Centrifugation techniques take a central position in modern biochemical, cellular and molecular biological studies. Depending on the particular application, centrifuges differ in their overall design and size. However, a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated. Particles of biochemical interest are usually suspended in a liquid buffer system contained in specific tubes or separation chambers that are located in specialised rotors. The biological medium is chosen for the specific centrifugal application and may differ considerably between preparative and analytical approaches. As outlined below, the optimum pH value, salt concentration, stabilising cofactors and protective ingredients such as protease inhibitors have to be carefully evaluated in order to preserve biological function. The most obvious differences between centrifuges are:
• the maximum speed at which biological specimens are subjected to increased sedimentation
• the presence or absence of a vacuum
• the potential for refrigeration or general manipulation of the temperature during a centrifugation run
• the maximum volume of samples and capacity for individual centrifugation tubes. Many different types of centrifuges are commercially available including:
• large-capacity low-speed preparative centrifuges • preparative high-speed ultracentrifuges
• refrigerated preparative centrifuges/ultracentrifuges
• analytical ultracentrifuges
• large-scale clinical centrifuges
• small-scale laboratory microfuges.
Some large-volume centrifuge models are quite demanding on space and also generate considerable amounts of heat and noise, and are therefore often centrally positioned in special instrument rooms in biochemistry departments. However, the development of small-capacity bench-top centrifuges for biochemical applications, even in the case of ultracentrifuges, has led to the introduction of these models in many individual research laboratories.
The main types of centrifuge encountered by undergraduate students during intro ductorypracticals may be divided into microfuges (so called because they centrifuge small volume samples in Eppendorf tubes), large-capacity preparative centrifuges, high-speed refrigerated centrifuges and ultracentrifuges. Simple bench-top centrifuges vary in design and are mainly used to collect small amounts of biological material, such as blood cells. To prevent denaturation of sensitive protein samples, refrigerated centrifuges should be employed. Modern refrigerated microfuges are equipped with adapters to accommodate standardised plastic tubes for the sedimentation of 0.5 to 1.5 cm 3 volumes. They can provide centrifugal fi elds of approximately 10 000×g and sediment biological samples in minutes, making microfuges an indispensable separation tool for many biochemical methods. Microfuges can also be used to concentrate protein samples. For example, the dilution of protein samples, eluted by column chromatography, can often represent a challenge for subsequent analyses. Accelerated ultrafiltration with the help of plastic tube-associated filter units, spun at low g-forces in a microfuge, can overcome this problem. Depending on the proteins of interest, the biological buffers used and the molecular mass cut-off point of the particular filters, a 10- to 20-fold concentration of samples can be achieved within minutes. Larger preparative bench-top centrifuges develop maximum centrifugal fi elds of 3000×g to 7000×g and can be used for the spinning of various types of containers. Depending on the range of available adapters, considerable quantities of 5 to 250 cm 3 plastic tubes or 96-well ELISA plates can be accommodated. This gives simple and relatively inexpensive bench centrifuges a central place in many high-throughput biochemical assays, where the quick and efficient separation of coarse precipitates or whole cells is of importance.
High-speed refrigerated centrifuges are absolutely essential for the sedimentation of protein precipitates, large intact organelles, cellular debris derived from tissue homogenisation and microorganisms. the initial bulk separation of cellular elements prior to preparative ultracentrifugation is performed by these kinds of centrifuges. They operate at maximum centrifugal fi elds of approximately 100 000×g. Such centrifugal force is not sufficient to sediment smaller microsomal vesicles or ribosomes, but can be employed to differentially separate nuclei, mitochondria or chloroplasts. In addition, bulky protein aggregates can be sedimented using high-speed refrigerated centrifuges. An example is the contractile apparatus released from muscle fibres by homogenisation, mostly consisting of myosin and actin macromolecules aggregated in fi laments. In order to harvest yeast cells or bacteria from large volumes of culture media, high-speed centrifugation may also be used in a continuous flow mode with zonal rotors. This approach does not therefore use centrifuge tubes, but a continuous flow of medium. As the medium enters the moving rotor, biological particles are sedimented against the rotor periphery and excess liquid removed through a special outlet port.
Ultracentrifugation has decisively advanced the detailed biochemical analysis of subcellular structures and isolated biomolecules. Preparative ultracentrifugation can be operated at relative centrifugal fi elds of up to 900 000×g. In order to minimise excessive rotor temperatures generated by frictional resistance between the spinning rotor and air, the rotor chamber is sealed, evacuated and refrigerated. Depending on the type, age and condition of a particular ultracentrifuge, cooling to the required running temperature and the generation of a stable vacuum might take a considerable amount of time. To avoid delays during biochemical procedures involving ultracentrifugation, the cooling and evacuation system of older centrifuge models should be switched on at least an hour prior to the centrifugation run. In contrast, modern ultra centrifuges can be started even without a fully established vacuum and will proceed in the evacuation of the rotor chamber during the initial acceleration process. For safety reasons, heavy armour plating encapsulates the ultracentrifuge to prevent injury to the user in case of uncontrolled rotor movements or dangerous vibrations. A centrifugation run cannot be initiated without proper closing of the chamber system. To prevent unfavourable fluctuations in chamber temperature, excessive vibrations or operation of rotors above their maximum rated speed, newer models of ultracentrifuges contain sophisticated temperature regulation systems, flexible drive shafts and an over-speed control device. Although slight rotor imbalances can be absorbed by modern ultracentrifuges, a more severe misbalance of tubes will cause the centrifuge to switch off automatically. This is especially true for swinging-bucket rotors. The many safety features incorporated into modern ultracentrifuges make them a robust piece of equipment that tolerates a certain degree of misuse by an inexperienced operator. In contrast to preparative ultracentrifuges, analytical ultracentrifuges contain a solid rotor that incorporates one counterbalancing cell and typically either three or seven analytical cells. A specialised optical system enables the sedimenting material to be observed throughout the duration of a centrifuge run. Using either an absorption optical system (based on ultraviolet/visible light absorption) or a Rayleigh interference optical system (based on light refraction), or a combination of both, concentration distributions of macromolecules in solution can be recorded at any time during ultracentrifugation. From these records, information about the purity/heterogeneity, sedimentation coefficient distribution, average molar mass and molar mass distributions, and ligand interaction information can be obtained.
