A centrifuge uses centrifugal force in order to separate different components from a fluid. This is done by spinning fluid at high speeds within a container. It can then separate fluids with different densities (e.g. Cream from milk, or liquids and solids. It causes particles and substances that are denser to move in the radial direction. Conversely, objects with a lower density are displaced and moved to the centre. A laboratory centrifuge using sample tubes causes the radial acceleration to cause denser particles settle to the bottom of a tube while lower-density substances rise up to the top. A centrifuge is a powerful filter that can separate contaminants from main fluid.
In manufacturing and waste treatment, industrial centrifuges are used to remove suspended solids or separate immiscible fluids. One example of this is the cream separator used in dairy plants. Ultracentrifuges and high-speed centrifuges that can provide high accelerations are capable of separating fine particles down to the nanoscale and molecules with different masses. For example, large centrifuges can be used to simulate high acceleration or gravity environments (for pilot training). For the extraction of water from fabrics, medium-sized centrifuges can be found in washing machines as well as at swimming pools. Gas centrifuges can be used to separate isotopes, such as enriching nuclear fuel for fissile elements.
Benjamin Robins (1707-1751), an English military engineer, invented a whirling-arm apparatus to measure drag. Antonin Prandtl, an 1864 inventor of the dairy centrifuge that separates cream from milk, proposed the idea. Alexander Prandtl, his brother, put the idea into practice and displayed a functioning butterfat extraction machine in 1875.
Definition of Centrifugation
Centrifugation is a process that involves the use of a centrifuge to separate particles from a solution based on their size, shape, density, or other physical properties. A centrifuge is a device that rotates a sample at high speeds to generate a centripetal force, which causes the heavier particles to sediment to the bottom of a tube or rotor while the lighter particles remain suspended.
There are several types of centrifuges, including high-speed centrifuges, low-speed centrifuges, and ultracentrifuges. High-speed centrifuges are used to separate cells and other biological materials, while low-speed centrifuges are used to separate non-biological materials such as suspensions of crystals or inks. Ultracentrifuges are used to separate very small particles or to measure the size, shape, and density of particles.
Centrifugation is a widely used technique in research, industry, and clinical laboratories, and it has many applications, including the purification of biological samples, the separation of cells and organelles, the analysis of proteins and nucleic acids, and the preparation of diagnostic specimens.
A centrifuge is a device that uses centrifugal force to separate particles from a solution based on their size, shape, density, or other physical properties. It consists of a motor, a rotating head or rotor, and a series of tubes or cups that hold the sample. When the rotor is rotated at high speeds, the centripetal force generated causes the heavier particles to sediment to the bottom of the tubes or cups while the lighter particles remain suspended.
There are several types of centrifuges, including high-speed centrifuges, low-speed centrifuges, and ultracentrifuges. High-speed centrifuges are used to separate cells and other biological materials, while low-speed centrifuges are used to separate non-biological materials such as suspensions of crystals or inks. Ultracentrifuges are used to separate very small particles or to measure the size, shape, and density of particles.
Centrifuges are widely used in research, industry, and clinical laboratories, and they have many applications, including the purification of biological samples, the separation of cells and organelles, the analysis of proteins and nucleic acids, and the preparation of diagnostic specimens.
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
The main objectives of centrifugation are to separate particles from a solution based on their size, shape, density, or other physical properties, and to purify or isolate specific components of the sample. Centrifugation is a widely used technique in research, industry, and clinical laboratories, and it has many applications, including the following:
- Separation of cells and organelles: Centrifugation can be used to separate cells from tissue samples, or to isolate specific organelles such as mitochondria, nuclei, or ribosomes.
- Purification of biological samples: Centrifugation can be used to remove contaminants or unwanted materials from a sample, such as cells, proteins, or nucleic acids.
- Analysis of proteins and nucleic acids: Centrifugation can be used to separate proteins or nucleic acids based on their size, charge, or other physical properties, allowing for the analysis of specific molecules.
- Preparation of diagnostic specimens: Centrifugation is used in clinical laboratories to prepare diagnostic specimens, such as blood or urine, for analysis.
- Clarification of suspensions: Centrifugation can be used to remove suspended particles from a solution, resulting in a clearer sample.
- Separation of immiscible liquids: Centrifugation can be used to separate immiscible liquids, such as oil and water, based on their densities.
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.
