Types of Centrifuge & Centrifugation (definition, principle, uses)

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Last Updated on September 4, 2020 by Sagar Aryal

Centrifuge definition

A centrifuge is a device used to separate components of a mixture on the basis of their size, density, the viscosity of the medium, and the rotor speed.

  • The centrifuge is commonly used in laboratories for the separation of biological molecules from a crude extract.
  • In a centrifuge, the sample is kept in a rotor that is rotated about a fixed point (axis), resulting in strong force perpendicular to the axis.
  • There are different types of centrifuge used for the separation of different molecules, but they all work on the principle of sedimentation.

Centrifugation definition

Centrifugation is the technique of separating components where the centrifugal force/ acceleration causes the denser molecules to move towards the periphery while the less dense particles move to the center.

  • The process of centrifugation relies on the perpendicular force created when a sample is rotated about a fixed point.
  • The rate of centrifugation is dependent on the size and density of the particles present in the solution.

Read More: Centrifugation- Principle, Types, and Applications

Types of Centrifuge and Centrifugation

Image created using biorender.com.

Relative Centrifugal Force (RCF)

  • Relative centrifugal force is the measure of the strength of rotors of different types and sizes.
  • This is the force exerted on the contents of the rotor as a result of the rotation.
  • RCF is the perpendicular force acting on the sample that is always relative to the gravity of the earth.
  • The RCF of the different centrifuge can be used for the comparison of rotors, allowing the selection of the best centrifuge for a particular function.

The formula to calculate the relative centrifugal force (RCF) can be written as:

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.

Centrifuge Rotors

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

Fixed angle rotors

Figure: Fixed angle rotors. Image Source: Beckman Coulter, Inc.

  • 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 or Horizontal rotors

Figure: Swinging bucket rotors/ Horizontal rotors. Image Source: Beckman Coulter, Inc.

  • 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

Figure: Vertical rotors. Image Source: Beckman Coulter, Inc.

  • 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.

Types of Centrifuge Rotors Video Lecture (nptelhrd)

Types of centrifuges

1. Benchtop centrifuge

Benchtop centrifuge

Figure: Thermo Scientific™ Sorvall™ ST 8 Small Benchtop Centrifuge. Image Source: Thermo Scientific.

  • Benchtop centrifuge is a compact centrifuge that is commonly used in clinical and research laboratories.
  • It is driven by an electric motor where the tubes are rotated about a fixed axis, resulting in force perpendicular to the tubes.
  • Because these are very compact, they are useful in smaller laboratories with smaller spaces.
  • Different variations of benchtop centrifuges are available in the market for various purposes.
  • A benchtop centrifuge has a rotor with racks for the sample tubes and a lid that closes the working unit of the centrifuge.

2. Continuous flow centrifuge

Continuous flow centrifuge Principle

Figure: Equipment arrangement during continuous flow centrifugation. Image Source: Beckman Coulter, Inc.

  • Continuous flow centrifuge is a rapid centrifuge that allows the centrifugation of large volumes of samples without affecting the sedimentation rates.
  • This type of centrifuge allows the separation of a large volume of samples at high centrifugal force, thus removing the tedious part of emptying and filling the tubes with each cycle.
  • They have a shorter pathlength which facilitates the process of pelleting out the solid part out of the supernatant, thus maintaining the speed of the process.
  • They also have larger capacities which saves time as the sample doesn’t have to be load and unloaded over and over again like in traditional centrifuges.
  • Up to 1 liter of samples can be centrifuged by this centrifuge at a time period of 4 hours or less.

3. Gas centrifuge

Gas centrifuge

Figure: Diagram of a gas centrifuge with countercurrent flow, used for separating isotopes of uranium. Image Source: Wikipedia (Inductiveload).

