The main difference between zonal and isopycnic centrifugation lies in the principle of separation. Zonal Centrifugation is a technique separates particles based on their size and shape while Isopycnic Centrifugation is a method separates particles based on their density.
In summary, zonal centrifugation focuses on size and shape, while isopycnic centrifugation focuses on density for the separation of particles.
Difference between Zonal and Isopycnic Centrifugation (With Table)
| Aspects | Zonal Centrifugation | Isopycnic Centrifugation |
| Principle of Separation | Separates particles based on size and shape.
|
Separates particles based on density. |
| Gradient | Uses a pre-formed density gradient. | The gradient forms during centrifugation. |
| Separation Mechanism | Centrifugation: Particles move through the gradient and are separated into zones. | Particles move to the position where their density matches the gradient’s density. |
| Types of Particles | Best for particles differing in size and shape (e.g., organelles, proteins). | Centrifugation: Best for particles differing in density (e.g., DNA, RNA). |
| Gradient Composition | Gradient is often made of sucrose or glycerol. | Gradient is often made of cesium chloride. |
What Is Zonal Centrifugation?
Zonal centrifugation, also known as gradient centrifugation, is a technique used to separate particles (such as cells, organelles, or macromolecules) based on their size and shape.
The process involves the following steps:
- A gradient of increasing density is created in a centrifuge tube. This gradient can be continuous or stepwise and is typically made using substances like sucrose or cesium chloride.
- The sample containing the particles to be separated is layered on top of the density gradient.
- The tube is then centrifuged at high speeds. During centrifugation, particles move through the gradient at different rates based on their size and shape. Larger and denser particles sediment faster than smaller and less dense ones.
- Particles migrate through the gradient until they reach a position where the centrifugal force and the buoyant force of the gradient are balanced. At this point, they form distinct bands or zones.
- After centrifugation, the gradient is fractionated, and the separated particles are collected from different zones or layers.
Zonal centrifugation is widely used in biochemistry and cell biology for tasks such as isolating organelles, purifying proteins, and separating nucleic acids. It is especially useful when dealing with complex mixtures of particles with varying sizes and shapes.
What Is Isopycnic Centrifugation?
Isopycnic centrifugation, also known as equilibrium density gradient centrifugation, is a technique used to separate particles based on their density.
Here is how it works:
- A density gradient is established in a centrifuge tube using a medium such as cesium chloride or sucrose. This gradient has a range of densities from low to high.
- The sample containing particles of different densities is mixed with the gradient medium or layered on top of it.
- The tube is centrifuged at high speeds for an extended period. During centrifugation, particles move through the gradient until they reach the point where their density matches the density of the surrounding medium. At this point, they no longer move and form a distinct band.
- The particles reach an equilibrium position where the centrifugal force and the buoyant force from the gradient are balanced. Each type of particle settles at the point in the gradient that corresponds to its density.
- After centrifugation, the gradient is fractionated to collect the separated particles from their equilibrium positions.
Isopycnic centrifugation is particularly useful for separating particles with very similar sizes but different densities, such as different types of nucleic acids (DNA and RNA), organelles, and certain viruses. This method allows for precise separation and isolation based on density alone, regardless of the size or shape of the particles.
Difference between Zonal and Isopycnic Centrifugation
Principle of Separation
Zonal Centrifugation: Separates particles based on size and shape.
Isopycnic Centrifugation: Separates particles based on density.
Gradient
Zonal Centrifugation: Uses a pre-formed density gradient.
Isopycnic Centrifugation: The gradient forms during centrifugation.
Separation Mechanism
Zonal Centrifugation: Particles move through the gradient and are separated into zones.
Isopycnic Centrifugation: Particles move to the position where their density matches the gradient’s density.
Types of Particles
Zonal Centrifugation: Best for particles differing in size and shape (e.g., organelles, proteins).
Isopycnic Centrifugation: Best for particles differing in density (e.g., DNA, RNA).
Gradient Composition
Zonal Centrifugation: Gradient is often made of sucrose or glycerol.
Isopycnic Centrifugation: Gradient is often made of cesium chloride.
Preparation
Zonal Centrifugation: Requires careful preparation of a pre-formed gradient.
Isopycnic Centrifugation: Gradient forms automatically during centrifugation.
Centrifugation Time
Zonal Centrifugation: Typically shorter centrifugation times.
Isopycnic Centrifugation: Typically longer centrifugation times to reach equilibrium.
Sample Application
Zonal Centrifugation: Sample is layered on top of the gradient.
Isopycnic Centrifugation: Sample can be mixed with the gradient or layered on top.
Resolution
Zonal Centrifugation: Higher resolution for particles with different sizes and shapes.
Isopycnic Centrifugation: Higher resolution for particles with different densities.
Fractionation
Zonal Centrifugation: Fractionation of zones after centrifugation.
Isopycnic Centrifugation: Fractionation of density bands after centrifugation.
Equipment
Zonal Centrifugation: Requires a zonal rotor.
Isopycnic Centrifugation: Can be performed in a fixed-angle or swinging-bucket rotor.
Uses
Zonal Centrifugation: Commonly used for separating cell organelles, viruses, and protein complexes.
Isopycnic Centrifugation: Commonly used for purifying nucleic acids, separating different types of cells, and isolating subcellular particles.
Gradient Stability
Zonal Centrifugation: Gradient can be unstable if not handled properly.
Isopycnic Centrifugation: Gradient is stable once formed.
Quantitative Analysis
Zonal Centrifugation: Allows quantitative analysis based on zone position.
Isopycnic Centrifugation: Allows quantitative analysis based on band position.
Typical Examples
Zonal Centrifugation: Sucrose gradient centrifugation for separating cellular components.
Isopycnic Centrifugation: Cesium chloride gradient centrifugation for purifying plasmid DNA.
Similarities between Zonal and Isopycnic Centrifugation
- Both methods use centrifugal force to separate components of a mixture. The samples are placed in a centrifuge, and the centrifugal force causes components to move at different rates based on their physical properties.
- Both techniques rely on the differences in physical properties of the substances to achieve separation. In zonal centrifugation, it’s primarily based on sedimentation rates, while in isopycnic centrifugation, it’s based on density.
- Both methods use centrifuge tubes where the sample is loaded. The separation occurs within these tubes as they spin at high speeds.
- Both techniques often involve the use of gradient media. In zonal centrifugation, a gradient (e.g., sucrose or density gradient) is used to separate particles by their sedimentation rates. In isopycnic centrifugation, a gradient (often a density gradient) is used to separate components based on their densities.
- Both methods are widely used in biochemical and molecular biology research to purify and analyze biological molecules, such as proteins, nucleic acids, and organelles.
- Both methods require high-speed centrifugation to achieve effective separation of components.
Conclusion
In conclusion, zonal and isopycnic centrifugation are distinct techniques with specific applications based on their separation principles.
Zonal centrifugation separates components based on their sedimentation rates through a pre-established gradient, allowing for the separation of particles that differ in size, shape, or mass.
Isopycnic centrifugation separates components based on their density, utilizing a density gradient that facilitates the equilibrium separation of particles.
Both methods leverage the principles of centrifugal force and gradient media but are tailored to different types of separations, making them complementary tools in biochemical and molecular biology research.