# Centrifugation

## What is Centrifugation?

Centrifugation is a process in which the centrifugal force is applied to separate particles from a solution on the basis of their density, size, shape and viscosity of the medium.

### Significance of Centrifugation

Centrifugation is an important separation technique that is based on the concept of centrifugal force and finds application in various branches of science. It can be schematically represented as follows:

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### Centrifugal Force

The centrifugal force is an apparent force felt by the body moving along a curved or circular path acting in the outward direction. Centrifugal force is a fictitious force and this force arises only when the system is not in reference with the frame of reference. Centrifugal force comes to play in the non-inertial frame of reference.

The examples of the centrifugal force are the revolution of the earth around the sun and the people on a merry go ride having the feeling of being pushed outward. The bodies of the people on the merry go round want to fly off the ride in a tangential path or a straight line path but their hands holding the bars of the ride, keep the riders in position.

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### General Applications of Centrifugation

Coming to the process of centrifugation, in simple words, it is used in separating skimmed milk from whole milk that is used to make ghee and butter. This process is used to separate water from clothes. The washing machines use this technique to spin the clothes at high speed after the washing and rinsing process is completed to dry the clothes. In the laboratories, centrifugation is used to separate cells from plasma present in the blood. Chalk powder can be removed from water using the process of centrifugation. Centrifugation stabilizes the wine. Particles from a high-velocity airflow can be separated. Centrifugation is used in the separation of proteins in various purification techniques.

### What is a Centrifuge?

A centrifuge is an equipment in which an object rotates around a fixed path as in a circle. It applies a force perpendicular to the axis of spin in the outward direction that is very strong. The centrifuge uses the sedimentation principle. The centrifugal acceleration causes denser substances and particles to move in the outward direction in a tangential path.  Objects having less density get displaced and move to the centre. In laboratories where the samples of blood, urine and various chemicals are tested, due to the radial acceleration, denser particles to settle to the bottom of the tube and particles of higher density reach the top of the tube.

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### Usage of a Centrifuge:

1. The centrifuge bottles or tubes are to be checked for cracks. Otherwise, the sample in the tubes can become aerosol. In case of a leak, the liquid will flow into the rotor causing malfunctioning of the equipment.

2. The specific cap or lid provided with the tube or bottle should never be interchanged. It will lead to the spilling of the liquid.

3. While testing biohazardous samples, the outer surface of the tube must be wiped with disinfectant.

4. Labelling the tube is very important, as after the experiment is done, telling apart the different samples is difficult.

5. Balancing the masses of the tube, not volume is the most crucial step to avoid mishaps at very high speeds of rotation.

6. The centrifuge must always be placed on a solid and stable surface that is sturdy.

7. The rotor speed, centrifuge machine, tube of the sample and speed of rotation must be in sync.

8. Balancing the tubes in multiple slots must be taken care of.

9. The centrifugation speed or the number of rotations per minute differs for different samples. Care should be taken to enter the right speed.

10. Imbalanced centrifuges or wobbling ones should be stopped immediately and a safe distance should be maintained from the equipment once it starts functioning.

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### Radial Acceleration

While discussing the topic of centrifugation, the students need to understand the concept of radial acceleration. Radial acceleration is derived by dividing the velocity squared by the radius. Radial acceleration = v²/r. Radial acceleration is caused to the change in direction of the velocity. Tangential acceleration is not used for calculating the total force. Only the radial acceleration keeps an object in the circular motion.

### Types of Centrifugation:

• Density gradient centrifugation

• Differential centrifugation

• Rate zonal centrifugation

• Isopycnica centrifugation

Differential centrifugation is the most common type of centrifugation. Tissues of human beings like the liver tissue are homogenized in a solution buffer. The homogenate is then placed in a centrifuge at a constant temperature. Sediment at the bottom of the centrifuge is known as pellet and the solution above is known as supernatant.

Density gradient centrifugation is used for purification purpose like for virus, membrane etc. The particles to be purified or tested are placed on top of the gradient in an ultracentrifuge. When the density of the sample matches the density of sucrose surrounding them, the process is completed.

Rate zonal centrifugation works on the concept of the sedimentation coefficient. During the centrifugation process, faster sediment particles of the sample move ahead and they are separated into various zones. Protein sediments based on different sedimentation coefficients are separated and their fractions are collected.

In Isopycnic centrifugation, the sample is mixed with a gradient forming solution. The sample and caesium salt is loaded in a centrifugal tube in the ultracentrifuge. The sample molecules move to the area where their density is equal to the density of the gradient.

### What is the gradient?

The density gradient is a variation that occurs spatially over a particular area. The matter or the sample has varying density but is applicable to any quantity.

### Principles of Centrifugation:

1. In a particular solution, particles with a higher density than the solvent will sink to the bottom and the lighter density particles will float on the top.

2.  If there is no difference in the density of the solute and the solvent, particles will stay put and if there is a greater difference in the density between those two, particles will move faster in such circumstances.

3. Gravity is replaced with the more powerful centrifugal force so that minute differences in the density can be used to separate particles in a solution.

4. A centrifuge is a piece of equipment that keeps an object in a circular motion about a fixed axis and a force tangential or perpendicular is applied in the outward direction to the axis of spin.

5. The centrifuge works on the sedimentation principle in which the centripetal acceleration makes the denser particles move out word in a radial direction and the low-density particles move towards the centre.

6.  In the laboratories that use sample tubes, the denser particles settle at the bottom of the tube and the lower density particles settle on the top due to radial acceleration.

Utilizing the various principles of Physics and nature, the centrifugation process has made experimentation and research at a micro-level easier and practical.

FAQ (Frequently Asked Questions)

1) What are the Factors that Influence Centrifugation?

Answer: The factors that influence the process of centrifugation are the density of both the samples and the solution, distance of the particles that have displaced from their original position, speed of the rotation of the centrifuge and the temperature and viscosity of the medium in which the sample particles are placed.

2) What are the Different Parameters of Centrifugation?

Answer:  For a successful centrifugal run, the following parameters are to be followed: The rotors in the laboratory centrifugation are of a fixed angle or swing bucket type of rotors. Applications and some special experimentation requite special kind of rotors like continuous flow rotors and drum rotors. Some rotors are also used to test the gravity of different people and various types of objects. Many rotors are built for the simplest form of centrifugation called the differential centrifugation or the differential pelleting.

3) What are the Different Types of Centrifuges?

Answer: Low-speed centrifuges are used for sedimentation of heavy particles. The speed of the centrifuges is 4000 to 5000 rotations per minute. These centrifuges have no temperature control. These are mostly used for the sedimentation of red blood cells separated by a process called decantation. The high-speed centrifuges have temperature controls and are used in sophisticated experimentations. Their speed is 15000 to 20000 rotations per minute.  Ultra centrifuges are the most modern instruments presently being used in the field of centrifugation. Their maximum speed is 65000 rotations per minute. Their spinning chambers must be refrigerated as heat is generated is high due to their speed. They are used for analytical purposes.

4) Give Advanced Applications of Centrifugation.

Answer: Centrifugation is used to separate the proteins in a process like salting out. It is used in the fractionation of membranes and subcellular level organelles. Centrifugation process is used in the purification of cells in the mammals.  Centrifugation is used to separate two miscible substances. The process of centrifugation is used to study and analyze the hydrodynamic properties of macromolecules.

5) Is there any Difference Between the Units of G-forces and RCF?

Answer: In the process of centrifugation while using different types of centrifuges, the question of units arises.  The relative centrifugal force, RCF and the units of the G-forces are the same. They are the same units of measurement and can be interchangeable as and when required. The force on the particles is exponential to speed of rotation.