Examples of Analytical Centrifugation
Analytical centrifugation is a technique that uses a centrifuge to measure the size, shape, and density of particles in a sample. Some examples of analytical centrifugation techniques are:
- Sedimentation velocity centrifugation: This technique uses a centrifuge to measure the size and shape of particles based on their sedimentation rate. The sample is placed in a tube or rotor and rotated at high speeds, causing the particles to sediment through a fluid. The sedimentation rate is measured using a detector, such as a laser or interferometer, and the data is used to calculate the size and shape of the particles.
- Sedimentation equilibrium centrifugation: This technique uses a centrifuge to measure the size, shape, and density of particles based on their sedimentation equilibrium. The sample is placed in a tube or rotor and rotated at high speeds, causing the particles to sediment through a fluid until they reach a state of equilibrium. The concentration of the particles is measured using a detector, such as a UV spectrophotometer, and the data is used to calculate the size, shape, and density of the particles.
- Analytical ultracentrifugation: This technique uses an ultracentrifuge to measure the size, shape, and density of very small particles, such as proteins or nucleic acids. The sample is placed in a tube or rotor and rotated at high speeds, causing the particles to sediment through a fluid. The sedimentation rate is measured using a detector, such as a laser or interferometer, and the data is used to calculate the size, shape, and density of the particles.
- Dynamic light scattering: This technique uses a laser to measure the size and shape of particles based on their scattering of light. The sample is placed in a cuvette and illuminated with a laser, and the scattered light is detected and used to calculate the size and shape of the particles.
- Electrophoretic light scattering: This technique uses a laser to measure the size and shape of particles based on their electrophoretic mobility. The sample is placed in a cuvette and illuminated with a laser, and the scattered light is detected and used to calculate the size and shape of the particles.
Advantages Analytical Centrifugation
- High resolution: Analytical centrifugation allows for the measurement of the size, shape, and density of particles with a high degree of resolution.
- Versatility: Analytical centrifugation can be used to measure a wide range of particles, including proteins, nucleic acids, cells, and organelles.
- Non-invasive: Many analytical centrifugation techniques are non-invasive, meaning they do not alter or damage the sample during the measurement process.
- High sensitivity: Analytical centrifugation techniques are highly sensitive and can detect even small amounts of sample.
Disadvantages Analytical Centrifugation
- Complexity: Analytical centrifugation techniques can be complex and require specialized equipment and expertise.
- Limited to measuring particles based on size, shape, and density: Analytical centrifugation is limited to measuring particles based on their size, shape, and density, and it may not be suitable for measuring other physical properties.
- Time-consuming: Analytical centrifugation techniques can be time-consuming, particularly if multiple measurements are required.
- Expense: Analytical centrifugation techniques require specialized equipment and consumables, which can be expensive.
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.
Examples of Density gradient centrifugation
Density gradient centrifugation is a type of centrifugation that uses a gradient of increasing density to separate particles based on their density or size. Some examples of density gradient centrifugation techniques are:
- Isopycnic centrifugation: This technique uses a density gradient to separate molecules based on their size and shape. The sample is layered on top of the gradient, and the molecules sediment through the gradient based on their density.
- Rate-zonal centrifugation: This technique uses a gradient of increasing density to separate molecules based on their size and shape. The sample is layered on top of the gradient, and the molecules sediment through the gradient based on their size and shape.
- Sedimentation velocity centrifugation: This technique uses a density gradient to separate molecules based on their size and shape. The sample is layered on top of the gradient, and the molecules sediment through the gradient based on their size, shape, and density.
- Sucrose gradient centrifugation: This technique uses a gradient of increasing sucrose density to separate organelles, proteins, or nucleic acids based on their size and shape. The sample is layered on top of the gradient, and the molecules sediment through the gradient based on their size, shape, and density.
- Percoll gradient centrifugation: This technique uses a gradient of increasing percoll density to separate cells or organelles based on their size and density. The sample is layered on top of the gradient, and the cells or organelles sediment through the gradient based on their size, shape, and density.
Advantages Density gradient centrifugation
- High resolution: Density gradient centrifugation allows for the separation of molecules with a high degree of resolution, making it useful for the purification of specific molecules or organelles.
- High efficiency: Density gradient centrifugation is highly efficient at separating molecules based on their size, shape, and density, allowing for the separation of large quantities of sample in a short amount of time.
- Gentle on samples: Density gradient centrifugation is relatively gentle on samples, as it does not rely on physical forces such as filtration or sedimentation.
- Versatility: Density gradient centrifugation can be used to separate a wide range of molecules, including proteins, nucleic acids, cells, and organelles.
Disadvantages Density gradient centrifugation
- Complexity: Density gradient centrifugation requires the preparation of a gradient, which can be time-consuming and requires specialized equipment and expertise.
