Centrifugation

Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures to separate the two miscible substances ,and also to analyze the hydrodynamic properties of macro molecule with a centrifuge , used in industry and in laboratory setting.

1. By Dr. Rachana Choudhary Asstt . Prof. Department of Microbiology SHRI SHANKRACHARYA MAHAVIDYALAY JUNWANI, BHILAI CENTRIFUGATION

2. SYNOPSIS INTRODUCTION CENTRIFUGE HISTORY BASIC PRINCIPLE OF CENTRIFUGATION /SEDIMENTATION TYPES OF ROTOR TECHNIQUE OF CENTRIFUGATION SEDIMENTATION COEFFICENT FACTOR AFFECTING SEDIMENTATION COEFFICIENT. PRECAUTION APPLICATION OF CENTRIFUGATION REFERENCE

3. INTRODUCTION Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures to separate the two miscible substances ,and also to analyze the hydrodynamic properties of macromolecule with a centrifuge , used in industry and in laboratory setting. More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis. The theoretical basis of this technique is the effect of gravity on particles (including macromolecules) in suspension. Two particles of different masses will settle in a tube at different rates in response to gravity. .

4. Device: Centrifuge is a piece of equipment, generally driven by a motor, that puts an object in rotation around a fixed axis , applying force perpendicular to the axis. It is device for separating particles from a solution according to there size shape , density and viscosity of the medium. Centrifuge uses centrifugal force to separate phases of different densities. The centrifugal force is proportional to the rotation rate of the rotor. Basic components of centrifuge: A metal rotor with holes (it accommodates vessel of liquid) A motor or alternative means of spinning- the rotor at a selected speed.

5. HISTOR Y English military engineer Benjamin Robins (1707-1751) invented a whirling arm apparatus to determine drag . In 1864, Antonin Prandtl proposed the idea of a dairy centrifuge to separate cream from milk. The potential of the centrifuge in the laboratory setting was first exploited by Friedrich Miescher in 1869 ,Miescher used a crude centrifuge system to isolate a cell organelle. In 1879,the first continuous centrifugal separator was demonstrated by Gustaf de lava & development made the commercialization of the centrifuge. In 1923 the Svedberg and Nicols employed it for increase the gravitational force.

6. Basic principle of centrifugation A particle, whether it is precipitate a macromolecule or a cell organelle is subjected to a centrifugal force when is rotated at high rate of speed. The centrifugal force F is denoted by equation: F= m  2 r F = intensity of the centrifugal force. m= effective mass of the sedimenting particle. = angular velocity of rotation. r= distance of the migrating particles from the central axis of rotation. A more common measurement of F in the terms of the earths gravitation force, g is the relative centrifugal force , RCF its defined by RCF=(1.11910ˉ 5 )(rpm 2 (r) T his equation relates RCF to revolutions per minute of the sample. Equation dictates that the RCF on a sample will vary with r, the sedimenting particles from the axis of rotation.

7. Types of rotors Basically there are four types of otors Fixed Angle Rotors Vertical Tube Rotors Swinging Bucket Rotors Zonal Rotors

8. fixed angle rotors Consist of metal with 6 to 12 holes within their body . The hole angle between( 14˚ & 40˚) to vertical axis. Particles travels short distance and strike with the wall.

9. VERTICAL TUBE ROTORS:- Have holes within their body lie parallel to the rotor shaft and not at an angle. As the rotor accelerates and centrifugal field is applied, the solution within the tube reorients through 90˚ (makes solution to the axis of rotation). As the rotor decelerates the solution orients back to the original position.

10. Swinging bucket rotors These rotor have bucket that swing out to a horizontal position when the rotor accelerates. The solution in the tube reorients to lie perpendicular to the axis of rotation and parallel to the applied centrifugal field. As the rotor decelerate the solution orient back to the original position.

11. Zonal rotors The zonal rotor is more or less sphere which has its interior subdivided into four equal quadrants (sector shaped) by means of a four –veined core. The zonal rotor is rotated while it is still empty i.e. without gradient. When the speed has been brought up to 3000- 4000rpm, the gradient is loaded into the rotor.

12. TECHNIQUES OF CENTRIFUGATION Preparative centrifugation Differential centrifugation Density gradient centrifugation. Rate –zonal centrifugation. Isopycnic centrifugation. Analytical centrifugation. Ultracentrifugation.

13. Preparative centrifugation :- Preparative centrifugation is con- cerned with the actual isolation of biological material for subsequent biochemical investigation. It can be divided into two main techniques depending upon the medium of suspension in which the separation is carried out in a sus-pending medium which is homo- genous are known as differential centrifugation . Those carried out in a suspending medium having density gradient are known as density gradient centrifugation .

