Centrifugation and its Types Biological Techniques

Centrifugation is a technique used to separate components of a mixture based on their size, shape, density, and viscosity. This method applies centrifugal force to separate particles suspended in a solution by spinning the mixture at high speeds. The components of the mixture move outward from the center of rotation, with denser particles moving faster and further outward than less dense ones. Centrifugation is commonly used in laboratories for various applications, including the separation of blood components, purification of cells, and isolation of DNA. There are several types of centrifugation, each suited for specific applications:

1. Differential Centrifugation
Differential centrifugation separates particles based on their size and density by spinning the sample at progressively higher speeds. It typically involves multiple rounds of centrifugation at increasing centrifugal forces:

Low-speed centrifugation: Larger and denser particles, like cell nuclei, sediment first.
Medium-speed centrifugation: Smaller organelles, such as mitochondria, sediment.
High-speed centrifugation: Even smaller particles, like microsomes, sediment.
Ultracentrifugation: Very small particles, including ribosomes and proteins, sediment.
2. Density Gradient Centrifugation
This technique involves layering the sample on top of a gradient of a dense substance, such as sucrose or cesium chloride, and then centrifuging it:

Rate-Zonal (Size) Centrifugation: Particles are separated based on their size and shape as they travel through a density gradient under centrifugal force. Larger particles move faster through the gradient.
Isopycnic (Density) Centrifugation: Particles are separated solely based on their buoyant density. Each particle moves until it reaches a point in the gradient where its density matches the surrounding medium.
3. Ultracentrifugation
Ultracentrifugation is a high-speed centrifugation technique used to separate very small particles, such as proteins, nucleic acids, and viruses:

Preparative Ultracentrifugation: Used to isolate and purify specific components of a mixture. It can operate at speeds up to 100,000 RPM.
Analytical Ultracentrifugation: Used to analyze the properties of the particles, such as their size, shape, and mass. It often involves monitoring the sedimentation process in real-time using optical systems.
4. Isopycnic Centrifugation
In isopycnic centrifugation, particles are separated based on their density. The sample is mixed with a density gradient medium, and during centrifugation, particles migrate to the point where their density matches the gradient.

5. Svedberg (S) Units
In biological sciences, the sedimentation rate is often expressed in Svedberg units (S), which reflect how fast a particle sediments when subjected to centrifugation. The Svedberg unit is a measure of time (10^-13 seconds), but it is commonly used to describe the rate of sedimentation.

1. Centrifugation and its Types Biological Techniques Lecture

2. Introduction Centrifugation is a process that uses centrifugal force to separate particles of different densities or sizes from a mixture. Principle : Centrifugation works on the principle of sedimentation, where particles heavier than the surrounding fluid move towards the bottom of the container, while lighter particles remain suspended.

3. Type of Centrifugation Principle Procedure Applications Differential Centrifugation Separates particles based on differences in sedimentation rates (size, shape, and density) Sample is placed in a centrifuge tube. Spun at low speed to pellet large particles. Supernatant is decanted and spun at higher speeds progressively to pellet smaller particles. Repeated until desired separation is achieved Separating cellular components (nuclei, mitochondria, lysosomes, ribosomes) Isolating organelles from cell homogenates. Density Gradient Centrifugation Separates particles based on buoyant density using a gradient medium. Gradient medium (e.g., sucrose, cesium chloride) is prepared in the centrifuge tube. Sample is layered on top of the gradient. Spun at high speed. Particles move through the gradient and form bands at positions corresponding to their densities Purification of nucleic acids (DNA, RNA). Separation of proteins, lipoproteins, viruses. Isolating subcellular organelles. Ultracentrifugation Uses extremely high centrifugal forces (up to 1 million x g) to separate very small particles and macromolecules. Samples placed in ultracentrifuge tubes or rotors. Operates under vacuum to reduce friction and heat generation. Spun at very high speeds, resulting in rapid sedimentation of particles Studying macromolecular complexes and interactions. Microcentrifugation Handles small volumes (up to 2 ml) and uses high centrifugal forces (up to 20,000 x g) to separate small particles or precipitate Small volumes placed in microcentrifuge tubes (e.g., Eppendorf tubes). Operates at high speeds to quickly sediment particles Routine lab procedures ( pelleting cells , DNA/RNA extraction, protein precipitation ).

4. Advantages and Disadvantages of Centrifugation Advantages Disadvantages Efficient separation: Centrifugation is a fast and efficient way to separate particles Requires specialized equipment: Centrifuges can be expensive and require maintenance High resolution: Can separate particles with very small differences in density Sample preparation: Samples may require preparation (e.g., dilution, labelling) before centrifugation Gentle on samples**: Centrifugation is a gentle process that preserves sample integrity Operator expertise: Requires trained operators to set up and run centrifuges Wide applicability: Can be used for various sample types (biological, chemical, pharmaceutical) Limited capacity: Centrifuges have limited capacity, requiring multiple runs for large samples.