CENTRIFUGATION

Centrifugation is a basic separation technique. A centrifuge is a device for separating particles in an applied centrifugal field in a solution. There are two different forces act on an object moving in a circular motion. Centrifugal force: Force directed outward from the center. E.g. While turning a bus in twist way, the passengers strike on the bus wall is due to centrifugal force. Centripetal force: The force exerted towards the center is now as centripetal force. E.g. the force acts on passengers by the turning car. Now, suppose a particle is exerted to sediment by centrifugal force, then The rate or velocity at which it sediments is proportional to the force applied

Sedimentation is more rapid when the force applied is greater than the gravitational force of the Earth Basis of separation is to exert a larger force than does the Earths gravitational force.

Basic Principle of Sedimentation The particles to be separated are suspended in a specific liquid media, held in tubes or bottles which are located in rotor in centrifuge machine, positioned centrally to the drive shaft. These particles are differing in size, shape and density. As we have already mentioned that, The rate of sedimentation is dependent upon the applied centrifugal field (G) G Where W: R: Angular velocity of revolving particle (Remember: one revolution of the rotor is equal to 2 radians) Radial distance from axis of rotation In terms of revolution per minute, we have W= 2 rev min-1/ 60 Therefore: G = W 2R = W2R equ (i)

It is expressed as a multiple of the earths gravitational field (g=981 cm s-2). Hence RCF, Relative Centrifugal Field =G/g = RCF = 1.119 x 10-5(rev min-1)2 R .equ (ii) = x g unit (number times g) It means, RCF is the ratio of the weight of the particle in the applied centrifugal field to the weight of the same particle when acted by gravity alone. Therefore the rotor speed, radial dimensions and time of the rotor must be quoted during the centrifugation. However: This is not the only case in Biochemical experiments as biological samples are always found in dissolved or suspended form in a solution. Thus, the rate of sedimentation not only depends on the centrifugal field but also on 1. Mass of particle 2. Density of particle 3. Density and viscosity of the medium used 4. The extent to which its shape deviates from spherical Now according to Newtons Second law of Motion, the centrifugal force (F) exerted on particle is = M. a = M. W2R .equ (iii) Where: M: mass of particle a: acceleration while in angular motion= W2R Increasing the sharpness of a turn, w and r decreases. Since r is linear, w has greater effect on the particle. It causes the molecules to sediment down the centrifuge tube. They start to move downward to sediment; however they encounter opposing force, a frictional resistance in their movement.

Frictional force

=f = 6. h. Rp. ) equ (iv)

Where: f: h: Rp: Frictional force Viscosity coefficient of medium Radius of sedimenting particle dr/dt: Rate of sedimentation expressed as the change in radius with time (velocity v)

The sedimenting molecule must also displace the solvent into which it sediments and give rise to a buoyant force Buoyant force Where: V: m: Specific volume of the molecule Density of the medium = mass x a = V. m W2R ..equ (v)

While sedimenting, the velocity of the particle increases until it equals the frictional force resisting its motion through the medium. This is an equilibrium state when the particles stop to move or sediment. From equations iii, iv and v. Centrifugal force M. W2R Rp3 p W2R Rp3 (p - m) W2R = Frictional force + Buoyant force

= 6. h. Rp. ) + V. m W2R = 6. h. Rp. ) + Rp3 m W2R = 6. h. Rp. )

= h Rp2 (p - m) W2R v Where: dr/dt: v, is the velocity of the sedimenting particle Mass: Density x Volume p: m: Density of particle Density of medium = h Rp2 (p - m) W2R ..equ (vi)

From above equation, it seems clear that velocity is proportional to its size, to the differences in density between the particle and medium and to the applied centrifugal field. It is zero when the density of the particle and medium are equal. It decreases when the viscosity of the medium increases. Since the Rp is in square form, the size of particle has greater influence on velocity. For a particle, h, Rp, p, m and W all are constants t Where t: Rt: Rb: The sedimentation time in seconds Radial distance from the axis of rotation to liquid meniscus Radial distance from the axis of rotation to bottom of tube = In

