PRADEEP VERMA, Centrifugation

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centrifugation & centrifuges.

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CENTRIFUGATION:

CENTRIFUGATION Presented by:- Pradeep Verma M.Phil. 2 nd Sem . Department of Zoology, C.C. S. University, Meerut (U.P.)

Contents :

Contents Introduction History Centrifugation and its types Ultracentrifugation Principle of centrifugation Material used in rotors construction Centrifugal rotors Geometry of rotors Different types centrifuge Gradient media Centrifugal fractionation Corrosion Precautions Biological applications Conclusion Bibliography Acknowledge.

Introduction:

Introduction Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge. Widely applicable technique in biochemistry, cell Biology, molecular biology, and medical fields. Current research and clinical applications rely on isolation of cells, subcellular organelles, and macromolecules, often in high yields. A centrifuge uses centrifugal force (g-force) to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis.

History:

History English military engineer Benjamin Robins (1707–1751) invented a whirling arm apparatus to determine drag . In 1864, Antonin Prandtl 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.

Centrifugation and its types:

Centrifugation and its types Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge , used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or " supernatant liquid ".

Cont….:

Cont…. The rate of centrifugation is specified by the angular velocity measured in revolutions per minute (RPM), or acceleration expressed as g . The conversion factor between RPM and g depends on the radius of the sample in the centrifuge rotor . 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 .

PowerPoint Presentation:

Preparative centrifugation This techniques are concerned with the actual separation, isolation and purification of, for example, whole cells, subcellular organelles, plasma membrane, polysomes, ribosomes, chromatin, nucleic acid, lipoprotein and viruses, for subsequent biochemical investigation. Analytical centrifugation T his techniques are devoted mainly to the study of pure or virtually pure macromolecules or particles. It is primarily concerned with the study of the sedimentation characteristics of biological macromolecules and molecular structure, rather than with the actual collection of particular fractions. Types of centrifugation

Ultracentrifugation:

Ultracentrifugation Ultracentrifugation makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and viruses. In addition to purification: Analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as sucrose , caesium chloride , or iodixanol .

Principle of Centrifugation:

Principle of Centrifugation Protocols for centrifugation typically specify the amount of acceleration to be applied to the sample, rather than specifying a rotational speed such as revolutions per minute (rpm). This distinction is important because two rotors with different diameters running at the same rotational speed will subject samples to different accelerations. During circular motion the acceleration is the product of the radius and the square of the angular velocity , and the acceleration relative to " g " is traditionally named "relative centrifugal force" (RCF).

Cont.….:

Cont.…. Where - Where -

Materials used in rotor construction:

Materials used in rotor construction One of following materials can be used in centrifugal rotors: Brass Steel Aluminum alloy Titanium alloy

Centrifugal rotors:

Centrifugal rotors Swinging bucket rotor Fixed angle rotor Vertical-tube rotor Central shaft Types of rotors

PowerPoint Presentation:

Centrifuge rotors Fixed-angle axis of rotation At rest Swinging-bucket g Spinning g Fixed Angle Rotor Sedimenting particles have only short distance to travel before pelleting. Shorter run time . The most widely used rotor type . Swinging Bucket Rotor Longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet.

Cont.….:

Cont .…. Vertical-tube Rotor In 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 path length. Applications include plasmid DNA, RNA, and lipoprotein isolations. Zonal Rotor the zonal rotor is a flattened sphere, the interior of which is divided into four sectors by four septa. These rotors are designed to minimize the wall effect which are encountered in swinging bucket and fixed angle rotors, and to increase sample size.

Cont… :

Cont… Continuous flow rotors Continuous flow rotors are designed for high speed separation of relatively small quantities of solid matter from large volumes of suspension and are particularly useful for the harvesting of cell and the large scale isolation of viruses. Elutriator rotors The elutriator rotor is a type of continuous flow rotor which contains a single conical shaped separation chamber and a bypass chamber on the opposite side of the rotor which serves as a counterbalance.

PowerPoint Presentation:

Geometry of rotors b c r max r av r min r max r av r min Sedimentation path length axis of rotation a r max r av r min

Different types of rotor head, corresponding RPM and RCF:

Different types of rotor head, corresponding RPM and RCF Rotor RPM RCF *g 12*1.5 ml Angle 20,000 26,600 10*8 ml Angle 16,000 21,750 6*30 ml Angle 12,000 12,880 20*5 ml Angle 17,000 22,620 9*1.5 ml Swing out 10,000 9,620 24*1.5 ml Angle 17,500 23,970 4*30 ml Angle 16,500 20,100 4*50 ml Angle 13,000 14,200 24*05 ml Angle 20,000 26,600

High-speed centrifuges:

High-speed centrifuges High-speed or super speed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30000 rpm). The rotors may come with different adapters to hold various sizes of test tubes , bottles, or microliter plates . High-speed centrifuges

Ultracentrifuges:

Ultracentrifuges Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocities in excess of 70000 rpm. Ultracentrifuges can separate molecules in batch or continuous flow systems. During the run, the particles or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution.