  • A gas centrifuge is a centrifuge explicitly used for the separation of gases based on heir isotopes.
  • This centrifuge is based on the same principle of centrifugal force as all other centrifuges where the molecules are separated on the basis of their masses.
  • This centrifuge is used mainly for the extraction and separation of uranium -235 and uranium-238.
  • The gas centrifuge works on eh design of the continuous flow of gas in and out of the centrifuge, unlike other centrifuge working on batch processing.
  • These centrifuges are arranged in cascades so that the gases are separated into two units based on their isotopes and then are passed onto the next centrifuge for further processing.
  • Gas centrifuges have replaced other gaseous diffusion methods as they provide a yield of higher concentration of the gases than the previous techniques.

4. Hematocrit centrifuge

Hematocrit centrifuge

Figure: AHN myLab® Hematocrit Centrifuge. Image Source: AHN Biotechnologie GmbH.

  • Hematocrit centrifuges are specialized centrifuges used for the determination of volume fraction of erythrocytes (RBCs) in a given blood sample.
  • This centrifuge provides hematocrit values that can be used for testing in biochemistry, immunity, blood test, and other general clinical tests.
  • Hematocrit centrifuges may be used to help diagnose blood loss, polycythemia (an elevation of the erythrocyte count to above-normal levels), anemia, bone marrow failure, leukemia, and multiple myeloma.
  • The microhematocrit centrifuge quickly attains speeds of 11,000 rpm and RCFs of up to 15,000 g to spin tube samples.
  • The components of a hematocrit centrifuge are similar to that of the benchtop centrifuge, but this centrifuge is specialized for the use of blood samples.

5. High-speed centrifuge

High-speed centrifuge

Figure: Avanti JXN-30 Series High-speed centrifuge. Image Source: Beckman Coulter, Inc.

  • High-speed centrifuge, as the name suggests, is the centrifuge that can be operated at somewhat larger speeds.
  • The speed of the high-speed centrifuge can range from 15,000 to 30,000 rpm.
  • The high-speed centrifuge is commonly used in more sophisticated laboratories with the biochemical application and requires a high speed of operations.
  • High-speed centrifuges are provided with a system for controlling the speed and temperature of the process, which is necessary for the analysis of sensitive biological molecules.
  • The high-speed centrifuges come with different adapters to accommodate the sample tubes of various sizes and volumes.
  • All three types of rotors can be used for the centrifugation process in these centrifuges.

6. Low-speed centrifuge

Low-speed centrifuge

Figure: Low-speed centrifuges – ScanSpeed 406 and ScanSpeed 416. Image Source: LaboGene.

  • Low-speed centrifuges are the traditional centrifuges that are commonly used in laboratories for the routine separation of particles.
  • These centrifuges operate at the maximum speed of 4000-5000 rpm.
  • These are usually operated under room temperature as they are not provided with a system for controlling the speed or temperature of the operation.
  • Swinging bucket and fixed angle type of rotors can be used in these centrifuges.
  • These are easy and compact centrifuges that are ideal for the analysis of blood samples and other biological samples.
  • The low-speed centrifuge works on the same principle as all other centrifuges, but the application is limited to the separation of simpler solutions.

7. Microcentrifuge

Microcentrifuge

Figure: Microfuge 16 and Microfuge 20. Image Source: Beckman Coulter, Inc.

  • Microcentrifuges are the centrifuges used for the separation of samples with smaller volumes ranging from 0.5 to 2 µl.
  • Microcentrifuges are usually operated at a speed of about 12,000-13,000 rpm.
  • This is used for the molecular separation of cell organelles like nuclei and DNA and phenol extraction.
  • Microcentrifuges, also termed, microfuge, use sample tubes that are smaller in size when compared to the standard test tubes used in larger centrifuges.
  • Some microcentrifuges come with adapters that facilitate the use of larger tubes along with the smaller ones.
  • Microcentrifuges with temperature controls are available for the operation of temperature-sensitive samples.

8. Refrigerated centrifuges

Refrigerated centrifuges

Figure: Allegra 64R Refrigerated Benchtop Centrifuge. Image Source: Beckman Coulter, Inc.

