Centrifugation is a widely used laboratory technique that separates components within a liquid mixture by applying a strong rotational force. This process relies on the principle of sedimentation, where particles move through a fluid under the influence of an external force. Since gravity is too weak for most microscopic particles, a centrifuge generates an outward pull known as centrifugal force. This force causes denser components to move away from the center of rotation, while lighter components remain closer to the axis.
The effectiveness of separation is measured by the relative centrifugal force (RCF), often expressed in multiples of g-force. The sedimentation rate depends on the particle’s size, shape, density, and the viscosity of the surrounding medium. Increasing the rotational speed exponentially increases the centrifugal force, accelerating the sedimentation rate and allowing scientists to isolate specific components.
Differential Centrifugation
Differential centrifugation is the simplest and most common method for separating biological mixtures, relying on sequential separation based purely on particle size. This technique involves a series of spins at progressively increasing speeds without using a specialized density medium. The process begins with a low-speed spin designed to pellet the largest and densest particles. The remaining liquid, called the supernatant, is then transferred to a new tube.
The supernatant is subjected to a higher centrifugal force, causing the next largest particles to sediment. Repeating this process with successively higher speeds fractionates the sample into components of decreasing size. For example, cell fractionation first pellets nuclei and debris, followed by higher speeds to pellet mitochondria, chloroplasts, and finally, smaller structures like ribosomes.
This technique is often used as a preparatory step to isolate specific cellular organelles or to harvest large volumes of cells. While effective for crude separations, differential centrifugation does not achieve high-resolution purification because the resulting pellets often contain a mix of different particle types.
Density Gradient Centrifugation
Density gradient centrifugation offers a higher degree of resolution than differential methods. It uses a medium like sucrose or cesium chloride that forms a stable density gradient within the tube. This gradient acts as a molecular sieve, allowing particles to separate based on finer differences in their physical properties. This technique is categorized into two distinct methods: rate-zonal and isopycnic centrifugation.
Rate-Zonal Centrifugation
Rate-zonal centrifugation separates particles primarily based on their size and shape. A shallow density gradient is pre-formed in the tube, with the sample carefully layered on top. This layering prevents the mixing of separated bands due to convection.
When the tube is spun, particles move through the gradient at a rate determined by their sedimentation velocity. Larger and heavier particles travel faster and farther down the tube than smaller ones. The spin is stopped before any particles reach the bottom, resulting in distinct bands or zones of separated components. This technique is effective for purifying specific viruses or separating sub-cellular components that vary significantly in size.
Isopycnic Centrifugation
Isopycnic centrifugation, also called equilibrium sedimentation, separates particles solely based on their buoyant density, independent of size or shape. This method uses a dense medium, such as a concentrated salt solution, to create a gradient spanning the entire range of densities found within the sample.
Particles travel down the tube until they reach the isopycnic point, where their density exactly matches the surrounding medium. Once reached, movement stops, even if centrifugation continues. This provides a highly stable and precise separation that is not dependent on the spin time. A classic application is the high-resolution separation of different forms of DNA, such as plasmid DNA from genomic DNA, using a cesium chloride gradient.
Preparative and Analytical Ultracentrifugation
Ultracentrifugation refers to any centrifugation process carried out at extremely high speeds, typically generating RCF values exceeding 100,000 times gravity. This high force is necessary to sediment very small particles like proteins, nucleic acids, and small viruses. Ultracentrifugation is divided into two categories based on purpose: preparative and analytical.
Preparative Ultracentrifugation
Preparative ultracentrifugation focuses on the physical isolation and collection of purified material. It applies the principles of differential or density gradient centrifugation at high speeds to separate microscopic components. The goal is to harvest sufficient quantities of the purified product, such as a specific protein or viral particles, for use in subsequent experiments.
Analytical Ultracentrifugation (AUC)
Analytical ultracentrifugation (AUC) focuses on the measurement and characterization of macromolecules rather than bulk collection. Specialized analytical ultracentrifuges are equipped with optical detection systems that monitor particle movement in real-time. These optics track the sedimentation boundaries, allowing researchers to calculate precise physical properties. AUC is used to determine molecular weight, purity, conformational changes of proteins, and binding affinity between molecules.