C E N T R I F U G A T I O N

CENTRIFUGATION * Centrifugation is one of the most important separation technique. * It exploits the inherent varied sedimenting property of substances for their isolation by the application of centrifugation field. * The resullting solution has 2 components namely the SEDIMENT (Pellet), and SUPERNATENT. C E N T R I F U G A T I O N - BASIC PRINCIPLES * This method is based on the principle of sedimentation. * When a particle sediments, it must displace some of the solution in which it is suspended, resulting in an upthrust on the particle equal to the weight of the liquid displaced. * If a particle is assumed to be a sphere of known volume and density, then the net force(f) is experienced when the centrifugal force at an angular velocity of ω radians/sec is given by: S = Volume x Density x ω2r Or, F = 4/3 Π rp 3 (ρp – ρm) ω2r ------------------ 1 Where, 4/3 Π rp 3 = Volume of sphere of radius ‘r’. ρp = Density of the particle. ρm = Density of the suspended medium. ν = Distance of the particle from the center of rotation. ω = Angular velocity of rotor. FRICTIONAL COEFFICIENT * Particles however generate friction as they migrate through the solution. * If the particle is spherical and is moving at a known velocity, then its frictional force appearing opposing motion is given by Stoke’s law. f0 = 6Πηrpv ------------------------------ 2 Where: f0 = Frictional coefficient of spherical particles. η = Coefficient of viscosity, and v = Velocity of the sedimenting particle. * A particle of known volume and density are present in a medium ofconstant density will therefore be accelerated in a centrifugal field, until the net force on the particle equals the force resisting its motion through the medium f = f0 or, 4/3 Π rp 3 (ρp – ρm) ω2r = 6Πηrpv ---------------------- 3 * In practice, the balancing of this force occurs quickly, with the result that the particles sediment at a constant ratio. Its rate of sedimentation (v) is given by: v = dx/dt = 2/9 rp2 (ρp – ρm) ω2r -------------------- 4η * The non-spherical molecules as in the case of rod like molecules, such as DNA and proteins like myosin experience considerable frictional resistance. * Frictional coefficient (f) of the molecules can be increased as much as 10 times of the frictional coefficient of the sphere (f0). * This results in the particles sedimenting at a lower rate and thus, equation 4 can be modified as: v = dr/dt = 2/9 rp2 (ρp – ρm) ω2r --------------------f/f0 * From equation 5, it is clear that the sedimentation rate is of a particle is dependant on the following factors: * It is directly proportional to the size of the particle, since the equation involves the square of the particle radius. It is apparent that the size of the particle has the greatest influence upon sedimentation. * It is directly proportional to the difference in density between the particle & the medium. It will be zero, when the density of the particle and the medium are equal. * It will decrease, when the viscosity of the medium increases (i.e., ω2r) v = 1/η. * The sedimentation rate (or) velocity decreases as the frictional coefficient ratio (f/f0) increases the ratio f/f0 is approximately 1 for a spherical molecule. * The rate of sedimentation depends on the applied centrifugal field (G) ω2r being directed radially outwards which is determined by the square root of angular velocity of the rotor and radial distance of the particle from the axis of rotation. * According to the equation, G= ω2r, since one revolution of the rotor is equal to 2Π radians, its angular velocity in rad/sec can be readily expressed in terms of revolutions per minute. The common way of expressing rotor speed is: ω = 2Π r.p.m / 60 (angular velocity (ω)) The centrifugal field (G) in terms of r.p.m is then: G = [ 4 Π2 (r.p.m)2r / 3600 ] G = ω2r It is generally expressed as multiples of earth’s gravitiational field, the ratio of the weight of the molecule in centrifugal field to the weight of the same particle when acted upon by gravity alone. It is referred to as RELATIVE CENTRIFUGAL FIELD (RCF). R.C.F = [ 4 Π2 (r.p.m)2r / 3600 x 980(g) ] = (ω2r/g)

