CENTRIFUGE, TYPES ,USES AND ITS APPLICATION.pptx

Definition
Types
Uses
Relative centrifugal force
Clinical application

1. CENTRIFUGE Dr .B. priyadharsini

2. PRINCIPLE The particles get separated from the solution according to their size, shape, density, viscosity of the medium and rotor speed. Involves principle of sedimentation in a centrifugal field.

3. BIOLOGICAL CENTRIFUGATION Process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. Key technique for Isolating and analysing cells , Subcellular fractions, Supramolecular complexes Isolated macromolecules ( Proteins or nucleic acids )

4. IN CLINICAL LABORATORY, Remove cellular elements from blood – Provide plasma or serum for analysis. Concentrate cellular elements & other components of biological fluids – Microscopic examination or chemical analysis. Separate protein-bound or antibody - bound ligand from free ligand in immunochemical & other assays. Separation of lipoproteins from one another. Isolation of genomic DNA in PCR technique

5. CENTRIFUGATION PROTOCOL B iological structures exhibit a drastic increase in sedimentation when they undergo acceleration in a centrifugal field. PROTOCOL The more dense a biological structure is , the faster it sediments in a centrifugal field. The more massive a biological particle is, the faster it moves in a centrifugal field.

6. PROTOCOL (CONTINUED) The denser the biological buffer system is , the slower the particle will move in centrifugal field ; The greater the frictional coefficient is, the slower a particle will move ; The greater the centrifugal force is , the faster the particle sediments ; The sedimentation rate of a given particle will be zero when the density of particle and the surrounding medium are equal.

7. CALCULATION OF RELATIVE CENTRIFUGAL FIELD Depends on rotor speed , radial dimensions and effective path length by sedimented particles . Rate of sedimentation is dependent upon the applied centrifugal field (G), determined by The radial distance, (r), of the particle from the axis of rotation The square of the angular velocity of the rotor ( ) G =  2 r

8. CENTRIFUGAL FIELD RCF =1.12× 10 -5 rpm 2 r Where 1.12× 10 -5 = an empirical factor r= radius in centimeters from the centre of rotation to the bottom of the tube in rotor cavity or bucket during centrifugation. rpm = the speed of rotation of the rotor in revolution per minute.

9. NOMOGRAPH

10. SVEDBERG UNIT Unit for sedimentation coefficient. Offers a measure of a particle size and shape based on its sedimentation rate. One Svedberg unit = 10 -13 seconds.

11. INSTRUMENTATION Rotor Motor Drive shaft Speed control Power switch Brake Refrigeration unit Tachometer Timer Alarm

12. CENTRIFUGE TUBES Recommended tubes – withstand the RCF. Polypropylene tubes – withstand RCF of upto 5000  g. Tapered bottom Size should fit into the rack to be centrifuged.

13. TYPES OF CENTRIFUGES A common feature for all centrifuge – Central motor that spins a rotor containing the samples to be separated. The most obvious difference between centrifuges are, Maximum speed of sedimentation ; Presence or absence of vacuum ; Potential for refrigeration & manipulation of temperature; Volume of sample & capacity for individual centrifugation tubes.

14. TYPES OF CENTRIFUGE Speed of the centrifuge Types of rotor

15. TYPES OF ROTORS Swinging – Bucket Rotors. Fixed- Angle Rotors Vertical Rotors.

16. SWINGING- BUCKET CENTRIFUGES Allows the tubes placed in cups of the rotor - Assume horizontal plane when the rotor is in motion - Vertical plane when it is at rest. Allow the sample to move through the density gradients. Features full pathlength, slow run time , & excellent resolution. Ideal for separating- large volume samples at low speed & when samples are to be resolved in density gradients.

17. SWINGING BUCKET ROTOR

18. FIXED ANGLE OR ANGLE HEAD CENTRIFUGE Fixed positions at angles from 25 to 40 to the vertical axis of rotation. Feature a medium pathlength & excellent resolution. Sedimentation parallel to the shaft of the tube. Shorter run time . Used when rapid sedimentation of small particles are needed.

19. FIXED ANGLE ROTOR

20. VERTICAL ANGLE ROTORS Tubes are vertical during the run. Feature the shortest pathlength of any rotor type, the fastest run time & the highest resolution Used for isopycnic separations – for the banding of DNA.

