_ PRINCIPLE AND‘APPLICATION OF L CENTRIFUGATION gre INTRODUCTION WHAT IS CENTRIFUGE? Centrifiage is a device for separating particles from a solution according to there size, shape, density, viscosity of the medium. | war IS CENTRIFUGATION? i Centrifugation is a process which involves the use of the centrifugal forg sedimentation of heterogencous mixtures with a centrifuge.Centrifugal force > Centrifugal force, word from Latin Centrum, meaning “centre”, means “to flee”, is the apparent force that draws a rotating body centre of rotation. > The term centrifugal force is used to refer to one of two distinct c inertial force observed in a non-inertial reference frame, and a r corresponding to a centripetal force. > The concept of centfifugal force is applied in rotating devices centrifuges, centrifugal pumps. The two different forces are equ: but centrifugal forces is opposite in direction to the centripetal fotPrinciple » The centrifuge involves principle of centrifugation, denser substances cause: acceleration at centripetal for along the radial direction at the bottom of the tube. > Ina solution, particles whose density is higher than thaf sink (sediment), and particles that are lighter than ‘it flog Relative Centrifugal Force (RCF) + RCE, is the ratio of the centrifugal acceleration ata specified speed to the standard acceleration of gravity. > Relative Centrifugal force is defined as F = Mw2 r Where,> F= intensity of centrifugal force > M= mass of particle >» w= angular velocity of rotation » R= distance of migrating particles from central axis of rotatio » The RCF value for any centrifuge may be Sey fr following equation: RCF = 1.18 * r x (rpm) 2 * 10- 5 Where, > 1.18 108 = An empirical factor > ris the radius in cm from the centre of the centrifuge external tip of centrifuge tube. > rpm is the number of revolution per minute of the centr! > The time required to separate particles depends on the rotor speed, q and effective path length travelled by sedimented particles. > The following formula useful for the calculation of the speed require! with a radius that differ from the radius with which a prescribed, 0 > RPM. = 1000x VRCF 118 xr > The length of time of centrifugation also can be calculated so, that ry alternative rotor of a different size is equivalent to running with a o1Types of Centrifuge depends on : > Speed of sedimentation (ultra centrifuge or high speed centrifuge) >» Presence /absence of vacuum (ultra centrifuge or small bench top) | > Temperature control refrigeration. > Volume of sample and capacity of centrifugation tubes » Depending on the particular application, centrifug | in their overall design and size. >» Acommon feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated. Small Bench top » With or. without refrigeration it ting an » Slow speed (eg up to 4000 RPM)~ Common in clinical lab (blood/plasma/serum separation ) » Can take approx (up to) 100 tubes, depending on diameter Micro centrifuge (“microfuge”, “Eppendrof”) > Sample volume is small in eppendrof tubes + Refrigerated with or without + Centrifuge maximum approx 10000 g + Take tube of small volume up to 2ml. + Commonly used of concentration protein High speed centrifuge > Refrigerated > Use for protein precipitates, large intact organelles cellular debris from tissue homogenization and microorganism» They operate maximal centrifugal force of approx 5 > Use for research applications » Differential separation of nucleus, mitochondrial, protein precipitate, etc. Ultra centrifuge + Refrigerated and evacuated + The detail biochemistry analysis of subcellular struc; and isolate biomolecules. > Operate at upto 90000 g Type of rotor > Fixed angle rotor » Swinging bucket rotor Fixed angle rotor > Ideally tool for pelleting » Isopycnic banding may form > Centrifugation tubes are held at at fixed angle of betwee!14 '-40' to vertical axis of rotation. armored chamberSwinging bucket rotors: > Sample tubes are loaded the rotor is at rest. » When the rotor begins to rotate Usefull when samples are to be resolved in density gradients. > The longer path length permits better separation of individual particle t mixture. This rotor is relatively inefficient for pelleting. into individual buckets that hang vertical the buckets swing out to a horizontal positi swinging-arm rotor Many cell fractionations are done Ina second type of rotor, a CENTRIFUGATION The metal buckets that hold the tubes are free to swing outward.as the rotor turns. swinging-arm rotor. fomost ie SUPERNATANT poneooure — CENTRIFUGATION Psat cane tuiiaiion) SENTRIFUGATION dense components: Peter larger and more done components BEFORECentrifuge Calibration Purpose This procedure provides accurate rotation speed, t verification and centrifuges that are temperature in a laboratory environment. ication of rotation speed: \ > Centrifuges used in the laboratory are to be considered as c0 should only be handled with gloves and other personal protectiy and/or thoroughly disinfected before calibration verification. | » Place a small section of black and white reflective tape that ¢o tachometer on the center spindle of the test centrifuge. \ + In order to measure the rotation speed, there must be a views top cover that will allow the tachometer line of sight to the reff > Place a normal well-balanced load using specimen covers intgthe > Start the centrifuge and allow it come to equilibrium at a normal speed, > Use the tachometer through the viewing port above the reflgs a rotation rate reading., Record the rotation speed indica or by a built in tachometer on th | Verification of timer: ed by the centrifuge either b he centrifuge. » Centrifuges used in the laboratory are to be cond contaminated and should only be handled with glovesia personal protective equipment and/or thoroughly disin' , calibration verification. ') > Set the centrifuge timer at a setting frequently used in pre start the stopwatch simultaneously. » Stop the stopwatch at the same time as the centrifuge ti > Calculate the difference between the two times. Interpretation of results: Verification of rotation speed: If the difference between the test centrifuge and the certifie is + 5% of the procedure speed, then the test centrifuge rot; libration is verified as acceptable, ci)ification of timer: ceptable difference between the test timer and the certifie be #2% of the total test timer setting. SAFETY MEASURES, The work surface must be level and firm. Balance the tubes in the rotor. Do not open the lid while the rotor is moving. Bucket is properly cushioned. If you see it wobbling or shaking, turn it off or pull the plug. A Tittle vi but excessive amounts can mean danger. > Wear a face shield and / or safety goggles if you have to work anywher that’s in use. \ > Examine tubes and bottles for cracks or stress marks before using them. > Discard any centrifuge tubes that have cracks in them > Clean the rotor and centrifuge well after each use Maintenance of Centrifuge Daily maintenance > Wipe the inside of the bow! with disinfectant solution and rinse thoroughly. ge must not be used ifthe interior is hot, ifunusual vibrations deterioration (corrosion of parts) is detected. > A.qualified service technician should be contacted. \ZMost vibrations are due toi ‘i = '0 improper balancing and ean be corrected by rebalan Monthly maintenance Glen the centrifuge housing, rotor chamber, rotors and rotor accessories with an Clean plastic and non-metal parts with a fresh solution of 0.5% sodium hypochlorit Application’ > Remove cellular elements from blood to provides cell free, plasma > Conc. Cellular element for microscopy. + Remove protein precipitate from analytic sample. > Isolation of macromolecules such as DNA, RNA, proteil Use in haematology lab for PCV determination, (3Reference > Principles and technique in biochemistry-L WALKER WILSON > Lynch's Medical Laboratory Technology (4th edition, le