RELATION BETWEEN R.C.F AND g The RCF is given by the equation: Or, r.p.m = R.C.F x 3600g 4Π2 x r It can be shortened as : r.p.m. = 9.549 RCF x gr By plotting the values of Π and g, r.p.m = 298.93 RCF / r Where, r = Radial distance from the axis of the rotor Sedimentation Rate * It is the sedimentation velocity of a particle in a centrifugal field and is denoted as v. * It is related to particle size, & its radial distance from the axis of the rotor and the applied centrifugal field by the following equation: v = dv/dt = 2/9 rp2 (ρp – ρm) ω2rη Sedimentation coefficient (S) * It is the sedimentation velocity (v) of a particle / unit centrifugal field and is denoted as ‘S’. S = v/ ω2r = [dv/dt]/ ω2r = [dv/dt] x (1/ ω2r) * Integration of the above equation between the limits r1, r2 and t1, t2, we have: S = ln t2/r1 ω2(t2’ - t1’) * S is determined by observing the particles at different positions and at different times help in the calculation of the molecular weight of the macromolecules and is related as follows: M = RTS / D(1-v ρ)S20W ► It is the standard sedimentation coefficient of a substance in water at 200C. ► Since sedimentation rate studies may be preformed using a wide variety of solvent, solute system. ► The measured value of sedimentation coefficient, which is affected by temperature, solution viscosity, and density is often connected to a value that could be obtained in a medium with viscosity and density of water at 200C and expressed as the sedimentation coefficient (or) S2Ow. ► It is given by the equation: S2Ow = S observed = 1-v ρ2Ow nT n 1-v ρT n20 n0 Where: S observed = Sedimentation coefficient observed in a particular medium. ρ2Ow = Density of water at 200C. v = Partial specific volume n/no = Derived velocity of the solvent to that of water. nT/n20 = Relative velocity of water at the temperature ‘T’, compared to that at 200C. ► In the above equation, the partial specific volume is assumed to be unaffected. Svedberg Unit ► It is the time dimensional constant of sedimentation coefficient. The basic unit of which is taken is 10-13s. It is denoted as ‘S’ ► For example, a molecule possessing a sedimentation coefficient of 5 x 10-13 secs, will have a value of 5S. ► This gives and idea about the size of the molecule. CENTRIFUGATION – CLASSIFICATION Centrifugation can be classified in 2 ways: • PREPARATIVE CENTRIFUGATION • ANALYTICAL CENTRIFUGATION PREPARATIVE CENTRIFUGATION • It is a separation technique, used for the preparation of cellular, sub cellular and molecular fractions, in a uniform (or) density gradient medium. • It separates various substances into fractions on the basis of sedimentation rate, which in turn depends on the size, shape, density of the molecule and viscosity of the medium. v = 2/9 rp 2 (ρp – ρm) ω2r ------------------- 1η (f/f0) Where, rp= Radius of the particle ρp = Density of the particle ρm = Density of the medium ω2r = Applied centrifugal field η = Viscosity of the medium f/f0 = Frictional coefficient. * Depending on the method of approach adapted for preparation of various fractions of substances, preparative centrifugation can be classified into: * DIFFERENTIAL CENTRIFUGATION * DENSITY GRADIENT CENTRIFUGATION DIFFERENTIAL CENTRIFUGATION

