet at the inside of the tube leaving smaller-sized structures in the supernatant. So, larger molecules are able to settle rapidly and have lower centrifugal forces , while smaller molecules require longer time and have higher forces. When particles that are not as dense in comparison to the surrounding medium they tend to flounder instead of sinking.
Steps of Differential centrifugation
- A homogenized sample is created the buffer that contains it.
- The sample is then put inside the centrifuge tube which is run at the same centrifugal force for the specified amount of time at a specific temperature.
- After this process the pellet will have formed in the middle of the tube. It is then separated by the supernatant.
- The supernatant is then added to the new centrifuge tube, where it is then centrifuged at a new speed for a specific period of period of time and at a specific temperature.
- In addition, the supernatant gets separate from the pellets that have formed.
- This process continues until the components are isolated from one the other.
- The particles are identified by looking for specific indicators that are specific to the particular particle.
Uses of Differential centrifugation
- Differential centrifugation is a common method to separate membranes and organelles of cells within the cell.
- It is also used to separate the nucleus.
- Because this method is able to separate particles based on the size it can be used to purify extracts that have larger impurities.
4. Isopycnic centrifugation
Isopycnic Centrifugation is a form of centrifugation in which the particles within a specimen are distinguished on basis of their densities when an applied force of centrifugal force to the specimen.
Principle of Isopycnic centrifugation
Isopycnic centrifugation can also be referred to as the equilibrium centrifugation because particle separation happens only on the basis their density, not the size of the particles. The particles travel toward the bottom, and the speed of movement is dependent on the dimensions of particles. It ceases to flow when the particle’s density is equal to that of the medium surrounding it. The density of the gradient grows as we travel downwards towards the bottom. This is why the particles with the highest density fall to the bottom, and are followed by smaller particles that form bands above more dense particles. It is thought to be an equilibrium because it is directly influenced by buoyant density, not the dimensions of particles.
Steps of Isopycnic centrifugation
- A gradient that is created using increasing intensity towards the lower end of the tube is created. A gradient that has been pre-formed can also be utilized.
- The biological samples solution as well as salt is evenly distributed inside the centrifuge tube before being put inside the centrifuge.
- When the centrifuge is turned on the concentration gradient of salt will be formed inside the tube.
- The particles travel through the tube, and are able to settle as they enter the area with their respective densities.
- The particles are separated and identified by various other methods.
Uses of Isopycnic centrifugation
- The use of isopycnic centrifugation may be used to purify huge amounts of biomolecules.
- This technique is used as a method for determination of densities for various particles.
5. Rate-zonal density gradient centrifugation/ Moving Zone Centrifugation
Rate-zonal density gradient centrifugation a form of centrifugation that is used to separate particles based on their size as well as shape. It follows the same principle of density gradient centrifugation , however it operates in a different manner. It’s also known as move zone centrifugation.
Principle of Rate-zonal density gradient centrifugation
Rate zonal centrifugation divides particles according to dimensions and shapes. The process involves layering the sample within a limited area on top of a density gradient that has been pre-poured. The gradient is then centrifuged. The particles all move into the gradient due to the density gradient only has densities smaller than the particles that are being centrifuged.
The particles are divided according to size or shape. The larger the particle in size, the faster it swells. The more spherically-symmetrical a particle appears, the faster it dissolves. The particles move through this gradient with a speed that is proportional to their sedimentation ratio. In contrast to differential centrifugation, where the sample is distributed across the medium in rate-zonal centrifugation the sample initially appears only on the top of the gradient in an extremely narrow band.
Steps of Rate-zonal density gradient centrifugation
- Density gradients are made in a centrifuge tube prior using the test sample.
- This is then laid over the surface of the gradient, in the shape of the band.
- When centrifugation is performed, the particles that move fast (larger in dimensions and with a circular shape) are able to move faster than slower ones, so that particles are separated into different bands that are located on different regions of the gradient.
- They are separated from each other on foundation of sedimentation coefficients and they are extracted from the tube’s bottom by the perforation.
Uses of Rate-zonal density gradient centrifugation
- Rate-zonal centrifugation differential centrifugation has been used to separate viruses because they have components of different dimensions and density, which are distinct to each virus.
- This technique is used to fractionate RNA using sucrose gradients.
- In addition, rate-zonal differential centrifugation can also be employed to separate from, purification and fractionation of DNA molecules, both bacteria and viruses.
- A fractionation process for polysomes as well as subunits of ribosomes is one of the first applications of this technique.
6. Differential velocity (Moving Boundary) centrifugation
Differential velocity centrifugation (DVC) is a kind of centrifugation technique where components are separated and placed into the centrifuge tube using an increasing number of velocities.
Principle of Differential velocity (Moving Boundary) centrifugation
Differential centrifugation relies on the variations in the rate that biological matter is dissolved with different dimensions and sizes. With the speed increasing, the rotors are applied, the initial sedimentation of larger molecules occurs. More particles settle down based on the speed and length of each centrifugation step and also the density and dimensions of the particles.
The most massive class of particles forms a solid pellet at one side of the centrifuge tube creating smaller-sized structures in the supernatant. The pellet is removed and the supernatant is centrifuged to produce smaller particles. So, larger molecules are able to settle rapidly and with lower velocity and the smaller molecules take longer and have higher velocity. When particles that are not as dense in comparison to the media, they are likely to float rather than settle.
Steps of Differential velocity (Moving Boundary) centrifugation
- It is then homogenized within the buffer that contains it.
- The sample is placed inside the centrifuge tube which is run with a slower rotor speed for a certain amount of period of time and at a certain temperature.
- At the end of this procedure the pellet will have formed in the middle of the tube. This is then separated from supernatant.