- Limited to separating molecules based on density: Density gradient centrifugation is limited to separating molecules based on their density, size, and shape, and it may not be suitable for separating molecules based on other physical properties.
- Limited to separating molecules with a narrow size range: Density gradient centrifugation is most effective at separating molecules with a narrow size range, and it may not be suitable for separating molecules with a wide size range.
- Expense: Density gradient centrifugation requires specialized equipment and consumables, which can be expensive.
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.
Types of centrifuge used in pharmaceutical industry
There are four types of centrifuges that are used in pharmaceutical production. They all have different characteristics.
A industrial centrifuge is an equipment that uses centrifugal force to separate fluids or particles. There are three types of centrifuges: liquid-liquid separation, liquid-liquid-solid separation and liquid-liquid–solid separation. This article will discuss four types of centrifuges used in the pharmaceutical industry.
1. Horizontal peeler centrifuges
The principle of centrifugal force is used to separate solids from liquids using density difference. You can use this method to separate salt from water, or urea from the unconverted carbamate mixture. High rotation speeds provide the centrifugal force necessary to allow suspended solids settle on the drum’s inner surface. All washing steps are done at the same speed and in the exact same centrifuge vessel.
Its unique feature is its constant basket speed. This allows for all operations such as feeding and drying, drying and discharging to be done at full basket speed. Horizontal peeler centrifuges are available in both batch and continuous mode.
This centrifuge’s peeler is controlled by a hydraulic piston and a peeler shaft. The blade and mounting arrangement are located above the discharge chute, so that scraped cakes can be directly deposited into the chute.
2. Top discharge centrifuges
They are an economical alternative to pharmaceutical production. Top discharge centrifuges are easily adaptable to product characteristics. They can handle a variety of products in small and large batches. The machine can discharge gentle solids without causing particle destruction.
This type usually has an automatic clean in-place (CIP), and a lossless filter cakes discharge. Cleaning is easy for machinery parts in direct contact with the product.
3. Vertical peeler centrifuges
Vertical peeler centrifuges operate under discontinuous filtration and are well-suited for the efficient separation of pharmaceutical and fine chemicals. This centrifuge can be used for small batches and products that change frequently.
Because the peeling process is done at a slower speed, there are no crystal breaks. The process housing opens/closes automatically, allowing for complete inspection of all parts within the area. The cleaning process is performed by a fully automated CIP system. It can also flood the process enclosure, if required.
This centrifuge machinery is suited for separating Amino acids, benzoic acid, benzene, sulphur, calcium hypochloride, hexachlorocyclohexane, insulin, penicillin, and starch, for example.
4. Inverting filter centrifuges
The filtration system can automatically discharge the particulates collected. This filtration system is ideal for filtering process waste or difficult-to-filter products. It has a gentle product discharge and leaves no residue heel. The particulates are kept in their original shape by the discharge method, which does not require pressing or scraping. Hydraulic systems can be contaminated by the mechanically controlled system. It spins at high speeds with maximum basket loads and consumes relatively little washing medium.
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.
Fixed-angle rotors are centrifuge rotors that have a fixed angle of inclination and are used in centrifuges to separate particles based on size and density. Fixed-angle rotors are typically used for high-speed separations, and are capable of reaching very high speeds.
There are several advantages to using fixed-angle rotors:
- High speed: Fixed-angle rotors are capable of reaching very high speeds, making them ideal for separating particles based on size and density.
- Efficient separation: Because the samples are suspended at a fixed angle, fixed-angle rotors are generally more efficient at separating particles compared to swing-bucket rotors.
- Variety of sizes: Fixed-angle rotors come in a range of sizes and designs, making them suitable for a variety of applications and sample types.
- Versatility: Fixed-angle rotors can be used for a wide range of applications, including separating proteins, nucleic acids, cells, and other particles.
- Durability: Fixed-angle rotors are generally more durable and long-lasting compared to other types of rotors.
It is important to choose the appropriate fixed-angle rotor for your specific application, as different rotors are optimized for different types of separations and sample volumes.
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.
Vertical rotors are designed for use in vertical centrifuges, which are laboratory instruments used to separate particles based on their size and density. Vertical rotors are typically smaller and more compact than horizontal rotors, and are designed for use with smaller volumes of samples.
There are several advantages to using vertical rotors:
- High speed: Vertical rotors are capable of reaching very high speeds, making them ideal for separating particles based on size and density.
- Compact size: Vertical rotors are typically smaller and more compact than horizontal rotors, making them ideal for use in smaller laboratories or when space is limited.
