Lecture 1: Centrifugation - BIOT7002 PDF

CENTRIFUGATION Bioanalytical Techniques BIOT7002 LECTURE 1 Centrifugation Centrifugation is one of the most important and widely applied preparatory & purification techniques in biochemistry, cellular & molecular biology. Current research & clinical applications...

CENTRIFUGATION Bioanalytical Techniques BIOT7002 LECTURE 1 Centrifugation Centrifugation is one of the most important and widely applied preparatory & purification techniques in biochemistry, cellular & molecular biology. Current research & clinical applications rely on the isolation of cells, subcellular organelles & macromolecules. Centrifugation A centrifuge uses centrifugal force to isolate suspended particles from their surrounding medium. Applications for Loading… centrifugation include sedimentation of cells & viruses, separation of subcellular organelles & isolation of macromolecules such as DNA, RNA, proteins or lipids learn On M/Q Centrifugation Any particles or cells in a liquid suspension will settle at the bottom of a container due to gravity (1 x g). Loading… Length of time for this is impractical When a particle is rotated at a certain speed or revolutions per minute (RPM), centrifugal force Gravity takes to long centrifuges applied causes the particles to move radially away from the axis of rotation.  Centrifugation -Push towas a Centripetal Force: A centrifuge works by Definitions: spinning mixtures around a central axis * on MCQ Centrifugal Force: The force that (centrifugal force). As the sample spins the tends to impel matter outward tendency of the inertia of the object is to move from a centre of rotation. It is in a straight-line path. However, due to its dependent on 3 variables: mass, confinement within the centrifuge, the path of the object must be bent into a circular one. - speed & radius. (shown in B) Much The body of the centrifuge, or the body of the away from centre container within the centrifuge, provides a normal force that pushes the object toward the center of the circular path of travel. This inward force is refered to as a centripetal force, and its magnitude and direction are exactly what is needed to keep the object moving in a circular path around the axis of rotation of the centrifuge (shown in A) what is the centrifugal force What is centrifugal dependent? force? what is the centripetal force? Centrifugation Revolutions per min (RPM): A unit for expressing the number of complete rotations per minute, a measure of speed. All comparisons between centrifuges must be in RCF Relative Centrifugal Force (RCF): a method of comparing the forces generated by various centrifuges, considering the speed of rotation & radius from the centre of rotation: 2 RCF = 1.12r (RPM/1000)2M r = radius in mm RPM = number of revolutions per minute Centrifugation Calculation Example 1: Calculate the corresponding RCF value Calculation in a centrifugation experiment from the Example 2: following data: RPM = 15,000 Calculate the Radius = 10cm (100mm) corresponding RCF = 1.12. r. (RPM/1000)2 RCF = 1.12 x 100 (15,000/1000)2 RPM value in a RCF = 25,200 centrifugation Rcf = 1.12r(rpm/1000)*2 Q1. Rpm = 15000 R= 10cm (100mm) 1.12(100)(15000/1000)*2 Q2. Rcf =15000 Radius = 10 cm 15000 = 1.12(100)(rpm/1000)*2 15000/112 = RPM/1000*2 134 = RPM/1000*2 ^134 = RPM/1000 11.5 = RPM/1000 11.5 x 1000 = RPM  Centrifugation When an RCF value specified Calculation example: in a procedure cannot be What is the adjusted run time obtained on a particular needed to achieve the centrifuge, can adjust the time following requirements in a of centrifugation to achieve centrifugation separation equivalent centrifugal force: experiment given that; Ts (run time specified) = 1hr T = Ts x RCFs / RCF RCF = RCF obtainable on gets hotel centrifuge = 12,000 g Where: Calculations RCF = 1.12R(RPM/1000)*2 T=Ts x RCFs/RCF  RCFs = RCF required in Classes of Centrifuges and their applications