Centrifugation

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1. CENTRIFUGATION Represented To: Dr. IU khan Represented By: Farzeen Javaid 0188-BH-CHEM-11 Course Title: Separation Techniques (Compulsory) Course Code: CHEM-4201 Govt. College University Lahore

2. History English military engineer Benjamin Robins (1707-1751) invented a whirling arm apparatus to determine drag. In 1864, Antonin Prandl invented the first dairy centrifuge in order to separate cream from milk. In 1879, Gustaf de Laval demonstrated the first continuous centrifugal separator, making its commercial application feasible.

3. Introduction WHAT IS CENTRIFUGE ? Centrifuge is device for separating particles from a solution according to there size, shape, density, viscosity of the medium. WHAT IS CENTRIFUGATION ? Centrifuge uses centrifugal force to separate phases of different densities.

4. Definition Centrifugation is a procedure that involves the use of centrifugal force for the sedimentation of mixture with a centrifuge used in industry and in laboratory settings. More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis.

5. Principle of centrifugation A particle whether it is a precipitate a macromolecule or a cell organelle is subjected to a centrifugal force when it is rotated at a high rate of speed. The centrifugal force F is denoted by equation F=mω 2 r Where F= intensity of the centrifugal force m= effective mass of the sedimenting particle ω= angular velocity of rotation r= distance of the migrating particles from the central axis of rotation 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 .”

6. A centrifuge is a piece of equipment, generally driven by an electric motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis to separate substances of different densities. Tubes in the centrifuge are tilted so centrifugal force can pull denser substances towards the bottom of the tube. Relative Centrifugal Force ( RCF) measures acceleration applied to the sample RCF=(r*ω 2 )/g r=distance from center or motor to sample ω= angular velocity (radians/ second) g=9.8m/s 2

7. General Idea Feed added to spinning bowl Sedimentation of particles occurs in centrifugal field Flow is upwards at a particular rate which determines residence time in device Separation happens if sedimentation velocity is high enough for particle to reach side of bowl within residence time Large particles have higher settling velocities than small particles Both large and small are still particles, have small Reynolds no.s (<1) and obey Stokes’ Law

8. Components of the centrifugation On and off control, operation time control (timer), rotation speed control, temperature control (in refrigerated centrifuges), vibration control (safety mechanism) and brake system Refrigeration system (in refrigerated centrifuges). Base Lid/cover Casing Electric motor Rotor. There are different types of rotors. The most common are the fixed angle, the swinging buckets, the vertical tube and the almost vertical tube types.

9. Instrumentation of centrifugation The basic centrifuge consists of two components one is Electric motor with drive shaft to spin the sample and a Rotor to hold tubes or other containers of the sample. A wide variety of centrifuges are available ranging from a low speed centrifuge used for routine pelleting of relatively heavy particles. There are three types of centrifuges 1 . Low –speed centrifuges . Its also called as clinical centrifuge . The common centrifuge has a maximum speed in the range of 4000 to 5000 rpm with RCF value approx. up to 3000Xg. These instruments usually operate at low temperature with no means of temperature control of the samples . Two types of rotors fixed angle and swinging bucket may be used in this instruments.

10. Low speed centrifuges are especially useful for the rapid sedimentation of coarse precipitates or red blood cells . The sample is centrifuged until the particles are packed into a pellet at the bottom of the tube. The upper liquid portion, the supernatant, is then separated by decantation . High Speed Centrifuges : High speed centrifuges for more sophisticated biochemical applications. Higher speeds and temperature control of the rotor chambers are essential. Rotor chambers in most instruments are maintained at or near 4 C. Three types of rotors are available for high speed centrifugation. a. Fixed Angel rotor. b. Swinging –bucket rotors c. vertical rotor High speed centrifuges are used to sediment Cell debris after cell homogenization. Ammonium sulfate precipitates of proteins and cellular organelles such as chloroplasts, mitochondria and nuclei.

11. 3. Ultracentrifuges : The most sophisticated of the centrifuges are the ultracentrifuges because of the high speeds attainable intense heat is generated in the rotor so the spin chamber must be refrigerated and placed under high vacuum to reduce friction. it is a high speed centrifuge that has fixed head rotors .It is mainly used in separation of lipoproteins .since the separation is long process there is generation of heat and thus are provided with internal cooling system. Ultracentrifuges can be used both for preparative work as well as for analytical. Preparative models its primarily used for separations and purification of samples for further analysis and analytical models which are designed for performing physical measurements on the sample during sedimentation.