- Efficient separation: Because the samples are suspended in a vertical position, vertical rotors are generally more efficient at separating particles compared to horizontal rotors.
- Variety of sizes: Vertical rotors come in a range of sizes and designs, making them suitable for a variety of applications and sample types.
- Versatility: Vertical rotors can be used for a wide range of applications, including separating proteins, nucleic acids, cells, and other particles.
It is important to choose the appropriate vertical rotor for your specific application, as different rotors are optimized for different types of separations and sample volumes.
4. Horizontal rotors
These rotors are designed for high-capacity separation and are often used for separating large volumes of samples.
Horizontal centrifuge rotors are designed for use in horizontal centrifuges, which are laboratory instruments used to separate particles based on their size and density. Horizontal rotors are typically larger and more robust than other types of rotors, and are designed for use with large volumes of samples.
There are several advantages to using horizontal centrifuge rotors:
- High capacity: Horizontal rotors are capable of separating large volumes of samples, making them ideal for use in high-throughput applications.
- Gentle separation: Because the samples are suspended in a horizontal position, horizontal rotors are generally less stressful on the samples compared to vertical rotors. This can be beneficial for sensitive samples, such as cells or proteins.
- Easy loading and unloading: Horizontal rotors typically have a front-loading design, which makes it easy to load and unload samples.
- Versatility: Horizontal rotors come in a range of sizes and designs, making them suitable for a variety of applications and sample types.
- Durability: Horizontal rotors are generally more durable and long-lasting compared to other types of rotors.
It is important to choose the appropriate horizontal rotor for your specific application, as different rotors are optimized for different types of separations and sample volumes.
5. Specialty rotors
There are many other types of specialty rotors available, including those designed for use with microtubes, tubes with large diameters, and tubes with irregular shapes.
Specialty centrifuge rotors are designed for specific applications or types of samples. Some examples of specialty rotors include:
- Microcentrifuge rotors: These rotors are designed for use with microtubes and are often used for high-speed separations of small volumes of samples.
- Large-capacity rotors: These rotors are designed for use with tubes with large diameters and are often used for separating large volumes of samples.
- Irregular-shape rotors: These rotors are designed for use with tubes with irregular shapes, such as round-bottom tubes or tubes with tapered ends.
- Preparative rotors: These rotors are designed for use in preparative centrifugation, which is a process used to purify or concentrate samples.
- Thermal rotors: These rotors are designed to maintain a constant temperature during the centrifugation process, which is important for certain types of samples, such as enzymes or proteins.
- Ultracentrifuge rotors: These rotors are designed for use in ultracentrifugation, which is a high-speed centrifugation process used to separate particles based on their size and density.
It is important to choose the appropriate specialty rotor for your specific application, as different rotors are optimized for different types of separations and sample volumes.
6. Other Types of Centrifuge rotor
- Drum rotors: Drum-rotors are specialized, high-centrifugal rotors. The samples are placed in a cassette, and then vertically into the rotating rotor. The samples will not move from their vertical position. The sedimentation process works in a similar way to swing-bucket, but at higher speeds. These rotors are very efficient at density separation, but they are not suitable for pelleting.
- Zonal Rotors: Zonel rotors can be either batch- or continuous-flow. They are more commonly used than the latter and are intended to reduce the wall effect in fixed angle and swinging bucket rotors and increase the sample size.
- Elutriator Rotors: The elutriator rotor is a type of continuous flow rotor. It has recesses that hold one conical shaped separation room, whose apex points away from the axis. There’s also a bypass chamber that acts as a counterbalance and provides the fluid outlet.
Care of Rotors
The anodized protective coating on the aluminium rotor is extremely thin and does not offer much protection against corrosion. Rotors should be treated with care to avoid scratching. It is important to thoroughly wash rotors with de-ionized water. Since moisture can cause corrosion, it is best to allow them to dry upside down in warm conditions. After drying, they should be kept dry in a dry, clean environment. Only the outer surfaces of rotors can be protected with lanolin or silicone polish.
However, swiping-bucket rotors should not be fully submerged in water. The bucket hanging system can be difficult to dry. The rotors made of titanium are resistant to corrosion. To avoid vibration-induced damage to the drive shaft of a centrifuge, it is important to balance the sample loads within the specified limits. The balance of the rotor should be maintained by not running the swiping-bucket rotor with any bucket or cap removed.
Metal fatigue can occur when the rotor is being decelerated or accelerates. This can be caused by cyclic stretching or relaxing of metal. To prevent overstressing the motor and ensure safe operation, it is important to keep a detailed record of all its usage. This includes speed, duration, and number of runs. The manufacturer can then either de-rate the motor after a specified number of runs, or replace it after a specific time.
