Instrumental technique. Manju C K

Transcription

1 Instrumental technique Manju C K

2 CENTRIFUGE

3 A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward). The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and move to the center. In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low-density substances rise to the top.

4 History English military engineer Benjamin Robins ( ) invented a whirling arm apparatus to determine drag. In 1864, the first dairy centrifuge was introduced to separate cream from milk by Antonin Prandtl. A working butterfat extraction machine was exhibited in A 19th-century hand cranked laboratory

5 What happens to a particle in a centrifugal field The particle (m) is acted on by three forces: FC: the centrifugal force FB: the buoyant force Ff: the frictional force between the particle and the liquid Equation that describes the motion of this particle as follows: F = ma where m is the mass of the particle and a is the acceleration.

6 The Physics of UltraCentrifugation 1.Centrifugal force:- The tube containing the suspension of particles is rotated at a high speed, which exerts a centrifugal force directed from the center of the rotor towards the bottom of the tube. Centrifugal Force: Where, M: mass of particle r: radius of rotation (cm) (ie distance of particle from axis of rotation) ω :Average angular velocity (radians/sec)

7 A more common measurement of F in terms of the earths gravitation force, g, is relative centrifugal force, RCF its defined by RCF = (1.119 x 10-5 (rpm) 2 (r) This equation relates RCF to revolutions per minute of the sample. Equation dictates that the RCF on a sample will vary with r, the distance of the sedimenting particles from the axis of rotation. The RCF value is reported as a number times gravity,g.

8 Instrumentation It consist of two components, an electric motor to spin the sample and a rotor to hold tubes.

9 LOW SPEED CENTRIFUGE Most laboratories have a standard low-speed centrifuge used for routine sedimentation of heavy particles. The low speed centrifuge has a maximum speed of rpm. These instruments usually operate at room temperatures with no means of temperature control. Two types of rotors are used in it, fixed angle and swinging bucket. It is used for sedimentation of red blood cells until the particles are tightly packed into a pellet and supernatant is separated by decantation.

10 HIGH SPEED CENTRIFUGE High speed centrifuges are used in more sophisticated biochemical applications, higher speeds and temperature control of the rotor chamber are essential. The operator of this instrument can carefully control speed and temperature which is required for sensitive biological samples. Three types of rotors are available for high speed centrifugation-fixed angle, swinging bucket, vertical rotors

11 ULTRA CENTRIFUGE It is the most sophisticated instrument Intense heat is generated due to high speed thus the spinning chambers must be refrigerated and kept at high vacuum. It is used for both preparative work and analytical work.

12 CENTRIFUGE Rotor A centrifuge rotor is the rotating unit of the centrifuge, which has fixed holes drilled at an angle. Test tubes are placed inside these holes and the rotor spins to aid in the separation of the materials.

13 Types of Rotor

14 Swing-bucket Rotor Swinging-bucket rotor are used for pelleting, isopycnic studies and rate zonal studies. Tubes are attached to the rotor body by hinge pins or a crossbar. The buckets swing out to a horizontal position. Isopycnic studies (separation as a function of density). Rate zonal studies (separation as a function of sedimentation coefficient).

15 Fixed-angle Rotor Fixed-angle rotors are general-purpose rotors that are especially useful for pelleting subcellular particles and in short column banding of viruses and subcellular organelles. Sedimenting particles have to travel only short time. Tubes are held at an angle (usually 20 to 45 degrees) to the axis of rotation in numbered tube cavities.

16 Vertical rotor Vertical rotors hold tubes parallel to the axis of rotation; therefore, bands separate across the diameter of the tube rather than down the length of the tube. Vertical rotors are useful for isopycnic and, in some cases, rate zonal separations when run time reduction is important.

17 Points to keep in mind

18 Ensure that centrifuge bowls and tubes are dry. Use matched sets of tubes, buckets and other equipment. Ensure that the rotor is properly seated on the drive shaft. Make sure that tubes or containers are properly balanced in the rotor. Do not exceed the rotor s maximum run speed. Close the centrifuge lid during operation.

19 Thank you