Centrifuge

BRIEF HISTORY

In 1864, Antonin Prandti discovered the first centrifuge-type machine, which was used in the dairy industry to separate milk and cream. The next evolution in centrifuge was Ultra-Centrifuge, discovered in 1920s by Swedish chemist Theodor Svedberg. This ultracentrifuge used for highly complex protein and viruses and determining of molecular weights of substances. In U.S, 1954 brought widely improvements to commercial centrifuge with new high speed motors and better rotors. In 1962 the German company Netheler & Hinz (now known as Eppendorf) created the first microcentrifuge for lab use. Eppendorf discovered personal centrifuges in 2000 and centrifuge technology has continued to improve commercially ever since, now used for density separation (Cash, 2017).

GENERAL PRINCIPLE

• Centrifugation is a process where the particles in a solution is separated on the basis of its size, shape and density.
• In a solution whose density higher, it remains as a sediment (bottom layer) and a substance whose density is lighter than its floats to the top.
• When the difference of density is greater than they move also faster in centrifuge machine.
• A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis.
• In a laboratory centrifuge that use same tubes, the radial acceleration (acceleration of the object along the radius directed towards the centre) cause denser particle settle down and low density particles rise to the top (Aryal et al., 2020).
• In centrifugation it is most important to differentiate between the speed of centrifugation (RPM= Revolution per minute) and relative centrifugal force (RCF or G). This centrifugal force generated by a centrifuge and easily calculated from the equation:

RCF= 11.8xRx (𝑅𝑃𝑀𝑥1000)² 

Where R= distance from the centre of rotation in centimeters

• The other very important point in optimizing centrifugal separation is the choice of rotors. There are some main types of rotors that are discuss below (Rickwood, 2001).

TYPES OF ROTORS IN CENTRIFUGE

Types or rotors are:

·    Swing Bucket Rotors

A swing bucket rotor usually supports samples ranging in volume from 36 ml to 2.2 ml, used for both: Rate zonal (mass and size based separation) and Isopycnic (density based separation). It is mainly used for high throughput protocols such as: batch harvesting of whole cells from growth media, blood collection tubes, tissue culture processing and small structural viruses.

·    Fixed Angle Rotors

This rotor carries range of solution from 0.2 ml to 1 ml. RCF and volume of the sample is a key factor in a fix angle rotor.

·    Vertical Rotors

Vertical rotor are highly specialized (Kingston, 2007) mostly used to band DNA in cesium chloride. Maximum range is 5-25 ml. Hence completed shortest path it is also used for the isolation of plasmid DNA.

·    Continuous Flow Rotors

It separates small quantity of matters from large suspension like: Mononuclear leucocytes in human blood, endothelial cells, rat brain cells and fat storing cells in liver.

·    Zonal Rotors

They can contain up to 2 liters of solutin and can work with tissues sample. Its capacity is: 300cm³ to 2000cm³ (“Types of rotors,” 2010).

TYPES OF CENTRIFUGE

There are three main types of centrifuge:

• Small bench centrifugation
• Desk top centrifugation
• High speed centrifuges
• Ultra centrifuges: - Preparative centrifugation  - Analytical centrifugation

·     Small Bench Centrifugation

1. Works with the speed of 8000-13000 rpm & RCF 10000g
2. For rapid sedimentation of small volumes (1-2 min)
3. Example: Blood

·     Desk top centrifugation

1. Very simple and small
2. Maximum speed is 3000 rpm
3. Do not have temperature regulatory systems
4. Used normally to collect rapidly sedimentation such as blood cells, yeast cells or bulky precipitated of chemical reactions.

·     High speed centrifuge

1. Maximum speed is 25000 rpm
2. Equipped with refrigeration to remove heat generated
3. Temperature is 0-4°C.
4. Used to collect microorganisms, cell debris, large cellular organelles, precipitates of chemical reactions.
5. Also useful for lysozyme, mitochondria and nuclei.

·     Ultracentrifuges

1. Operate at the speed of 75,000 rpm, providing centrifugal force 500,000g.
2. Rotor chamber is sealed and operate by means of pump for attain vacuum.
3. Centrifugation for isolation and purification is known as preparatory centrifugation and carried out with desire for characterization is known as analytical centrifugation.

• Preparative centrifugation: actual isolation of biological material for biochemical investigations. It divides into two main techniques depending on suspension medium in which separation occur like homogenous medium (differential centrifugation) and density gradient medium (density gradient centrifugation).
• Analytical centrifugation: speed is 70000 rpm, RCF is 5 lakh kg. light optical system that possess light absorption. Motor, rotor and chambers that is refrigerated and operate by optical system. It is used for analytical cells (Yadav, 2016).

OTHER TYPES OF CENTRIFUGATION

·     Differential Centrifugation

1. It is the most common type of centrifugation.
2. Liver tissue at 32°C in a sucrose solution that contains buffer.
3. The homogenate is then placed in a centrifuge after sometimes sediments form at the bottom of a centrifuge called pellet and upper solution called supernatant.

·     Density gradient centrifugation

1. It is used for purify viruses, ribosomes, membranes.
2. Particle of interest placed on top of gradient such as sucrose and cesium chloride and centrifuge in ultracentrifuge.
3. Molecules are banded in gradient and can be collected as a pure fraction.

·     Rate zonal centrifugation

1. Zonal centrifugation is also known as band or gradient centrifugation.
2. In this technique, a density gradient is created in a test tube with sucrose and high density at the bottom.
3. With centrifugation, faster sediments particles in sample move a head of slower ones.
4. Hence protein forms sediments and the fractions are collected by creating a hole at the bottom.

·     Isopynic Centrifugation

1. The sample is loaded into the tube with the gradient forming solution.
2. The solution of the biological sample and cesium salt is distributed in a centrifuge tube and rotated in an ultracentrifuge
3. After rotation, due to centrifugal force the cesium salts redistribute to form a density gradient from top to bottom.
4. Sample molecules move to the region where their density equals the density of gradient.

 

APPLICATIONS OF CENTRIFUGATION

• Analyze hydrodynamic properties of macromolecules.
• Separate two different substances.
• Isolation of sub cellular organelles.
• Separating chalk powder from water.
• Removing fat from milk to obtain skimmed milk.
• Separation of urine components and blood components in forensic and research laboratories.
• Dirt and water removed from oils for example: purification of olive oil this method is use.
• Bacterial enzymes are prepared by removing bacteria (Aryal et al., 2018).

By: Fatimah Khursheed

REFRENCES

1. Aryal, S., Wasiq, rosemary, M., Rawat, P., Ray, A., Kumar, K.S. (2020, January 2). Centrifugation, principle, types and applications: instrumentation. https://microbenotes.com/centrifugation-principles-types-and-applications/-
2. Cash,       K.        (2017,       February       16). https://www.marshallscientific.com/the history_of_the_centriguge_a/349.htm.
3. Kingston, S. (2019, March 2). Types Of Rotor Centrifuges. https://sciencing.com/types- rotor-centrifuges-5912175.html.
4. Rickwood, D. (2001). Centrifugation Techniques. Encyclopedia of Life Sciences. https://doi.org/10.1038/npg.els.0002704
5. Types             Of            Centrifuges            Rotors.            (2020,                      April        11). https://www.broadlearnings.com/lesson/types-centrifuge-rotors/.
6. Yadav,     D.     A.     (2016,     January     25).     Centrifugation    principle    and         types. https://www.slideshare.net/mobile/anurag_yadav/centrifugation-principle_and_types.