Chapter 3 Centrifugation

Chapter 3 Centrifugation-PDF Download

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General Steps in Biochemical Separation, 2, Separation of Macromolecules. Chromatography precipitation, Electrophoresis ultracentrifugation. 3, Densities of biological material, Material Density g cm3 . Microbial cells 1 05 1 15, Mammalian cells 1 04 1 10. Organelles 1 10 1 60, Proteins 1 30, DNA 1 70, RNA 2 00.
4, Introduction MBM 3 1 ,Principles of centrifugation. 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 , 5, Centrifugation, A centrifuge is used to separate particles or macromolecules . Cells, Sub cellular components, Proteins, Nucleic acids. Basis of separation , Size, Shape, Density,Methodology .
Utilizes density difference between the, particles macromolecules and the medium in which these are. dispersed, Dispersed systems are subjected to artificially induced. gravitational fields 6, Type 1 Preparative Centrifugation. Collect isolation material , cell subcellular structure membrane vesicles. 1 Handle larger liquid volumes i e , 1 to several thousand litres .
2 Range of designs, 3 Typical rotating speed 500 2000. rpm, Immunofluorescent imaging of, human cells U2OS with pan. Cadherin antibody 7, Type 2 Analytical Ultracentrifugation AUC . Determine the mass shape and stoichiometry ratio of non . covalent association of macromolecules protein protein . small molecule protein quaternary structure , , 1 Rotates at high speeds e g . 30000 rpm, 1 , 2 The high speeds used in, such devices generate.
, considerable amounts of, heat, 3 Therefore cooling. arrangements are required, in ultracentrifuges, http www cgmh org tw chldhos intr c4a90 new page 50 htm 8. 3 2 Basic Principle of Sedimentation AB 3 4 3 ,Relative centrifugal force F M 2 r. M mass of particle, r radius of rotation cm ie, distance of particle from axis. of rotation , Average angular velocity, radians sec .
2 rev min 1, , 60, Rev revolution per minute, r p m . 1 revolution 2 radians, 360 9, Centrifugal Field, G r 2 depends on the radical distance of the particle from the. rotation axis and the square of the angular velocity. G , 4 2, rev min r, 1 2, 3600, 10, Angular Velocity. 2 rev min 1, rev revolution per minute r p m , 60. 11, Because rotors are, Relative Centrifugal Force different from various.
manufactures we use, f c M 2 r RCF , RCF 2 r g 1 RCF value RCF to represent the. fg Mg No x g centrifugation force , 2 rmp, 2 multiples of earth s gravitational force . , RCF r g, 1, 60 Radius Radius Radius, Min Ave Max. RCF ,1 12 x 10 5 x rpm 2 x r, RPM x1000 Radius,rpm . revolution per minute,r radius of rotor RCF x1000 .
12, Relative centrifugal force, RCF , 1 12 x 10 5 x rpm 2 x r. rmin, rmax, 13, Interacting Forces in Centrifugation. Sedimenting force mp 2r is opposed by Fcentrifuge, mp the mass of equal volume of solvent. 1 Frictional Resistance against Ffriction Fbuoyancy. particle moving through fluid , f v, f frictional coefficient of particle in the solvent. v particle velocity,2 Flotation Force F ms r 2, BALANCE between the sedmenting force and counteracting force.
Net force m, M p M, p 2r fvfv, ms r , s, 2, 14, Sedimentation Coefficient s . w 2r mp ms f v 0,Theodor Svedberg 1884 1971 ,Chemist from Sweden. 1926 Nobel prize, 1908 He described a new method, ultracentrifuge of producing colloid particles. and gave convincing evidence of the validity of, the theory on the Brownian movements 15. 3 2 Basic Principle of sedimentation 3 3 Types care and safety of centrifuges 3 4 Preparative centrifugation 3 5 Analytical centrifugation Analytical Biochemistry AB 3 4 3 Ultracentrifugation Koolman Color Atlas of Biochemistry 2nd edition

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