14. Differential centrifugation :- Differential centrifugation is a common procedure in microbiology & cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first homogenized to break the cell membranes & mix up the cell contents. The homogenate is then subjected to repeated centrifugations, each time removing the pellet & increasing the centrifugal force.

15. DENSITY GRADIENT CENTRIFUGATION Also known as equilibrium sedimentation . It employs medium which has gradients. Separation under centrifugal force is depend upon buoyant densities of the particles. The gradients, apart from exerting their separating effect, eliminate mixing of separated components due to convection & mechanical vibrations, hence gives much better separation than Types of differential centrifugation. Rate-zonal centrifugation. Isopycnic centrifugation .

16. 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. The particles will begin sedimen - ting in separate zones according to their size shape and density .

17. Isopycnic centrifugation In, isopycnic separation, also called equilibrium separation 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 on to the position in the centrifuge tube at which the gradient densities 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.

18. Difference between Rate-zonal & isopycnic centrifugation. Rate zonal isopycnic synonym Sedimentation velocity. Sedimentation equilibrium. gradient Shallow, gradient continuous. steep, continuous or discontinuous gradient. centrifugation Incomplete sedimentation. Complete sedimentation. seperations RNA-DNA hybrids. DNA, plasma etc.

19. Analytical ultracentrifugation The aim of analytical ultracentri - fugation is use to study molecular interactions between macro- molecules or to analyze the pro- perties of sedimenting particles such as their apparent molecular weight. Which through rapid spinning imposes high centrifugal forces on suspended particles , or even molecules in solution, and cause separations of such matter on the basis of difference in weight. Can run at about 120,000-150,000rpm. RCF as high as 625,000 g., Volume a single tube accomodated-0.2-5.5ml. Separate particles as less as 10micron.

20. ultracentrifugation Svedberg coined the term “ ultracentrifugation ”. It is the most sophisticated instrument. Operate at speed of 75,000 rpm providing the centrifugal force of 500,000g. The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds , capable of generating acceleration as high 19600km/s2. Intense heat is generated due to high speed thus the spinning chambers must be refrigerated and kept at high vaccum .

21. Sedimentation coefficient The velocity of a particle per unit centrifugal field is referred to as its sedimentation coefficient. It is expressed in Svedberg unit, S. Mathematically expression s=v/ 2 r . where, v= velocity of sedementing particle = angular velocity of rotation r= distance of migrating particle from the central axis of the rotation The term s is most often defined under standard conditions, 20˚C and water as the medium, and it is denoted by s20,w. The s value is a physical characteristic used to classify biological macromolecules and cell organelles. 1S = 1 x 10 -3 second.Range 1 x 10 -13 to 10,000 x 10 -13 second.

22. 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 friction coefficient, slower particle 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.

23. factor affecting sedimentation coefficient Molecular shape : Large, rigid and extend molecule face more friction then spherical molecules. A rigid molecule face more friction affecting its sedimentation. Double stranded DNA is rigid due to hydrogen bonds. If temperature of double stranded DNA sample is increased the hydrogen bonds break decreasing its rigidity& making its flexible & increasing the sedimentation coefficient.

24. Molecular weight : Sedimentation coefficient also depend on molecular weight of the molecules. The relationship between sedimentation coefficient s and molecular weight. M of DNA is S 20.w= 2.8 +0.0083M 0.497 For protein molecules S  M 2/3 It is easier to characterize a protein by finding its sedimentation coefficient rather than its molecular weight.

25. Solute binding Binding of small solute molecules to large macromolecules affects their S value . If binding of the solute increases molecular weight of the macromolecule its sedimentation coefficient will increase. If binding of the solute changes the shape of macromolecule, it will also affect the sedimentation coefficient.

26. PRECAUTIONS Centrifuge tube must be of strong glass and tube must not be too long. Tube on opposite side must be balanced well. Speed/velocity must be increased slowly. Instrument be kept clean. Disinfectant the instrument regularly.

27. application In laboratory, centrifugation is used to ; Remove cellular elements from blood to provide cell free plasma or serum for analysis. Remove chemically precipitated protein from an analytical specimen. Separate protein bound from free ligand in immunochemical and other assay. Separation of the subcellular organelle , RNA ,DNA. Separate lipid components.

28. reference Modern and experimental biochemistry – RODNEY BOYER. Principles and technique in biochemistry – WILSON & WALKER Biophysical chemistry principle and techniques – UPADHYAY & UPADHYAY NATH. Google Search Slideshare

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