SEPERATION OF BIOMOLECULES Centrifugation Sedimentation density gradient Chromatography

SEPERATION OF BIOMOLECULES Centrifugation (Sedimentation, density gradient) Chromatography (Elementary idea of thin layer, gel filtration & ion exchange- Principles & applications) • In the field of cytology, molecular biology separation of biomolecules is an integral part of study their physical, chemical & biological properties. • Particles get separated according to their size, shape, density, velocity of the medium and rotor speed.

• Centrifugation: • Centrifugation is based on centrifugal forces and a device centrifuge used for separate the particles in solution according to their shape size, density, viscosity of the medium and speed applied. • The particles in the solution have density is higher than that of the solvent sink and lighter particles float to the top. In case of no density difference the particle remain steady.

• Process of Centrifugation: • For Centrifugation a device is used called Centrifuge which rapidly spin. • Different types of centrifuges are available now for different purposes. • Generally they are classified into five groups: 1. Bench centrifuges, 2 Large capacity refrigerated centrifuges, 3. High speed refrigerated centrifuges 4. Continuous flow centrifuges 5. Ultracentrifuges. (Preparatory, Analytical, Airfuge)

1. Bench centrifuges: • These are simplest and cheapest types of centrifuges used in pathological labs & college labs. • They are used for rapid sediment material like erythrocytes, yeast cells, coarse particles. • Maximum speed is 4000 to 6000 r. p. m. and maximum relative centrifugal force is 3000 to 7000 g (gravitation). • Relative centrifugal force = M r ω2 Where M = Mass of the particle r = radius of rotation (distance of paricle from axis of rotation) ω = Average angular velocity

• Operation is carried out at ambient temperature. 2. Large capacity refrigerated centrifuges: • The speed & centrifugal force are 6000 r. p. m. and 6500 g. • The rotor chambers refrigerated to keep cool and hence proteins are protected from denaturation due to heat of motion. • Sample tube capacity may be 10, 50 or 100 ml to 4 - 4 liters. • It is used to collect rapid sedimentation samples like RBCs , yeast, nuclei & chloroplast. • High speed refregerated centrifuges are used to collect microorganisms, cell organelles, cell debris.

4. Continuous flow centrifuge: • It is high speed centrifuge & has a long rotor through which the suspension of particles flow continuously at a rate of about 1 -1. 5 liters /minute. • The sediment is collected on the wall of rotor and clear fluid passes as overflow through an outlet. • Used to collect the bacteria, yeast from large volumes of culture media.

5. Ultracentrifuges: • It is a special type of centrifuge in which rotor rotates at much higher speed than standard centrifuge. • Rotation speed ranges from 30000 to 50000 r. p. m. • The high rotating speed used in these devices can generate high amount of heat. • Cooling arrangements are required. • Used for seperating macromolecules from solvents. • Equipped with angled spin tube, titanium rotor that provides mechanical integrity. • Can go up to 500, 000 to 100, 000 g. • Used to separate very small particles.

• When spins the larger particle in the mixture in to test tubes would get flung out further and smaller particles would stay closer to the center. • Even the centrifuge is spinning but the particle in the test tube keep straight so they get flung outward. The larger the particle further gets flung. • Due to the particles end up in different places in the centrifuge the mixture gets separated. For example, if milk spines in the centrifuge the heavier cream spins separates out. • If blood spines in the centrifuge, the RBC, WBC and platelets would separates from the plasma. The heavier particles go the bottom and lighter go to the top.

• Principle of Centrifugation: • The centrifugation based on the principle of sedimentation. • The acceleration of centripetal force causes separation of denser particles along the radial direction at the bottom and lighter particles will move to the top of the tube. • The particles have higher density settles at bottom (sediment) and lighter float to the top. • The concept of centrifugal force is applied in rotating devices. • Centrifugal force is the force that draws a rotating body away from the center of rotation.

• The two different forces are equal in magnitude, but centrifugal forces is opposite in direction to the centripetal force. Centrifuge

• Differential Centrifugation (Sedimentation): • It is tendency for substances in suspension to settle out of the solution in which they are entrained and come to rest against a barrier. • This is happened due to their motion through the solution in response to the forces due to centrifugal acceleration and gravity act on them. • Sedimentation is the terminal settling process of suspended particles.

• Biological components in tissues and cells are separated. Sub cellular components and substances like cytoplasm fluid, nucleus, mitochondria, Golgi bodies are separated, and proteins based on density in cells and tissues is separated. • Nucleic acids DNA, RNA etc are also separated by this method. The separation is based on size of the particle, lesser the size of particle more the particle will be towards the base. • The shape of particle like circular particles will settle down easily in comparison to polygonal shape particles. Density is a main characteristic, denser the object lowers the settling.

• Density Gradient Centrifugation: - Density gradient centrifugation is used to separate macromolecules that differ only slightly in size or density. - Two techniques commonly used are… - 1. Zonal centrifugation - 2. Isopycnic centrifugation

1. Zonal cenrifugation: - It is also known as Rate Zonal Centrifugation. It is a technique employed to effectively separate particles of different sizes. - Zonal technique is used to separate wide range of particles and macromolecules, E. g. Nuclei, Mitochondria, Ribosome and Proteins. - The tube is first filled with different concentrations of sucrose or another solute establishing layers with different densities and viscosities density gradient, within which the particles to be resolved are added.

• The larger particles will be able to travel to the bottom layer because they are more massive. • The greater mass allows the particles to travel through layers with a greater viscosity, while the smaller particles will remain at the top, as they lack the mass to travel through the more viscous layers. • Once the centrifugation is over, fractions are collected.

2. Isopycnic Centrifugation: • The word ‘isopycnic’ means equal density. • Each particle moves to the region corresponding to its own buoyant density. • This utilizes a specific medium that gradually increases in density from top to bottom of the centrifuge tube. • Particles move under centrifugal force through the medium & density gradient & stop at a point in which the density of the particle equals the density of the surrounding medium. • The medium used are Caesium Chloride, Sucrose, Dextran, methyl glucamine salts.

• It is the technique used to separate molecules soley on the basis of ‘buoyant density’. • It is independent of size and shape & time of centrifugation of the particles. • Buoyant density is a self generating density gradient established via equilibrium. • Fractionation of DNA from a mixture of caesium Chloride is done through this method. • A high speed centrifuge is applies about 100000 g.