  • Refrigerated centrifuges are the centrifuges that are provided with temperature control ranging from -20°C to -30°C.
  • A different variation of centrifuges is available that has the system of temperature control which is essential for various processes requiring lower temperatures.
  • Refrigerated centrifuges have a temperature control unit in addition to the rotors and racks for the sample tubes.
  • These centrifuges provide the RCF of up to 60,000 xg that is ideal for the separation of various biological molecules.
  • These are typically used for collecting substances that separate rapidly like yeast cells, chloroplasts, and erythrocytes.
  • The chamber of refrigerated centrifuge is sealed off from the outside to meet the conditions of the operations.

9. Ultracentrifuges

Ultracentrifuges

Figure: Ultracentrifuges. Image Source: Beckman Coulter, Inc.

  • Ultracentrifuges are the centrifuges that operate at extremely high speeds that allow the separation of much smaller molecules like ribosomes, proteins, and viruses.
  • It is the most sophisticated type of centrifuge that allows the separation of molecules that cannot be separated with other centrifuges.
  • Refrigeration systems are present in such centrifuges that help to balance the heat produced due to the intense spinning.
  • The speed of these centrifuges can reach as high as 150,000 rpm.
  • It can be used for both preparative and analytical works.
  • Ultracentrifuges can separate molecules in large batches and in a continuous flow system.
  • In addition to separation, ultracentrifuges can also be used for the determination of properties of macromolecules like the size, shape, and density.

10. Vacuum centrifuge/ Concentrators

Vacuum centrifuge or Concentrators

Figure: Savant™ SpeedVac™ SPD120 Vacuum Concentrator and Kits. Image Source: Thermo Scientific.

  • Vacuum centrifuge utilizes the centrifugal force, vacuum and heat to speed up the laboratory evaporation of samples.
  • These centrifuges are capable of processing a large number of samples (up to 148 samples at a time).
  • This type of centrifuge is used in chemical and biological laboratories for the effective evaporation of solvents present in samples, thus concentrating the samples.
  • These are commonly used in high throughput laboratories for samples that might have a large number of solvents.
  • A rotary evaporator is used to remove the unnecessary solvents and eliminate solvent bumping.
  • The centrifuge works by lowering the pressure of the chamber, which also decreases the boiling point of the samples.
  • This causes the solvents to be evaporated, concentrating the particles to be separated.

Types of centrifugation

1. Analytical Centrifugation

Analytical centrifugation is a separation method where the particles in a sample are separated on the basis of their density and the centrifugal force they experience. Analytical ultracentrifugation (AUC) is a versatile and robust method for the quantitative analysis of macromolecules in solution.

Principle of Analytical Centrifugation

  • Analytical centrifugation is based on the principle that particles that are denser than others settle down faster. Similarly, the larger molecules move more quickly in the centrifugal force than the smaller ones.
  • Analytical ultracentrifugation for the determination of the relative molecular mass of a macromolecule can be performed by a sedimentation velocity approach or sedimentation equilibrium methodology.
  • The hydrodynamic properties of macromolecules are described by their sedimentation coefficients. They can be determined from the rate that a concentration boundary of the particular biomolecules moves in the gravitational field.
  • The sedimentation coefficient can be used to characterize changes in the size and shape of macromolecules with changing experimental conditions.
  • Three optical systems are available for the analytical ultracentrifuge (absorbance, interference, and fluorescence) that permit precise and selective observation of sedimentation in real-time.

Steps of Analytical Centrifugation

  • Small sample sizes (20-120 mm3) are taken in analytical cells to be placed inside the ultracentrifuge.
  • The ultracentrifuge is then operated so that the centrifugal force causes a migration of the randomly distributed biomolecules through the solvent radially outwards from the center of rotation.
  • The distance of the molecules from the center is determined through the Schlieren optical system.
  • A graph is drawn from the solute concentration versus the squared radial distance from the center of rotation, based on which the molecular mass is determined.

Uses of Analytical Centrifugation

  • Analytical centrifugation can be used for the determination of the purity of macromolecules.
  • It can also be used for the examination of changes in the molecular mass of supramolecular complexes.
  • Besides, it allows the determination of the relative molecular mass of solutes in their native state.