It is now clear that a mixture of heterogeneous approximately spherical particles can be separated by centrifugation on the basis of their densities, their sizes and etc. t

It means, higher the size particles, faster is the sedimentation (Short time for sedimentation) of it and smaller the size slower is the sedimentation (takes longer time). CENTRIFUGATION: RCF CALCULATION The relative centrifugal force (RCF) can be calculated from the following equation: RCF = (1.119 x 10-5) (rpm)2(r) Where rpm is the speed of rotation expressed in revolutions per minute and r (radius) is the distance from the axis expressed in cm. The RCF units are "x g" where g represents the force of gravity. RCF can also be determined from the NOMOGRAPH below. Place a straight edge to intersect the radius and the desired RCF to calculate the needed rpm. Alternatively place the straight edge on the radius and the rpm to calculate the g-force. For example, spinning a sample at 2500 rpm in a rotor with a 7.7 cm radius results in a RCF of 550 x g.

Figure 1: Nomograph showing relationship between RCF, RPM and Radius

Centrifuges and their uses

1. Low Speed Centrifuge Least expensive and simplest in many design Maximum rotor speed of 4000-6000rpm (3000-7000 X g) a) Small bench centrifuges To collect small amounts of materials (250mm3) that is rapidly sediment (1-2 min)

No special cooling system Ambient air flows around the rotor to cool the system Use to rapid sedimentation of blood samples b) Large capacity refrigerated centrifuges Refrigerated rotor chambers for cooling the sample Large volumes 10, 50 and 100 cm3 processing depending upon the rotors and tubes Maximum capacity of 1.25 dm3 Rotors are mounted on a rigid suspension Erythrocytes, coarse or bulky precipitates, yeast cells, nuclei and chloroplasts

2. Microcentrifuge Maximum rotor speed of 12000rpm with RCF of 10000g Have total capacity of 1.5ml over very short time (0.05-5 min) Use to sediment large particles like cell ppt

3. High speed refrigerated centrifuge Maximum rotor speed of 25000rpm with RCF of 60000g Have total capacity of 1.25 dm3 Interchangeable fixed angle and swinging buckets rotors Use to collect microorganisms, cellular debris, larger cellular organelles and proteins precipitates by ammonium sulphate

Not use for viruses and smaller organelles like ribosome

4. Continuous flow centrifuge Relatively simple and high speed centrifuge Special design rotor (long and tubular) with non interchangeable system Have total capacity of 1-1.25 dm3/min with continuous flow Particles sediment at wall and excess clarified medium overflows through an outlet port Use to collect bacterial and yeast cells from their mass culture of about 100-500 dm3

5. Ultracentrifuge

Powerful with speed 2 types a) Preparative ultracentrifuge

Maximum rotor speed of 30000-80000 rpm with RCF of 600000 x g Highly sophisticated with refrigerated, sealed and evacuated to minimize excess heat generate More sophisticated temperature monitoring system employing an infrared temperature sensor Overspeed control system to prevent operation of rotor above its max rated speed Vibration minimize system (a flexible drive shaft system) during unequal loading of the centrifuge tubes Enclosed in heavy armour plating Airfuse for some biochemical applications requiring high centrifugal force Use for sediment macromolecule/ligand binding kinetic studies, steroid hormone receptor assays, separation of major lipoprotein from plasma and deproteinisation of physiological fluids for amino acid analysis b) Analytical ultracentrifuge

Maximum rotor speed of 70000 rpm with RCF of 500000 x g Highly protective chambers with refrigerated and evacuated system also have an optical system to enable the sedimenting material to be observed throughout the process. Three types of optical system, a light absorption system, alternative Schlieren system and Rayleigh interferometric system (both measures refractive index of solution)