Types of Centrifuges:

Types of Centrifuges High Speed centrifuges Ultracentrifuges – 15,000 – 20,000 RPM – large sample capacity depending on rotor – normally refrigerated – research applications → 65,000 RPM (100,000’s x g) – limited lifetime – expensive – require special rotors – care in use – balance critical! – research applications

Microcentrifuges:

Microcentrifuges Microcentrifuges are used to process small volumes of biological molecules, cells , or nuclei . Microcentrifuge tubes generally hold 0.5 - 2 ml of liquid, and are spun at maximum angular speeds of 12000–13000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a refrigeration function. This is the important process.

Small Bench Centrifuge:

Small Bench Centrifuge Simplest centrifuges that are used to separate erythrocytes, Blood samples, coarse precipitates and cells are known an bench or laboratory centrifuges. They have a speed ranging from 4000 – 6000 RPM and a relative centrifugal force of 3000 – 7000 g. Small samples are sedimented now a days with microfuge that after a speed of 8000-13000 RPM and relative RCF of approximately 10000 g. They sediment small volume (250 mm to 1.5 cm ) of material in 1 or 2 min. 3 3

Cont.…..:

Cont.….. Small Bench top Microcentrifuges (“microfuge”, “Eppendorf”) – with or without refrigeration – slow speed (eg up to 4000 RPM) – common in clinical labs (blood/plasma/serum separation) – can take approx (up to) 100 tubes, depending on diameter – take tubes of small vols (up to 2 mL) – very common in biochemistry/molecular biology/ biological labs – can generate forces up to ~15,000 x g – with or without refrigeration

Gradient Media:

Gradient Media Gradient media Their uses Sucrose and Ficoll Helps in preserving the morphology and activity of subcellular fractions. Cesium chloride Useful in isopycnic density gradient centrifugation technique. Potassium bromide Useful in isopycnic density gradient centrifugation technique. Percoll Because of its low osmolarity, low viscosity and large particle size, is suitable for separating cells, bacteria, viruses and sub cellular organelles Metrizamide For the isolation of membrane fractions by floatation Nycodenz For the isolation of membrane fractions by floatation Renografin For cell fractionation There are several gradient materials used in centrifugation:-

Centrifugal fractionation of cell organelles:

Centrifugal fractionation of cell organelles Cell Organelle Required centrifugal force Nuclei 800 – 1000 g Mitochondria 20,000 – 30,000 g Chloroplasts 20,000 – 30,000 g Lysosomes 20,000 – 30,000 g Microbodies 20,000 – 30,000 g Rough ER membranes (microsomes) 50,000 – 80,000 g Plasma Membranes, Smooth ER membranes 80,000 – 1,00,000 g Free Ribosome particles 1,50,000 – 3,00,000 g

Corrosion:

Corrosion Many rotors are made from either titanium or aluminium alloy, chosen for their advantageous mechanical properties. While titanium alloys are quite corrosion-resistant, aluminium alloys are not. When corrosion occurs, the metal is weakened and less able to bear the stress from the centrifugal force exerted during operation. The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress levels than an uncorroded rotor.

Precautions:

Precautions A centrifuge user should strictly observe the following precautions : Manufacturer’s manual should be strictly followed. Rotor should be stored in proper containers. Attention should be given to imbalance detectors. Rotor speed should not exceed the assigned speed. Lid of the rotor chamber should remain locked during operation. To avoid the rotor failure, manufactures instructions regarding rotor care and use should always be followed.

Applications in Biological Sciences:

Applications in Biological Sciences To separate cellular and subcellular components Separating one cell type from another. Removing cells or other suspended particles from their surrounding milieu on either a batch or a continuous-flow basis. Isolating viruses and macromolecules, including DNA, RNA, proteins, and lipids or establishing physical parameters of these particles from their observed behaviour during centrifugation. To study the effects of centrifugal forces on cells, developing embryos, and protozoa. These techniques have allowed scientists to determine certain properties about cells, including surface tension, relative viscosity of the cytoplasm, and the spatial and functional interrelationship of cell organelles when redistributed in intact cells.

Conclusion:

Conclusion The centrifugation is a modern & easy technique of separation and sedimentation on the basis of shape, size and density of macromolecules and other particles. In the centrifugation there are different types of forces are applied like as centrifugal force, gravitational force and centripedal force etc. and also different types of rotors are to be used that is; Swinging Bucket Rotor and fixed angle rotors at different RPM/RCF.

Bibliography :

Bibliography Sobti, R.C. & Pachouri, S.S., Essentials Of Biotechnology. Ane Books Pvt. Ltd. 2009. Rana, S. V. S., Biotechniques. Rastogi Pub. Meerut, Walker And Wilson, Principle and Techniques In Biochemistry. John Willey & Sons. Gurumani, N., Research Methodology, MJP Publishers, Chennai.

Acknowledgement:

Acknowledgement I acknowledge with sense of deep and sincere gratitude the inspirational, encouraging and erudite guidance from Prof. S. S. Lal department of zoology, C.C.S.University, Meerut. I am highly indebted him for providing the meticulous guidance and encouragement during the topic of study. I wish to express my sincere appreciation and heartful thanks to Dr. S. K. Bharadwaj, Prof. H. S. Singh, Dr. A. K. Chaubey and Dr. Dushyant for their regular and encouragement support. I am also thankful to my seniors Mr. Istkhar, Ms. Tanu and my all batch mates for providing me immense help in a number of ways to completing the topic.

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