- The supernatant gets added to an unopened centrifuge tube in which it is then centrifuged at a new speed for a specific period of duration and at a certain temperature.
- The supernatant is separate from the pellets that have formed.
- The process is repeated until the elements are separate from one another.
- The particles can be identified by testing for signs specific to the particular particle.
Uses of Differential velocity (Moving Boundary) centrifugation
- Differential centrifugation is widely used to separate cell membranes and organelles within the cell.
- It is also used to separate low resolution the nucleus.
- Since this method separates particles according to their size and shapes, it is a good tool to determine the size and compare of particles with different dimensions.
7. Equilibrium density gradient centrifugation
Equilibrium Density Gradient Centrifugation is an altered and specific form of centrifugation using a density gradient.
Principle of Equilibrium density gradient centrifugation
Equilibrium Density Gradient Centrifugation is built on the idea the particles within a solution can be separated based on their density. In this instance particles travel along the gradient of density and end in a place in which there is a density equivalent to that of the particle. In this case the force of centrifugal action against the particle similar with the force buoyant that pushes the particles upwards. This means that the particles stop moving and are divided into layers. The density of the gradient rises as we travel toward the bottom. This is why particles with higher density are able to settle at the bottom. They are then followed by smaller particles that form bands above more dense particles.
Steps of Equilibrium density gradient centrifugation
- A gradient with an increase in intensity towards the lower end of the tube is created. An already-performed gradient may also be utilized.
- The biological samples solution as well as salt is evenly distributed within the centrifuge tube, and then is then placed in the centrifuge.
- After the centrifuge has been operated the concentration gradient of salt is created in the tube.
- The particles travel along the tube, then are able to settle as they enter the area with their respective density.
- The particles are separated and identified using various other methods.
Uses of Equilibrium density gradient centrifugation
- The Equilibrium Density Gradient Centrifugation could be utilized to improve the purity of large quantities of biomolecules.
- This technique is used as a method for determination of the densities of different particles.
- Examples of Equilibrium centrifugation with a density gradient
- This technique has been employed in studies conducted by Meelson and Stahl to measure the density of various DNA molecules according to the point at which they landed in the gradient of density.
8. Sucrose gradient centrifugation
Sucrose gradient centrifugation is one kind of density gradient centrifugation in which it is made from sucrose by altering the sucrose concentration.
Principle of Sucrose gradient centrifugation
Sucrose gradient centrifugation works on the idea that molecules will settle under the force of a centrifugal until they find an environment with a density identical to their own. In this instance the medium has sucrose gradient is used or has smaller density towards the top, or a greater density on the lower end. Molecules within a sample are moved through the medium when the sample rotates producing an axial force.
The heavier molecules start moving toward the bottom as they traverse the densities gradient. The molecules are then suspended at a point which the density of particles is greater than that of the medium. In this manner molecules of various densities are separated in various layers. They can be recovered using different methods.
Steps of Sucrose gradient centrifugation
- A sucrose density gradient is created by gently placing the sucrose with a lower concentration over the higher concentrations of sucrose in the centrifuge tube.
- The sample is placed on top of the gradient after which the tubes will be then placed inside an ultracentrifuge.
- The particles move through this gradient till they arrive at a point where their density is equal to with the denseness of medium around them.
- The particles are separated and separated, giving the particles in separate units.
Uses of Sucrose gradient centrifugation
- Sucrose gradient centrifugation can be described as a efficient method to separate of macromolecules such as DNA and the RNA.
- It has also been utilized to study protein complexes as well as to measure the density and the size of other macromolecules.
Rotors in centrifuges are the motor devices that house the tubes with the samples. Centrifuge rotors are designed to generate rotation speed that can bring about the separation of components in a sample. There are three main types of rotors used in a centrifuge, which are:
1. Fixed angle rotors
These rotors hold the sample tubes at an angle of 45° in relation to the axis of the rotor. In this type of rotor, the particles strike the opposite side of the tube where the particles finally slide down and are collected at the bottom. These are faster than other types of rotors as the pathlength of the tubes increases. However, as the direction of the force is different from the position of the tube, some particles might remain at the sides of the tubes.
2. Swinging bucket rotors/ Horizontal rotors
Swinging bucket rotors hold the tubes at an angle of 90° as the rotor swings as the process is started. In this rotor, the tubes are suspended in the racks that allow the tubes to be moved enough to acquire the horizontal position. In this type of rotors, the particles are present along the direction or the path of the force that allows the particles to be moved away from the rotor towards the bottom of the tubes. Because the tubes remain horizontal, the supernatant remains as a flat surface allowing the deposited particles to be separated from the supernatant.
3. Vertical rotors
Vertical rotors provide the shortest pathlength, fastest run time, and the highest resolution of all the rotors. In vertical rotors, the tubes are vertical during the operation of the centrifuge. The yield of the rotor is not as ideal as the position of the tube doesn’t align with the direction of the centrifugal force. As a result, instead of settling down, particles tend o spread towards the outer wall of the tubes. These are commonly used in isopycnic and density gradient centrifugation.
Applications of Centrifugation
- To separate two miscible substances
- To determine the macromolecules’ hydrodynamic properties
- Mammalian cell purification
- Fractionation of organelles that are subcellular (including membrane fractions and membrane fragments) The membranes of vesicles undergo a fractionation
- The separation of chalk and water
- Eliminating fat from milk in order to create skimmed milk
- Separating air-flow particles from the particles through the process of cyclonic separation
- Stabilization and clarification of the wine.
- Separation of urine components from blood components in research and forensic labs
- It aids in the separation of proteins through purification techniques like salting out e.g. ammonium sulfate precipitation.