Centrifuge.pptx. Centrifuge.pptxCentrifuge.pptx

Centrifuge.pptx

1. Principles and applications of centrifuge Lab Instruments Lecture 8 Fundamentals of Lab Instructor Naeem Bukhari

2. Definition “An apparatus that rotates at high speed and by centrifugal force separates substances of different densities.” 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.

3. 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" 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 The supernatant liquid is then either decanted from the tube without disturbing the pellet, or withdrawn with a Pasteur pipette.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 ).

4. Operation The particles' settling velocity in centrifugation is a function of their size and shape, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity.

5. Rotor objectives Generally spoken, there are two main types of rotors: Fixed-angle rotor The rotor (mainly made of aluminum) is very compact. There are boreholes with a specific angle (like 45°) within the rotor. These boreholes are used for the sample tubes.

6. 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 adapter for every tube shape is mandatory. Rotor objectives

7. 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.

8. 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). Design Rotor objectives

9. Centrifuge tubes Centrifuge tubes or centrifuge tips are tapered tubes of various sizes made of glass or 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). Micro-centrifuges typically accommodate micro-centrifuge tubes with capacities from 250 μl to 2.0 ml  These are exclusively made of plastic.

10. 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

11. Centrifuge tubes Three micro centrifuge tubes: 2 mL, 1.5 mL and 200 μL (for PCR ). Four screw-top micro-centrifuge tubes.

12. INSTRUMENTATION FOR CENTRIFUGATION It consist of two components, an electric motor to spin the sample and a rotor to hold tubes. Here we describe 2 types, the low speed, the high speed.

13. LOW SPEED CENTRIFUGES Most laboratories have a standard low-speed centrifuge used for routine sedimentation of heavy particles The low speed centrifuge has a maximum speed of 4000-5000rpm 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.

14. LOW SPEED CENTRIFUGES

15. HIGH SPEED CENTRIFUGATION High speed centrifuges are used in more sophisticated biochemical applications, higher speeds and temperature control of the rotor chamber are essential. Speeds of 20000 to 25000rpm The operator of this instrument can carefully control speed and temperature which is required for sensitive biological samples. T hree types of rotors are available for high speed centrifugation-fixed angle, swinging bucket, vertical rotors

16. HIGH SPEED CENTRIFUGATION

17. ULTRA CENTRIFUGE It is the most sophisticated instrument. Attain the speed of 75000rpm. 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.

18. ULTRA CENTRIFUGE

19. ANALYTICAL TECHNIQUES All analytical techniques require use of an ultra centrifuge and can be classified as 1)DIFFERENTIAL CENTRIFUGATION 2)DENSITY GRADIENT CENTRIFUGATION

20. 1)DIFFERENTIAL CENTRIFUGATION

21. 2)DENSITY GRADIENT CENTRIFUGATION

22. CARE OF CENTRIFUGE AND ROTORS Carefully read the manual before using centrifuges Select proper operating conditions   Check rotor for cleanliness and for damage   Select proper rotor of definite size   Be sure the rotor is clean and undamaged   keep accurate record of centrifuge and rotors   carefully clean rotors after centrifugation.

23. Safety aspects Centrifuge servicing includes full strip down, chemical cleaning and mechanical breakdown so that each individual component is cleaned and re-calibrated before re-installation. Regular servicing and maintenance ensures that your bench top centrifuge works effectively and provides accurate mixing of samples which can affect results in clinical trials .   Centrifuge rotors should never be touched while moving , because a spinning rotor can cause serious injury. Modern centrifuges generally have features that prevent accidental contact with a moving rotor as the main lid is locked during the run.

24. Safety aspects The load in a laboratory centrifuge must be carefully balanced . Small differences in mass of the load can result in a large force imbalance when the rotor is at high speed. This force imbalance strains the spindle and may result in damage to centrifuge or personal injury. Some centrifuges have an automatic rotor imbalance detection. The control software immediately discontinue the run when imbalance appears. Before starting a centrifuge, an accurate check of the rotor lockage as well as the lid lockage is mandatory.

25. Applications The centrifugation is a modern and easy technique of separation. Due to centrifugation it is easy to separate cellular and sub cellular components. It is used to study the effects of centrifugal forces on cells

26. “Never take a person's dignity: it is worth everything to them, and nothing to you” Thank You!!! Frank Barron quotes