2. Density gradient centrifugation

Density gradient centrifugation is the separation of molecules where the separation is based on the density of the molecules as they pass through a density gradient under a centrifugal force.

Principle of Density gradient centrifugation

  • Density gradient centrifugation is based on the principle that molecules settle down under a centrifugal force until they reach a medium with the density the same as theirs.
  • In this case, a medium with a density gradient is employed, which either has to decrease density or increasing density.
  • Molecules in a sample move through the medium as the sample is rotated creating a centrifugal force.
  • The more dense molecules begin to move towards the bottom as they move through the density gradient.
  • The molecules then become suspended at a point in which the density of the particles equals the surrounding medium.
  • In this way, molecules with different densities are separated at different layers which can then be recovered by various processes.

Steps of Density gradient centrifugation

  • A density gradient of a medium is created by gently laying the lower concentration over the higher concentrations in a centrifuge tube.
  • The sample is then placed over the gradient, and the tubes are placed in an ultracentrifuge.
  • The particles travel through the gradient until they reach a point at which their density matches the density of the surrounding medium.
  • The fractions are removed and separated, obtaining the particles as isolated units.

Uses of Density gradient centrifugation

  • Density gradient centrifugation can be applied for the purification of large volumes of biomolecules.
  • It can even be used for the purification of different viruses which aids their further studies.
  • This technique can be used both as a separation technique and the technique for the determination of densities of various particles.

Examples of Density gradient centrifugation

  • This method was used in the famous experiment, which proved that DNA is semi-conservative by using different isotopes of nitrogen.
  • Another example is the use of this technique for the isolation of the microsomal fraction from muscle homogenates and subsequent separation of membrane vesicles with a differing density.

3. Differential centrifugation

Differential centrifugation is a type of centrifugation process in which components are separately settled down a centrifuge tube by applying a series of increasing centrifugal force.

Principle of Differential centrifugation

  • Differential centrifugation is based upon the differences in the sedimentation rate of biological particles of different size and density.
  • As the increasing centrifugal force is applied, initial sedimentation of the larger molecules takes place.
  • Further particles settle down depending upon the speed and time of individual centrifugation steps and the density and relative size of the particles.
  • The largest class of particles forms a pellet on the bottom of the centrifuge tube, leaving smaller-sized structures within the supernatant.
  • Thus, larger molecules sediment quickly and at lower centrifugal forces whereas the smaller molecules take longer time and higher forces.
  • In the case of particles that are less dense than the medium, the particles will float instead of settling.

Steps of Differential centrifugation

  • The sample solution is homogenized in the medium containing buffer.
  • The sample is then placed in the centrifuge tube, which is operated at a particular centrifugal force for a specific time at a particular temperature.
  • By the end of this operation, a pellet will be formed at the bottom of the tube, which is separated from the supernatant.
  • The supernatant is added to a new centrifuge tube where it is centrifuged at another speed for a particular time and particular temperature.
  • Again, the supernatant is separated from the pellets formed.
  • These steps are continued until all particles are separated from each other.
  • The particles can then be identified by testing for indicators that are unique to the specific particles.

Uses of Differential centrifugation

  • Differential centrifugation is commonly used for the separation of cell organelles and membranes found in the cell.
  • It can also be used for low-resolution separation of the nucleus.
  • As this technique separates particles based on their sizes, this can be used for the purification of extracts containing larger-sized impurities.

4. Isopycnic centrifugation

Isopycnic centrifugation is a type of centrifugation where the particles in a sample are separated on the basis of their densities as centrifugal force is applied to the sample.

Principle of Isopycnic centrifugation

  • Isopycnic centrifugation is also termed the equilibrium centrifugation as the separation of particles takes place solely on the basis of their densities and not on their sizes.
  • The particles move towards the bottom, and the movement is based on the size of the particles. And, the flow ceases once the density of the particle becomes equal to the density of the surrounding medium.
  • The density in the gradient increases as we move down the tube towards the bottom. As a result, the particles with higher densities settle down at the bottom, followed by less dense particles that form bands above the denser particles.
  • It is considered as a true equilibrium as this depends directly on the buoyant densities and not the sizes of the particles.