* It is a type of preparative centrifugation, in which the substance is separated as fraction by applying increasing centrifugal field for different time durations, in a uniform medium, keeping ρ m a nd η constant, and assuming f/f0 as 1. * From eqn 1 it is clear that the sedimentation velocity will depend on vp, ρp and ω2r as: v α vp 2, ρp, ω2r Or, v = 2/9vp 2 ρp ω2r ------------ 2 * Thus at different magnitudes of the centrifugal field, different fractions having different fractions, having different size and density are separated. * From equation 2 the rate of sedimentation depends on size, than on density of the particle. * Moreover the density of the protein particles of different sizes and density are made to sediment as pellet by applying varying centrifugal force for varying time periods. * This can be done by varying the speed of the centrifuge as: Centrifugal field = ω2r = (2Πn)2r= 4Π2(r.p.m)2r Where, r=Distance from the rotor. * Thus by making the centrifuge revolve at different speeds, different magnitude of centrifugal field is created which makes the particles of particular dimension to sediment at a particular time as pellet, and the unsedimented particles remain in the supernatent. * They can also be made to sediment by applying greater centrifugal force by increasing the speed. ISOLATION OF SUBCELLULAR ORGANELLES * It is also known as cell fractionation. * The procedure involves 2 main types * HOMOGENIZATION * DIFFERENTIAL CENTRIFUGATION * Homogenization is the 1st step in cell fraction and involves disorganization of tissues & breaking of cell wall/cell membrane, leading to the release of the cellular contents. * The tissue in this process gets converted into a homogentate. HOMOGENIZATION * The animal is sacrificed, and the tissue is excised & washed in ice cold buffer or sucrose. * The tissue is weighed & mixed into small pieces and transferred into a rotor tube. * To the tissue, appropriate amounts of buffer or sucrose are added and are homogenized by a Teflon pestle. * The pestle is allowed to revolve at a speed of 2000rpm such that the tissues are ruptured and but the cell organelles are intact and the homogenate obtained is subjected to differential centrifugation. DIFFERENTIAL CENTRIFUGATION - Assay of purity * The subcellular organelles are subjected to differential centrifugation. * Assay of purity of the separated components is carried out by: * MICROSCOPIC EXAMINATION OF ISOLATED FRACTION * CHEMICAL ANALYSIS 1. DNA content 2.Enzyme activity (Widely used) 3. Immunological precipitation 4. Spectrophotometry DENSITY GRADIENT CENTRIFUGATION * It is a type of preparative centrifugation. * It involves the separation of various fractions by sedimenting them in a density gradient medium. * Depending on the approach adopted it can be further classified as: RATE ZONAL CENTRIFUGATION ISOPYCNIC CENTRIFUGATION RATE ZONAL CENTRIFUGATION * This involves the separation of fractions on the basis of their molecular size or molecular traces or sedimentation rate. v = 2/9 rp 2 (ρp – ρm) ω2r (ρp > ρm) η (f/f0) * Here, the substance to be separated is carefully layered over a preformed layer of density gradient. * This is done in a preformed density gradient. The highest density in the gradient should not be greater than the densest particle to be separated. * The density gradient is so established that it gives a sharp reduced separation of fraction in the form of bands. * This separation is achieved by centrifugation for definite period of time where the applied centrifugal field makes the particle to sediment at different rates. * This is due to the fact that different particles will exhibit different sedimentation velocity, which mainly depends on their size.

RATE ZONAL CENTRIFUGATION – CHARACTERISTICS * It separates the particles on the basis of their size dimensions and sedimentation rate. * The highest density in the gradient should not be greater than the densest particle to be separated. * It is time dependant. ISOPYCNIC CENTRIFUGATION * It is also known as equilibrium density centrifugation. * It separates the fractions on the basis of their characteristic buoyant density in a preformed density gradient. * The maximum density of the gradient exceeds the density of the densest particle in a time independent fashion. v = 2/9 rp 2 (ρp – ρm) ω2r (ρp < ρm) η (f/f0) * Sedimentation occurs at their isodensity, and no further sedimentation occurs irrespective of the time. * This is because the sedimented particles keep floating on a cushion of the medium having greater density than its own. FORMATION OF DENSITY GRADIENT * The 2 main types of density gradient are: – Continuous density formation – Discontinuous density formation * Continuous density ity formation is done by careful layering of the gradient medium of different densities one over the other. * This gradient is not very sharp, and it rather forms in a linear to the bottom of the tube. * It is formed by gradient formers. * Discontinuous density formation , also known as step gradient is characterized by marked variation by dissolving the gradient in various proportions and layered one over the other. * Layering is done by using a pipette, and the high density region is at the bottom of the tube and the lowest at the top. CHOICE OF GRADIENT * An ideal gradient must possess the following characteristics: – Permit the desired type of separation. – Be stable in the solution. – Be inert to biological material. – Be non-toxic – Must be inexpensive and easily available – Have negligible osmotic pressure and cause minimum change in pH, ionic strength, & viscosity. – Should not absorb light of wavelength used for the spectrophotometric assay. – Should not interfere with other assaying procedures. * No material however fulfill all the conditions strictly. * The commonly used gradients in biochemical separations are: * Sucrose (Not preferred due to increase in viscosity at density greater than 1.1-1.2 g/cm3 and it exerts very high osmotic effect at low concentrations) * Ficoll * Malrizamide * Percol * Cscb * Ludose SAMPLE APPLICATION * The mixture of the substance is carefully layered on top of the gradient. * This is done with the help of a pipette or a hypodermic syringe FRACTION COLLECTION * The various fractions are collected after the completion of the centrifugation procedure. * This can be done in 2 ways: – Careful sucking – Puncturing the bottom of the tube. Density gradient centrifugation – Applications * Rate zonal centrifugation is used for the separation of DNA, RNA, enzymes, hormones, hybrids, ribosomal subunits, and other subcellular fractions. * Isopycnic centrifugation is used in the separation of subcellular organelles, and isotopic studies involving macromolecules. --------------------------------------------------------------------------------------------------------------------------------------------Basics of Centrifugation The purpose of this tutorial is to introduce basic concepts of centrifugation, including vocabulary, centrifuge and rotor types, separation techniques, and even gradient selection. For further details regarding centrifugation, please refer to the Sorvall® and Heraeus recommended reading the literature cited in the reference section. Contents. I. Introduction II. Increasing the effect of gravity: the centrifuge