21. BASED ON SPEED LOW SPEED CENTRIFUGE HIGH SPEED CENTRIFUGE ULTRACENTRIFUGE

22. LOW SPEED CENTRIFUGE ROTOR -Steel or brass. 4000 to 5000rpm. Room temperature, No means of temperature control Uses fixed angle and swinging bucket rotors. Sedimentation of RBC

23. HIGH SPEED CENTRIFUGE ROTOR- Aluminium/ titanium reinforced fibres Temperature control is a must Uses 10000-15000rpm Uses fixed angle, swinging bucket & vertical rotors Differentially separate nuclei, mitochondria. Handling of large volume of samples.

24. ULTRACENTRIFUGE ROTOR-Titanium alloy. 70000 to 80000rpm. Overspeed control device is a must. Generate considerable heat as a result of friction at high speeds Requires refrigerated chamber. Available in both preparative and analytical models.

25. ULTRACENTRIFUGE Flexible drive shafts Over speed control device

26. MICROFUGES Spin small ( 2 ml or less ) liquid samples at high speeds of approximately 10000 g. Small, compact benchtop centrifuges in refrigerated or non- refrigerated models.

27. CENTRIFUGE CLASSES LOW SPEED HIGH SPEED ULTRA CENTRIFUGE MAXIMUM SPEED (rpm × 10 3 ) 10 20 100/150 MAXIMUM RCF(× 10 3 ) 7 100 800/900 ROTORS Steel/brass Aluminium/titanium Titanium alloy covered with polyurethane layer BACTERIA yes yes yes ANIMAL & PLANT CELLS yes yes yes NUCLEI yes yes yes PRECIPITATES Some Most Yes MEMBRANE FRACTIONS Some Some Yes RIBOSOMES/ POLYSOMES - - Yes MACROMOLECULES - - Yes VIRUSES - Most yes

28. CENTRIFUGATION TECHNIQUES PREPARATIVE CENTRIFUGATION ANALYTICAL CENTRIFUGATION

29. PREPARATIVE CENTRIFUGATION DENSITY GRADIENT CENTRIFUGATION DIFFERENTIAL CENTRIFUGATION

30. PREPARATIVE CENTRIFUGATION DENSITY GRADIENT DIFFERENTIAL CENTRIFUGATION Biological particles of similar size but differ in their densities. Sedimentation of particles depend on density, shape & size. Rate zonal & Isopycnic centrifugation Subcellular fractionation

31. DIFFERENTIAL CENTRIFUGATION

32. SUBCELLULAR FRACTIONATION

33. DENSITY GRADIENT CENTRIFUGATION

34. RATE ZONAL & ISOPYCNIC CENTRIFUGATION

35. PREPARATIVE & ANALYTICAL CENTRIFUGE PREPARATIVE CENTRIFUGATION ANALYTICAL CENTRIFUGATON High -velocity centrifuge used in separation of small submicroscopic particles. High-velocity centrifuge used in the analytical processes. Used in pelleting small materials such as membranes & organelles. Used to determine the mass & shape of macromolecules such as protein complexes & rate of sedimentation of molecules. A large sample size can be processed. Only small volume less than 1 mL can be processed. Fractions can be collected after the run ; no optical read out. Progress can be observed visually. No cooling arrangements are used. Cooling arrangements are made ( High speed generates more heat )

36. ANALYTICAL CENTRIFUGATION

37. POTENTIAL HAZARDS PHYSICAL HAZARDS Mechanical stress , metal fatigue & corrosion – Mechanical failure over time. Unbalanced & other mechanical failures – centrifuge explosion . EXPOSURE HAZARDS Materials being centrifuged can become aerosolized & if materials are hazardous can expose the user to hazardous air when centrifuge is opened.

38. RECOMMENDATIONS Speed check every 3 months Temperature check-monthly. Not to vary more than 2°C CLSI propose an RCF of 1000 to 1200g for 10 ± 5 minutes. Log book maintenance Proper balancing of rotor. Regular preventive maintenance.

39. BALANCED SWINGING BUCKET ROTOR

40. CENTRIFUGING HAZARDOUS MATERIALS  Always wear appropriate PPE for the hazard.  Let the centrifuge stand for at least 10 minutes after the rotor has stopped to allow any aerosols to settle before opening the lid.  If centrifuging materials from a Biosafety Level 2 or above, rotors must have aerosol containment or be used inside a biosafety cabinet. o Rotors must be loaded and unloaded inside the biosafety cabinet.  If centrifuging radioactive materials, the centrifuge must be kept behind an appropriate shield.  If centrifuging hazardous chemicals, open rotor and/or containers inside a fume hood if necessary