Centrifugation is a common technique used for the separation of heterogeneous mixtures. The force of gravity on matter is the core principle of centrifugation. Matter naturally separates based on density, with the most dense particles precipitating out of solution first, and the less dense particles falling out later. Some particles prefer to remain in colloidal solution under normal gravitational force, and will not separate naturally within a reasonable time frame.Centrifugation can be used to rapidly separate those mixtures. The spinning centrifuge creates centripetal forces much greater than gravity, which can force particles to separate. The centrifugal protocol must be optimized for each experiment in order to efficiently separate the mixtures into desired layers. The most basic separation results in a higher density pellet at the bottom of the tube, and a lower density supernatant. Differential centrifugation is a process whereby the pellet and supernatant are separated during multiple centrifugation steps. Density gradient centrifugation utilizes a density gradient matrix within the tube to aid in separation. Another useful tool for centrifugation is a centrifugal filter. The filter helps to separate particles by size as well as density in one swift motion. The centrifugal filter is used to isolate RNA or DNA, to consolidate proteins, to separate molecules by size, or to remove contaminants from a liquid.
Centrifugal filter principle and design
In the simplest form, the centrifugal filter works on the same principle as a standard filter. Particles larger than the pore size are unable to pass through, and are concentrated together. The benefit of the centrifugal filter is that the separation is faster due to the centripetal forces that are much greater than gravity. The particles collect on the membrane and form a “cake” while the remaining mixture flows through the filter. The cake can be dried by excessive centrifugation as the liquid is forced out through the membrane. This is a common source of error when using centrifugal filters. In some cases, when the filter cake is excessively dried, it becomes adsorbed to the filter material itself and cannot be recovered with good efficiency. This is a common problem with filtration of proteins at low concentrations. In these situations, the concentration of the proteins is so low that it is impossible to visibly see a filter cake and it is therefore impossible to know how much of the protein sample is stuck on the filter material. Instead of spinning the centrifugal filter until it is dry, the recommended procedure is to spin until a small amount of liquid remains, add rinsing buffer to the filter, and spin again. This process is often repeated at least 5 times.
Centrifugal filters are made of a variety of materials, and are commercially available for many applications. These filters can be small (milliliters) for small laboratory research and development scale work, or can be large (liters) for industrial production of pharmaceutical products or even for the purification of oil in diesel engines. Some examples of centrifugal filter material available for small-scale biotechnology applications are cellulose actetate, PTFE, and nylon. There are numerous proprietary polymer membranes on the market as well. It is important to consider what kinds of non-specific interactions or adsorption can occur between your target and the membrane. The membrane should be selected to decrease the non-specific interactions as much as possible to avoid losing sample to the membrane material throughout the filtration process.
New centrifugal filter developments
As with any technology, the centrifugal filter is ammenable to modification and fine-tuning. One new development is a centrifugal filter that incorporates immuno-binding for the detection of circulating tumor cells (CTCs) in blood. CTCs are cells that detach from a primary tumor site and circulate throughout the body. They are indicative of cancer progression and metastasis. Therefore, the ability to quickly identify their presence in patent blood samples is great interest, and CTCs are the target of many new biotechnological advancements, including magnetic separation.
Thisnewly reported centrifugal filter with immuno-binding capability is concentric, with varying pore sizes moving outward from the center. The sample is placed at the center of the filter, and particles move toward the outer edges during centrifugation. The pores of the inner filter are 20 μm, but the outer filter pores are 10 μm. The target binds to an immuno-bead to form a complex larger than 20 μm. This target is then trapped at the interior of the filter while all other non-target particles are forced outward.
The centrifugal filter adds a layer of specificity to the process of heterogeneous mixture separation by centrifugation. With careful attention to detail, this simple concept can be extended to efficiently and rapidly separate complicated mixtures.