How To Operate The Centrifuge Tubes
Centrifuge tubes are cylindrical-shaped, calibrated glass containers that can be used to analyze and separate various materials in the laboratory. For the experiments, they had to fit in the slots of the centrifuge.
These devices are used by lab technicians to test liquids and solid-liquid samples. They first deposit the substances in the tubes, then secure them within the slots. The lab technicians then spin the tubes at the right speeds to ensure that the denser particles of each substance collect at the point closest to the axis, and vice versa.
There are many options for laboratory centrifuge tubes in plastic, copolymers and polypropylene.
You can find everything from small tubes with caps for micro-centrifuges to large tubes that can hold 50,000 grams in frigerated centrifuges.
Types of centrifuge tubes
Different types of centrifuge tubes are widely used for various purposes, such as:
- Conical Bottom Tubes
- Microcentrifuge Tubes- Snap Cap
- Microcentrifuge Tubes – Screw Cap and So On
How to use a centrifuge safely
These are the key guidelines to follow when operating a centrifuge tube to avoid damage and potential injuries to lab technicians or doctors.
- You should always work on an even surface. You should ensure that the surface is level and firm.
- Balance the centrifuge tubes within the rotor is safe. For example, if you need to run a centrifuge with 20mL liquid, you can put another tube with 20mL water in the opposite hole. If the liquid is of a different density than water, it is best to balance the tubes using mass and not volume.
- Do not open the lid when the rotor is in motion. This can sometimes prove to be dangerous. There are centrifuges with safety shutoffs. The lid can be opened to prevent the rotor from spinning. Because of its inertia, the rotor will continue to spin so it is best to be cautious.
- The centrifuge tubes rotate, so there is some vibration. However, excessive wobbling could be dangerous. Double check that the tubes are properly balanced. You should not continue to run a centrifuge that is shaking, wobbling, or spinning while it is spinning.
- It is always a good idea to wear safety glasses or a face shield when working in a laboratory. It is also a good idea to wear safety glasses if you work near a centrifuge.
- While the centrifuge is still spinning, do not touch, bump, or move it.
- To avoid any disruptions, it is better to clean your centrifuge and rotor after every use.
Applications of Centrifugation
- Separations in Laboratory: There are many laboratory-scale centrifuges that can be used to separate suspended liquids or immiscible liquids in biology, chemistry, and clinical medicine.
- Separation of Isotope: There are other centrifuges that separate isotopes. The first being the Zippe type centrifuge. These centrifuges can be used in nuclear power or nuclear weapon programs.
- Industrial centrifugal separator: This coolant filtration system is used to separate particles from liquids like grinding coolant or machining coolant. It is used to separate non-ferrous particles such as silicon, glass and ceramic. It does not use any consumable parts such as filter bags. This saves the earth.
- Geotechnical centrifuge modeling: Geotechnical Centrifuge Modelling is used to test models that involve soils. To scale models, centrifuge acceleration is used to create prototype scale stresses. Problems like building foundations, earth dams and tunnels, as well as slope stability.
- Materials Synthesis: The high gravity conditions created by the centrifuge can be used in the chemical industry, casting and material synthesis. Gravitational conditions can greatly affect convection and mass transfers. Researchers found that high-gravity levels can have a significant impact on the product’s phase composition and morphology.
- Commercial applications:
- Hand-washed clothes can be dried in a standalone centrifuge, usually with a water outlet.
- To get rid of water from laundry loads, washing machines can be used as centrifuges.
- In Mission: SPACE at Epcot, Walt Disney World, centrifuges are used to propel riders. This attraction uses a combination of a centerifuge and motion simulator to give the illusion of going into space.
- Centrifuges use centrifugal acceleration in soil mechanics to match soil stresses in scale models to real-life conditions.
- Many large industrial centrifuges can be used to dry sludges in wastewater and water treatment. The resultant dry product is commonly called cake. After most solids have been removed, the centrifuge leaves behind what is known as centrate.
- For the removal of solids from drilling fluid, large industrial centrifuges can also be used by the oil industry.
- Some companies use disc-stack centrifuges to separate bitumen from water and other solids.
- Centrifuges can be used to separate cream from milk (remove fat). See Separator (milk).
- Serwer, BACTERIOPHAGES: SEPARATION OF, Editor(s): Ian D. Wilson, Encyclopedia of Separation Science, Academic Press, 2000, Pages 2102-2109,
- Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology. Eighth edition. Cambridge University Press: New York.