CENTRIFIGUATION

DR. SHUCHI SHARMA

ASSISTANT PROFESSOR

DEFINITION�

• Centrifugation is a technique used for the separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
• The particles are suspended in a liquid medium and placed in a centrifuge tube. The tube is then placed in a rotor and spun at a define speed.
• Separation through sedimentation could be done naturally with the earth gravity, it would take Centrifugation is making that natural process much faster.
• Rotation of the rotor about a central axis generates a centrifugal force upon the particles in the suspension.�

• Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge, used in industry and in laboratory settings.
• This process is used to separate two immiscible liquids.
• More-dense components of the mixture migrate away from the axis of the centrifuge, while less- dense components of the mixture migrate towards the axis. Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge, used in industry and in laboratory settings.
• This process is used to separate two immiscible liquids. More-dense components of the mixture migrate away from the axis of the centrifuge, while less- dense components of the mixture migrate towards the axis.

HISTORY

• The first analytical ultracentrifuge was developed by Svedberg in 1920

PRINCIPLE

• In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top.
• The greater the difference in density, the faster they move. If there is no difference in density (isopyknic conditions), the particles stay steady.
• To take advantage of even tiny differences in density to separate various particles in a solution, gravity can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.
• If the particles suspended in a liquid are so small or have a density so close to that of the liquid, then the force of gravity fails to sediment the particles into a separate layer.

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• So the basis of centrifugation techniques is to exert a larger force than the gravitational force to enhance the effective sedimentation force for the separating such particles from the liquid.��

• A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top.
• The greater the difference in density, the faster they move. If there is no difference in density (isopyknic conditions), the particles stay steady.
• To take advantage of even tiny differences in density to separate various particles in a solution, gravity can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.

• A particle,whether it is precipitate,a macromolucle or cell organelle when rotated at high speed is subjected to a centrifugal force.
• Centrifugal force is defined as F=mw2r
• Where F=intensity of centrifugal force
• m=effective mass of sedimenting particle

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• w=angular velocity of rotation r=distance of migrating particles from central axis of rotation
• A more common measurement of F,in terms of Gravitational force g,is Relative Centrifugal Force RCF, Is given as RCF=(rpm)2(r) Thus this equation indicates that RCF varies with r,(the distance of the sedimenting particles from axis of rotation Thus it gives i

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• A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
• In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top.
• The greater the difference in density, the faster they move. If there is no difference in density (isopyknic conditions), the particles stay steady.

CENTRIFUGE

•  A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward).
• The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction.
• At the same time, objects that are less dense are displaced and move to the center. In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low- density substances rise to the top.

THE FACTORS ON WHICH THESE WORKS ARE

• More dense a biological structure, faster it sediments in centrifugal force.
• More massive biological particle, faster it moves in centrifugal field.
• Dense the buffer system, slower particle moves.
• Greater the frictional coefficient, slower a particle will move
• Greater the centrifugal force, faster particle sediments Sedimentation rate of a given particle will be zero when density of particle and the surrounding medium are equal.
• Sedimentation rate of a given particle will be zero when density of particle and the surrounding medium are equal.

TYPES OF CENTRIFUGE

•  High Speed Centrifuges:
• High speed centrifuges for more sophisticated biochemical applications.
• Higher speeds and temperature control of the rotor chambers are essential.
• Rotor chambers in most instruments are maintained at or near 40 C. Three types of rotors are available for high speed centrifugation.
• a. Fixed Angel rotor. b.
• Swinging –bucket rotors
• c. vertical rotor High speed centrifuges are used to sediment Cell debris after cell homogenization. Ammonium sulfate precipitates of proteins and cellular organelles such as chloroplasts, mitochondria and nuclei.

ULTRACENTRIFUGE

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• The most sophisticated of the centrifuges are the ultracentrifuges because of the high speeds attainable intense heat is generated in the rotor so the spin chamber must be refrigerated and placed under high vacuum to reduce friction..
• it is a high speed centrifuge that has fixed head rotors .It is mainly used in separation of lipoproteins .since the separation is long process there is generation of heat and thus are provided with internal cooling system.
• Ultracentrifuges can be used both for preparative work as well as for analytical. Preparative models its primarily used for separations and purification of samples for further analysis and analytical models which are designed for performing physical measurements on the sample during sedimentation.

