T.Elsarnagawy 11 Laboratory Centrifuges MLI-mde 210, 311

Centrifugation 1. Introduction: Centrifugation is a technique in which solutes are separated by their different rate of travel (or sedimentation) in a centrifugal field. 2. Centrifugation is widely used in biological separation. The solutes are usually cells, Sub- cellular organelles,viruses, large molecules such as proteins & nucleic acids. 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. Theodor Svedberg

MLI-mde 210, 3113 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 hover. 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. Type 1: Analytical Centrifugation : Applications: Measure the shape or mass of super molecular molecules Type 2: Preparative Centrifugation : Application: Separation of cell, sub cellular structure, membrane vesicles

MLI-mde 210, 3114 Components of a centrifuge: It has two main Parts: Head and Rotor Head: It is a revolving disc having four metal cups or four holding tubes. The sample to be centrifuged is filled in the tube. Rotor: It is a high speed motor which rotates the head to create centrifugal pull. The speed of rotation is controlled by a speed control switch. Working of centrifuge: A homogenous particulate matter is prepared. It is poured into the centrifuge tube and it is placed in the metal cups of the head. The speed of the rotor is set according to the requirement and allowed to run for a required period. Then the particulate matter settled down according to its density at the bottom of the tube.

T.Elsarnagawy 5 A laboratory centrifuge ◦ is laboratory equipment, ◦ driven by a motor, ◦ spins liquid samples at high speed. ◦ There are various types of centrifuges, depending on the size and the sample capacity. ◦ They vary widely in speed and capacity ◦ work by the sedimentation principle, where the centripetal acceleration is used to separate substances of greater and lesser density. ◦ comprise a rotor containing two, four, six, or many more numbered wells within which the samples containing centrifuge tips may be placed. Centrifuges are used in ◦ chemistry, biology, and biochemistry for isolating and separating suspensions. MLI-mde 210, 3115

T.Elsarnagawy 6 Operation Operation Increasing the effective gravitational force will more rapidly and completely cause the precipitate to gather on the bottom of the tube as a "pellet". The remaining solution is called the "supernate" or "supernatant". ◦ The supernatant liquid is then either  decanted from the tube without disturbing the pellet, or  withdrawn with a Pasteur pipette. The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or relative centrifugal force (RCF). MLI-mde 210, 3116

T.Elsarnagawy 7 Operation The particles' settling velocity in centrifugation is a function of their ◦ size and shape, ◦ centrifugal acceleration, ◦ the volume fraction of solids present, ◦ the density difference between the particle and the liquid, and the viscosity. MLI-mde 210, 3117

T.Elsarnagawy 8 Rotor objectives Rotor objectives Generally spoken, there are two main types of rotors: Fixed-angle rotor ◦ The rotor (mainly made of aluminium) is very compact. ◦ There are boreholes with a specific angle (like 45°) within the rotor. ◦ These boreholes are used for the sample tubes. MLI-mde 210, 3118

T.Elsarnagawy 9 Rotor objectives Rotor objectives Swing-out rotor (horizontal rotor) ◦ The rotor looks like a cross with bucket. ◦ Within these buckets, different tubes can be centrifuged. ◦ For a safe centrifugation, a specific adadpter for every tube shape is mandatory. MLI-mde 210, 3119

T.Elsarnagawy 10 Design Rotor objectives The rotor is closed by a rotor lid. The rotor is located in a rotor chamber which is covered by a metal centrifuge lid. The open lid prevents the motor from turning the rotor when the rotor chamber is open. During the run, the lid is locked. The lid protects the user from being injured by touching a rapidly spinning rotor. The rotor chamber and the lid of high quality centrifuges are robust enough to survive a rotor failure at full speed. MLI-mde 210, 31110

T.Elsarnagawy 11 Design Rotor objectives After a rotor crash, a centrifuge should not be reused as the enormous forces during a crash may have damaged essential parts of the device. The rotor must be balanced by placing samples or blanks of equal mass opposite each other. Since most of the mass is derived from the solvent, it is usually sufficient to place blanks or other samples of equal volume. As a safety feature, some centrifuges may stop turning when wobbling is detected (automatic imbalance detection, see Safety). MLI-mde 210, 31111

T.Elsarnagawy 12 Theory During circular motion  the acceleration is the product of the radius and the square of the angular velocity and it is traditionally named "relative centrifugal force" (RCF). MLI-mde 210, 31112

2. Theory of centrifugation Sedimentation rate of solutes is determined by their size, shape, and density, and the density and viscosity of media. The ability of an solute to centrifugal (or gravitational) field can be described by sedimentation coefficient (S = v/C): v linear velocity of a solute, C: centrifugal acceleration. Iso-density S2S2 = S1S1 r2r2 r1r1 ( ) 2 * d 2 –d 0 d 1 –d 0 Sedimentation coefficients of two solutes in a centrifugal field. S = 2/9 r 2 (d-d o )/  S is the sedimentation coefficient and is usually expressed in Svedbergs (S) or sec. r: radius of the solute, d: density of the solute, d 0 : density of media, and  :viscosity of the media. For a spherical solute: Centrifugal force

T.Elsarnagawy 14 MLI-mde 210, 31114

T.Elsarnagawy 15 Centrifuge tubes Centrifuge tubes or centrifuge tips are tapered tubes of various sizes made of glass or plastic.glass plastic They may vary in capacity from tens of mm, to much smaller capacities used in micro- centrifuges used extensively in molecular biology laboratories. The most commonly encountered tubes are of about the size and shape of a normal test tube (~ 10 cm long).test tube Micro-centrifuges typically accommodate micro- centrifuge tubes with capacities from 250 μ l to 2.0 ml  These are exclusively made of plastic. μ lml MLI-mde 210, 31115

T.Elsarnagawy 16 Centrifuge tubes Glass centrifuge tubes can be used with most solvents, but tend to be more expensive. They can be cleaned like other laboratory glassware, and can be sterilized by autoclaving. Plastic centrifuge tubes, especially micro- centrifuge tubes tend to be less expensive. Water is preferred when plastic centrifuge tubes are used. They are more difficult to clean thoroughly, and are usually inexpensive enough to be considered disposable MLI-mde 210, 31116

T.Elsarnagawy 17 Centrifuge tubes MLI-mde 210, Three microcentrifuge tubes: 2 mL, 1.5 mL and 200 μL (for PCR).PCR Four screw-top micro-centrifuge tubes.