Centrifugation is a technique of separation of mixtures e.g. subcellular organelles that involves the centrifugal force using a CENTRIFUGE(A centrifuge uses centrifugal force to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis).
A centrifuge is used to separate particles or macromolecules:
- Cells
- Sub-cellular components
- Proteins
- Nucleic acids
Basis of separation for a centrifuge are:
- Size
- Shape
- Density
A centrifuge utilizes density difference between the particles/macromolecules and the medium in which these are dispersed and Dispersed systems are subjected to artificially induced gravitational fields.
TYPES OF CENTRIFUGATION
Centrifugation
Preparative Analytical
Differential Density-gradient
Rate zonal Isopycni
Preparative Centrifugation; it is used to separate organelles and molecules and it can handle larger liquid volumes. It does not required any optical read-out.
Separation methods used in preparative ultracentrifugation are:
- Differential Centrifugation; In this, separation is based on the size of the particles. This is commonly used in simple pelleting and in obtaining partially-pure preparation of subcellular organelles and macromolecules. For the subcellular organelles, tissue or cells are first disrupted to release their internal contents. This crude disrupted cell mixture is called as a homogenate. During centrifugation of a cell homogenate, larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation. When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken cells and heavy nuclei pellet to the bottom of the tube.
The supernatant can be further centrifuged at 10,000 x g for 20 minutes to pellet subcellular organelles of intermediate velocities such as mitochondria, lysosomes, and microbodies. Some of these sedimenting organelles can obtained in partial purity and are typically contaminated with other particles. Repeated washing of the pellets by resuspending in isotonic solvents and re-pelleting may result in removal of contaminants that are smaller in size (Figure 1). Obtaining partially-purified organelles by differential centrifugation serves as the preliminary step for further purification using other types of centrifugal separation (density gradient separation).
Density gradient centrifugation; this method is preferred to purify subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories.
- Rate-zonal (size) separation.
- Isopycnic (density) separation.
Rate zonal (size) separation; also known as sucrose density gradient centrifugation,it uses particle size and mass instead of particle density for sedimentation. Figure 2 illustrates a rate-zonal separation process and the criteria for successful rate-zonal separation. Certain types of rotors are more applicable for this type of separation than others. Examples of common applications include
- separation of cellular organelles such as endosomes or
- separation of proteins, such as antibodies.
For instance, Antibody classes all have very similar densities, but different masses. Thus, separation based on mass will separate the different classes, whereas separation based on density will not be able to resolve these antibody classes.
Isopycnic separation; also known as caesium chloride density gradient centrigugation,in this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle. A common example for this method is separation of nucleic acids in a CsCl gradient. Figure 3 illustrates the isopycnic separation and criteria for successful separation