Steps of Isopycnic centrifugation

  • A gradient prepared with an increasing density towards the bottom of the tube is prepared. A pre-performed gradient can also be used.
  • The solution of the biological sample and salt is uniformly distributed in the centrifuge tube and placed inside the centrifuge.
  • Once the centrifuge is operated, a density gradient of the salt is formed in the tube.
  • The particles move down the tube and settle down as they reach the region with their respective densities.
  • The particles are then separated and identified using different other processes.

Uses of Isopycnic centrifugation

  • Isopycnic centrifugation can be applied for the purification of large volumes of biomolecules.
  • This technique can be used as a technique for the determination of densities of various particles.

5. Rate-zonal density gradient centrifugation/ Moving Zone Centrifugation

Rate-zonal density gradient centrifugation is a type of centrifugation that separates particles on the basis of their shape as size and works on the same principle of density gradient centrifugation but works in a different way. It is also called the moving zone centrifugation.

Principle of Rate-zonal density gradient centrifugation

  • Rate zonal centrifugation fractionates particles by both size and shape.
  • The procedure is to layer a sample in a restricted zone on top of a pre-poured density gradient. The density gradient is then centrifuged.
  • All particles migrate into the density gradient because the density gradient has only densities much lower than the densities of the particles being centrifuged.
  • The particles are fractionated primarily by size and shape. The larger a particle is, the more rapidly it sediments.
  • The more spherically symmetrical a particle is, the more rapidly it sediments.
  • The particles sediment through the gradient at a rate that is a function of their sedimentation coefficient.
  • Unlike differential centrifugation where the sample is distributed throughout the medium, in rate-zonal centrifugation, the sample is initially present only on top of the gradient as a narrow band.

Steps of Rate-zonal density gradient centrifugation

  • A density gradient is prepared in a centrifuge tube before applying the sample.
  • The same is then layered on the top of the gradient in the form of a band.
  • During centrifugation, fast-moving particles (larger in size and circular in shape) move ahead of slower particles so that different particles are separated as various bands on different parts of the gradient.
  • The particles are separated on the basis of their sedimentation coefficients, and they are obtained from the bottom of the tube through a perforation.

Uses of Rate-zonal density gradient centrifugation

  • Rate-zonal differential centrifugation has been used for the separation of viruses as they have components that are of different size and density that are unique to each virus.
  • This method has been employed for the fractionation of RNA on sucrose gradients.
  • Besides, rate-zonal differential centrifugation has also been used for the separation, purification and fractionation of DNA molecules from both viruses and bacteria.
  • The fractionation of polysomes and ribosome subunits has been one of the earliest applications of this method.

6. Differential velocity (Moving Boundary) centrifugation

Differential velocity centrifugation is a type of centrifugation process in which components are separately settled down a centrifuge tube by applying a series of increasing velocities.

Principle of Differential velocity (Moving Boundary) centrifugation

  • Differential centrifugation is based upon the differences in the rate of sedimentation of biological particles of different size and density.
  • As the increasing speed of the rotors is applied, initial sedimentation of the larger molecules takes place.
  • Further particles settle down depending upon the speed and time of individual centrifugation steps and the density and relative size of the particles.
  • The largest class of particles forms a pellet on the bottom of the centrifuge tube, leaving smaller-sized structures within the supernatant.
  • The pellet is then removed, and the supernatant is further centrifuged to obtain smaller particles.
  • Thus, larger molecules sediment quickly and at lower velocities, whereas the smaller molecules take longer time and higher velocities.
  • In the case of particles that are less dense than the medium, the particles will float instead of settling.