• Ultracentrifuges can be used both for preparative work as well as for analytical.
• Centrifugation for isolation and purification of components is known as preparatory centrifugation, while that carried out with a desire for characterization is known as analytical centrifugation.
• Preparative models its primarily used for separations and purification of samples for further analysis and analytical models which are designed for performing physical measurements on the sample during sedimentation.
• Analytical ultracentrifugation is an analytical technique which combines an ultracentrifuge with optical monitoring systems

• In an analytical ultracentrifuge (commonly abbreviated as AUC), a sample’s sedimentation profile is monitored in real time by an optical detection system.
• The sample is detected via ultraviolet light absorption and/or interference optical refractive index sensitive system.
• With modern instrumentation, these observations are electronically digitized and stored for further mathematical analysis.
• The information that can be obtained from an analytical ultracentrifuge includes the gross shape of macromolecules, conformational changes in macromolecules, and size distributions of macromolecules. With AUC it is possible to gain information on the number and subunit stoichiometry of non-covalent complexes and equilibrium constant constants of macromolecules such as proteins, DNA, nanoparticles or other assemblies from different molecule classes.

DIFFERENTIAL CENTRIFUGATION

• Differential  centrifugation works by a stepwise increase in the centrifugation speed. Lower speeds at the beginning are used to eliminate the heavier particles from the sample, and the speed is then increased until the targets themselves are pelleted.
•  Separation is achieved based in the size of particles in differential centrifugation.
• Commonly used in simple pelleting and obtaining the partially pure separation of subcellular organelles and macromolecules.
• Used for study of subcellular organelle, tissues or cells (first disrupted to study internal content)
• During centrifugation, larger particles sediment faster than the smaller ones.
• At a series of progressive higher g-force generate partially purified organelles.

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• During centrifugation, larger particles sediment faster than the smaller ones.
• At a series of progressive higher g-force generate partially purified organelles.
• Inspite of its reduced yield differential centrifugation remains probably the most commonly used method for isolation of intracellular organelle from tissue homogenates.
• This technique is a common procedure in microbiology & cytology.
• The main essence of this centrifugation is to separate certain cell organells from a cell to study & analyse the specific parts of the cell.
• In this process, a tissue sample is first homogenised to break the cell membrane and mix up the cellular contents.

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DENSITY GRADIENT CENTRIFUGATION

• Due to the heterogeneity in biological particles, differential centrifugation suffers from contamination and poor recoveries. Contamination by different particle types can be addressed by resuspension and repeating the centrifugation steps (i.e., washing the pellet
• A method where the components of a sample are separated on the basis of their density, in a dense medium or density gradient, in a centrifuge, according to the centrifugal force they experience.
• A procedure for separating particles (such as viruses or ribosomes or molecules such as DNA )in which the sample is placed on a preformed gradient such as sucrose or caesium chloride. Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are 'banded' in the gradient and can be collected as a pure fraction.
• Density gradient centrifugation are of two types: Rate zonal centrifugation Isopycnic centrifugation

DENSITY GRADIENT CENTRIFUGATION�

• This type of centrifugation is mainly used to purify viruses, ribosomes, membranes, etc.
• A sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher concentrations in centrifuge tubes
• The particles of interest are placed on top of the gradient and centrifuge in ultracentrifuges.
• The particles travel through the gradient until they reach a point at which their density matches the density of surrounding sucrose.
• The fraction is removed and analyzed.

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RATE ZONAL CENTRIFUGATION

• In Rate zonal centrifugation the solution have a density gradient. The sample has a density i.e. greater than all the layers in the solution.
• The sample is applied in a thin zone at the top of the centrifuge tube on a density gradient.
• Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s) .
• The run must be terminated before any of the separated particles reach the bottom of the tube. Rate zonal centrifugation (Sedimentation velocity zone centrifugation)
• This method is useful for separating particles which differ in size but not in density Extremely useful for the separation of proteins possessing nearly identical densities but differing only slightly in their molecular weights

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• Sample is layered as a narrow zone on the top of a density gradient . Under centrifugal force, particles move at different rates depending on their mass .
• The speed at which particles sediment depends primarily on their size and mass instead of density. As the particles in the band move down through the density medium, zones containing particles of similar size form as the faster sedimenting particles move ahead of the slower ones. Because the density of the particles is greater than the density of the gradient, all the particles will eventually form a pellet if centrifuged long enough.