III. Types of centrifugal separation IV. Rotor categories V. Selection of centrifuge tubes VI. Common centrifuge vocabulary and formulas VII. Reference and suggested readings I. Introduction. Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular and molecular biology, and in medicine. Current research and clinical applications rely on isolation of cells, subcellular organelles, and macromolecules, often in high yields. A centrifuge uses centrifugal force (g-force) to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis. Applications for centrifugation are many and may include sedimentation of cells and viruses, separation of subcellular organelles, and isolation of macromolecules such as DNA, RNA, proteins, or lipids. II. Increasing the effect of gravity: the centrifuge. Many particles or cells in a liquid suspension, given time, will eventually settle at the bottom of a container due to gravity (1 x g). However, the length of time required for such separations is impractical. Other particles, extremely small in size, will not separate at all in solution, unless subjected to high centrifugal force. When a suspension is rotated at a certain speed or revolutions per minute (RPM), centrifugal force causes the particles to move radially away from the axis of rotation. The force on the particles (compared to gravity) is called Relative Centrifugal Force (RCF). For example, an RCF of 500 x g indicates that the centrifugal force applied is 500 times greater than Earth’s gravitational force. Table 1 illustrates common centrifuge classes and their applications. Table 1. Classes of centrifuges and their applications.

III. Types of Centrifugal Separations. 1. Differential centrifugation. Separation is achieved primarily based on the size of the particles in differential centrifugation. This type of separation is commonly used in simple pelleting and in obtaining partially-pure preparation of subcellular organelles and macromolecules. For the study of subcellular organelles, tissue or cells are first disrupted to release their internal contents. This crude disrupted cell mixture is referred to as a homogenate. During centrifugation of a cell homogenate, larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation. A cell homogenate can be centrifuged at a series of progressively higher g-forces and times to generate pellets of partially-purified organelles. When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken cells and heavy nuclei pellet to the bottom of the tube. The supernatant can be further centrifuged at 10,000 x g for 20 minutes to pellet subcellular organelles of intermediate velocities such as mitochondria, lysosomes, and microbodies. Some of these sedimenting organelles can obtained in partial purity and are typically contaminated with other particles. Repeated washing of the pellets by resuspending in isotonic solvents and re-pelleting may result in removal of contaminants that are smaller in size (Figure 1). Obtaining partially-purified organelles by differential centrifugation serves as the preliminary step for further purification using other types of centrifugal separation (density gradient separation). 2. Density gradient centrifugation. Density gradient centrifugation is the preferred method to purify subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media (Table 2) such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories. 2a. Rate-zonal (size) separation. 2b. Isopycnic (density) separation.

2a. Rate zonal (size) separation Rate-zonal separation takes advantage of particle size and mass instead of particle density for sedimentation. Figure 2 illustrates a ratezonal separation process and the criteria for successful rate-zonal separation. Examples of common applications include separation of cellular organelles such as endosomes or separation of proteins, such as antibodies. For instance, Antibody classes all have very similar densities, but different masses. Thus, separation based on mass will separate the different classes, whereas separation based on density will not be able to resolve these antibody classes. Certain types of rotors are more applicable for this type of separation than others. Please See rotor categories (below) and Table 2 Criteria for successful rate-zonal centrifugation: • Density of the sample solution must be less than that of the lowest density portion of the gradient. • Density of the sample particle must be greater than that of the highest density portion of the gradient. • The pathlength of the gradient must be sufficient for the separation to occur. • Time is important. If you perform too long runs, particles may all pellet at the bottom of the tube. 2b. Isopycnic separation In this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle. A common example for this method is separation of nucleic acids in a CsCl gradient. Figure 3 illustrates the isopycnic separation and criteria for successful separation. A variety of gradient media can be used for isopycnic separations and their biological applications are listed in Table 2. Criteria for successful isopycnic separation: • Density of the sample particle must fall within the limits of the gradient densities. • Any gradient length is acceptable. • The run time must be sufficient for the particles to band at their isopycnic point. Excessive run times have no adverse effect.