Steps of Differential velocity (Moving Boundary) centrifugation

  • The sample solution is homogenized in the medium containing buffer.
  • The sample is then placed in the centrifuge tube, which is operated at a lower rotor speed for a particular time at a particular temperature.
  • By the end of this operation, a pellet will be formed at the bottom of the tube, which is separated from the supernatant.
  • The supernatant is added to a new centrifuge tube where it is centrifuged at another speed for a particular time and particular temperature.
  • Again, the supernatant is separated from the pellets formed.
  • These steps are continued until all particles are separated from each other.
  • The particles can then be identified by testing for indicators that are unique to the specific particles.

Uses of Differential velocity (Moving Boundary) centrifugation

  • Differential centrifugation is commonly used for the separation of cell organelles and membranes found in the cell.
  • It can also be used for low-resolution separation of the nucleus.
  • As this technique separates particles based on their sizes, this can be used for the identification and comparison of particles of different sizes.

7. Equilibrium density gradient centrifugation

Equilibrium density gradient centrifugation is a modified and specialized form of density gradient centrifugation.

Principle of Equilibrium density gradient centrifugation

  • Equilibrium density gradient centrifugation is based on the principle that particles in a solution are separated on the basis of their densities.
  • In this case, the particles move through the density gradient and stop in a region where the density of the medium is equal to the density of the particle.
  • At this point, the centrifugal force acting on the particle is equal to the buoyant force pushing the particles up. As a result, the particles cease to move and can be separated into different layers.
  • The density in the gradient increases as we move down the tube towards the bottom. As a result, the particles with higher densities settle down at the bottom, followed by less dense particles that form bands above the denser particles.

Steps of Equilibrium density gradient centrifugation

  • A gradient prepared with an increasing density towards the bottom of the tube is prepared. A pre-performed gradient can also be used.
  • The solution of the biological sample and salt is uniformly distributed in the centrifuge tube and placed inside the centrifuge.
  • Once the centrifuge is operated, a density gradient of the salt is formed in the tube.
  • The particles move down the tube and settle down as they reach the region with their respective densities.
  • The particles are then separated and identified using different other processes.

Uses of Equilibrium density gradient centrifugation

  • Equilibrium density gradient centrifugation can be applied for the purification of large volumes of biomolecules.
  • This technique can be used as a technique for the determination of densities of various particles.

Examples of Equilibrium density gradient centrifugation

  • This has been used in experiments performed by Meelson and Stahl to determine the densities of different DNA molecules based on where they reached on the density gradient.

8. Sucrose gradient centrifugation

Sucrose gradient centrifugation is a type of density gradient centrifugation where the density gradient is formed of sucrose by changing the concentration of sucrose.

Principle of Sucrose gradient centrifugation

  • Sucrose gradient centrifugation is based on the principle that molecules settle down under a centrifugal force until they reach a medium with the density the same as theirs.
  • In this case, a medium with sucrose gradient is employed, which either has a lower density at the top and higher density at the bottom.
  • Molecules in a sample move through the medium as the sample is rotated creating a centrifugal force.
  • The more dense molecules begin to move towards the bottom as they move through the density gradient.
  • The molecules then become suspended at a point in which the density of the particles equals the surrounding medium.
  • In this way, molecules with different densities are separated at different layers which can then be recovered by various processes.

Steps of Sucrose gradient centrifugation

  • A density gradient of sucrose is created by gently laying the lower concentration of sucrose over the higher concentrations in a centrifuge tube.
  • The sample is then placed over the gradient, and the tubes are placed in an ultracentrifuge.
  • The particles travel through the gradient until they reach a point at which their density matches the density of the surrounding medium.
  • The fractions are removed and separated, obtaining the particles as separated units.

Uses of Sucrose gradient centrifugation

  • Sucrose gradient centrifugation is a powerful technique for the separation of macromolecules like DNA and RNA.
  • This has also been used for the analysis of protein complexes and to determine the density as well as the size of various other macromolecules.

Theory of Centrifugation Video (Dr. Alankar Shrivastava)

Reference

  1. Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology. Eighth edition. Cambridge University Press: New York.
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