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Centrifugation: Principle, Types and Applications

1. Definition of Centrifugation

Centrifugation is a technique employed for separating substances via the application of centrifugal force. This method facilitates the segregation of particles from a solution based on several factors including size, shape, density, the viscosity of the medium, and the rotor speed.

2. Principle of Centrifugation

The principle behind centrifugation relies on the behavior of particles in a solution: denser particles will sediment at the bottom, while lighter particles float to the surface. The process is influenced by the differences in density; particles move faster with greater density discrepancies. Under isopycnic conditions, where there is no density difference, particles remain suspended. A centrifuge replaces gravity with a much stronger force, allowing for effective separation by spinning objects at high speeds around a fixed axis, creating a powerful centrifugal force.

3. Mechanism of Centrifugation

Centrifugation works on the sedimentation principle where centripetal acceleration causes denser substances to move outward radially, whereas less dense substances are displaced toward the center. In laboratory settings, when sample tubes are spun, denser particles settle at the bottom while lighter ones collect at the top, facilitating their separation based on density differences.

4. Types of Centrifuge

• Desktop Centrifuges: Commonly used for low-speed applications.

• High-Speed Centrifuges: Used for biochemical applications with higher speeds up to 20,000 RPM, enabling temperature control for sensitive samples.

• Ultracentrifuges: The most advanced, capable of speeds up to 65,000 RPM, requiring vacuum and refrigeration due to intense heat generated during operation.

5. Types of Rotors

• Fixed Angle Rotors: Tubes held at an angle (14-40°), causing particles to move radially outward. Useful for differential centrifugation but may result in reorientation during acceleration/deceleration.

• Swinging-Bucket Rotors: These rotors swing out to a horizontal position and allow for longer travel distance during sedimentation, improving separation efficiency. Commonly used in density-gradient centrifugation.

• Vertical Tube Rotors: Hold tubes vertically, suitable for certain applications but less common than the other types.

6. Application of Low-Speed Centrifuge

Low-speed centrifuges operate typically between 4000-5000 RPM and are widely utilized for sedimenting heavy particles, such as red blood cells, into a pellet, which is then separated by decantation.

7. Application of High-Speed Centrifuges

High-speed centrifuges, operating between 15,000 to 20,000 RPM, are essential for sophisticated biochemical applications where precise control over speed and temperature is required, allowing for the study of delicate biological samples.

8. Application of Ultracentrifuges

Ultracentrifuges operate under extreme conditions, offering maximum speeds and centrifugal forces, suitable for isolating and purifying biological components. This includes preparative work for biological materials and analytical work to characterize their properties.

9. Types of Centrifugation Techniques

• Differential Centrifugation: Separates components based on size and density, where larger, denser particles sediment faster than smaller ones. It is one of the most common methods for organelle isolation.

• Density Gradient Centrifugation: Utilizes a sucrose density gradient for purifying components like viruses and organelles, facilitating separation based on particle density as they migrate to a position where their density matches that of the surrounding medium.

  • Rate-Zonal Centrifugation: This method separates particles based on size and mass, where larger particles sediment ahead of smaller ones through a density gradient.

  • Isopycnic Centrifugation: Particles sink until they reach a point in the gradient where their buoyant density equals that of the surrounding solution, achieving a state of equilibrium.

10. Applications of Centrifugation

Centrifugation has numerous applications, including:

• Analyzing hydrodynamic properties of macromolecules.

• Purifying mammalian cells.

• Isolating subcellular organelles and membrane fractions.

• Clarifying wine, separating particles from air and liquid mixtures, and in forensic laboratories for blood and urine component separation.

• Aiding in protein purification techniques such as salting out.

In summary, centrifugation is a versatile technique fundamental to various scientific and medical applications, relying on the principles of density and sedimentation to efficiently separate and purify biological substances.