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ISOPYCNIC CENTRIFUGATION�

• In isopycnic separation, also called buoyant or equilibrium separation, particles are separated solely on the basis of their density. Particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium.
• The density of the gradient medium must be greater than the density of the particles to be separated. By this method, the particles will never sediment to the bottom of the tube, no matter how long the centrifugation time.
• In this type of centrifugation , the solution contains a greater range of densities.
• The density gradient contains the whole range of densities of the particles in the sample.
• Each particle will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density.
•  In Isopycnic centrifugation separation of particles occurs into zones on the basis of their density differences, independent of time.

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DISCONTINUOUS GRADIENTS OVERLAYERING TECHNIQUE

• The most widely used method for producing discontinuous gradients is to start with the densest solution and layer successively lower densities on top from a pipette. The higher the viscosity of the medium, the easier it is to achieve a relatively slow and smooth flow of liquid from the pipette.
• Low density solutions, having very low viscosities are more difficult to layer in this manner
• A syringe with a wide-bore metal filling cannula (i.d. 1-2 mm) can be used as an alternative to a pipette, but make sure that the barrel can move easily and smoothly when a small pressure is applied.

UNDERLAYERING TECHNIQUE

• Although the overlayering technique is probably the most widely used, the method in which successively denser solutions are underlayered under lighter ones is certainly the easier and the recommended one. The only important requirement is that no air bubbles are introduced which may disturb the lower density layers

ISOPYNIC CENTRIFUGATION�

• The sample is loaded into the tube with the gradient-forming solution (on top of or below pre-formed gradient, or mixed in with self-forming gradient)
• The solution of the biological sample and cesium salt is uniformly distributed in a centrifuge tube and rotated in an ultracentrifuge.
• Under the influence of centrifugal force, the cesium salts redistribute to form a density gradient from top to bottom.
• Particles move to point where their buoyant density equals that part of gradient and form bands. This is to say the sample molecules move to the region where their density equals the density of gradient.
• It is a “true” equilibrium procedure since depends on bouyant densities, not velocitie

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APPLICATIONS OF CENTRIFUGATION�

• To separate two miscible substances
• To analyze the hydrodynamic properties of macromolecules
• Purification of mammalian cells
• Fractionation of subcellular organelles (including membranes/membrane fractions)
• Fractionation of membrane vesicles
• Separating chalk powder from water
• Removing fat from milk to produce skimmed milk
• Separating particles from an air-flow using cyclonic separation
• The clarification and stabilization of wine
• Separation of urine components and blood components in forensic and research laboratories
• Aids in the separation of proteins using purification techniques such as salting out, e.g. ammonium sulfate precipitation.

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• This technique, in combination with other purification techniques, is extremely helpful while separating proteins.
• Centrifuges are widely used in the field of forensic chemistry. In this field, the technique is employed for the separation of blood components from blood samples. Furthermore, the technique is also employed in certain laboratories for the separation of urine components from urine samples.
• Differential centrifugation, a distinct type of centrifugation, is known to have applications in the identification of organelles.

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SWING-BUCKET ROTOR FIXED-ANGLE ROTOR VERTICAL ROTOR�

• A swing-bucket rotor usually supports samples ranging in volume from 36 mL to 2.2 mL. Swing-buckets can support two types of separations: rate-zonal and Isopycnic.
• Swing-buckets are preferred for rate-zonal separations, because the distance between the outside of the meniscus and the outside of the bottom of the tube is long enough for separation to occur

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• Fixed-angle rotors are usually used for pelleting applications to either pellet particles from a suspension and remove the excess debris, or to collect the pellet. Rotor cavities range from 0.2 mL to 1 mL.
• The most important aspect in deciding to use a fixed-angle rotor is the K factor. The K factor indicates how efficient the rotor can pellet at maximum speed. The lower the K factor, the higher the pelleting efficiency.

VERTICAL ROTORS

• Vertical rotors are highly specialized.
• They are typically used to band DNA in cesium chloride.
• Vertical rotors have very low K factors, which is useful if the particle must only move a short distance until it pellets. Run time on vertical rotors is short.