12. Types of Centrifugation a) Preparative 1 . Differential Centrifugation 2 . Density gradient Centrifugation 2a . Rate-Zonal 2b . Isopycnic b) Analytical

13. a) Preparative Centrifugation I t is used to separate organelles and molecules It can handle larger liquid volumes no optical read-out Separation methods used in preparative ultracentrifugation: 1 . Differential Centrifugation- pelleting, 2 . Density Gradient Centrifugation

14. 1.Differential Centrifugation based on the size of the particles used for simple pelleting, for the separation of subcellular organelles and macromolecules first , sample must be homogenised ultra centrifugation sedimentation depends on mass, shape and partial specific volume of a macromolecule, as well as solvent density, rotor size, rate of rotation. Usually uses a fixed angle rotor

15. 2.Density Gradient Centrifugation method to purify subcellular organelles and macromolecules . density gradients generated by placing layer after layer of gradient media Density gradient centrifugation classified into two: 2a . Rate-Zonal separation (size) 2b . Isopycnic Separation (Density)

16. 2a.Rate- Zonal Centrifugation use of continuous density gradient of solvent such as sucrose . density increases towards the bottom of the tube sample layered on the top molecules form discrete bands after centrifugation separation based on size of the molecules Swinging bucket rotors

17. 2b. Isopycnic Separation based on the density of the molecules Mix gradient material with the sample molecule ( CsCl ) molecules move to the position where their density is same as the gradient material ( isopycnic position) inorder to generate a gradient, we select a CsCl concentration that will give a range of densities that includes the range of molecules that have to be separated. used for the separation of DNA Swinging bucket or fixed angle rotor

18. b) Analytical Centrifugation uses small size samples built-in optical system uses relatively pure sample

19. Types of Centrifuges Types centrifuges depends upon Maximum speed of sedimentation Presence /absence of vacuum Temperature control refrigeration. Volume of sample and capacity of centrifugation tubes Depending on the particular application, centrifuges differ in their overall design and size. a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated

20. 1. Small Benchtop With or without refrigeration Slow speed ( eg up to 4000 RPM) Common in clinical lab (blood/plasma/serum separation) can take approx (up to) 100 tubes, depending on diameter

21. 2. Microcentrifuge (“microfuge”, “ eppendorf ”) Sample volume is small in tubes Refrigerated with or without can generate forces up to ~15,000 x g Take tube of small volume up to 2ml. Commonly used of concentration protein very common in biochemistry/molecular biology/biological labs

22. 3. High Speed centrifuges ( 15,000 – 20,000 RPM) Refrigerated use for protein precipitates, large intact organelles cellular debris from tissue homogenisation and micoorganism They operate maximal centrifugal force of approx 10000g Use for research applications differntial sepration of nucleus, mitrochondrial , protein precipitate,etc .

23. 4. Ultracentrifuge ( 65,000 RPM) Refrigerated and evacuated The detail biochmestry analysis of subcellular structures and isolate biomolecules . Operate at upto 90000 g limited lifetime expensive require special rotors care in use – balance critical! research applications

24. 5. Large-capacity preparative centrifuge Centrifugal fields of 3000 to 7000g. Efficient separation of coarse precipitates or whole cells.

25. Types of rotors Fixed angle Swinging bucket rotors (Vertical/ Horizontal)

26. 1. 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.

27. 2. Swinging bucket rotors a. Vertical rotors Vertical rotors Sample tubes are held in vertical position during rotation This type of rotor is not suitable for pelleting applications but is most efficient for isopycnic (density) separations due to the short pathlength . Applications include plasmid DNA, RNA, and lipoprotein isolations. b. horizontal rotors 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.

28. 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.

29. 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

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

31. Calculation of C entrifugal field Centrifugal field from the center of rotation may be calculated using the equation G=ω 2 r Calculation of Angular velocity Angular velocity ω may be calculated by using this equation ω= 2 π   rev min -1 60

32. Applications of Centrifugation Separate solids from solution Used during urinalysis to separate disease identifying components Separate blood into plasma Purification of mammalian cells Fractionation of subcellular organelles ( including membranes / membrane fractions) Fractionation of membrane vesicles

33. Application in Water Treatment

34. Commercial applications Centrifuges with a batch weight of up to 2,200 kg per charge are used in the sugar industry to separate the sugar crystals from the mother liquor. Standalone centrifuges for drying (hand-washed) clothes – usually with a water outlet. Large industrial centrifuges are also used in the oil industry to remove solids from the drilling fluid.

35. Preventive Maintenance Lubricate and clean motor. Clean case. Inspect power cords and plugs. Inspect controls and switches. Ensure appropriate menu settings for proper use. Ensure tightness of rotor. Check lights and indicators. Verify that alarms are operating properly. Ensure safety switch is functioning. If refrigerated, ensure temperature reading is working. Replace/repair gaskets, seals, and vacuum pump (if applicable).

36. Common Failures Machine will not start. Power supply is faulty. Safety switch or timer not working properly. Motor not working properly. Motor making noise. Rotor is broken. Lid will not open or close.

37. Thank you!