1.9. Centrifuges

Cells can readily be harvested from liquid cultures by using different centrifuges (Figure 1.15). Similarly, any suspension or floating colloidal particles (e.g. precipitated proteins) in a solution can be separated into fractions by spinning the sample in a centrifuge (see also Chapter 5 for more detail). The resulting fractions are referred to as “supernatant” (i.e. the solution) and “pellet” (i.e. the particles collected at the bottom of the centrifuge tube, pressed together into a compact mass). Centrifuges are relatively simple devices having stationary and rotary parts. The rotation generated by the electric motor of the centrifuge is transmitted to the rotor harbouring the samples contained within appropriate centrifuge tubes. Many biochemical samples are heat sensitive; for instance, proteins denature at elevated temperatures. Such samples require refrigerated centrifuges in which low temperature can be maintained during centrifugation. Centrifuges are available in different sizes ranging from simple bench-top centrifuges to preparative devices with much higher capacities (volumes up to several litres). Eppendorf tubes or Falcon tubes fit into some rotors of bench-top centrifuges, thereby simplifying the processing of many samples. Most centrifuges can be used in conjunction with several different rotors. This versatility allows users to adapt their centrifuges easily according to the actual requirements. Centrifuge tubes (Figure 1.16) must be chosen according to the manufacturer’s instructions. Importantly, the filled rotor must be counter-balanced during operation. To achieve this, tubes with equal weights must be placed into opposite buckets or holes of the rotor. The weight balance should always be checked by simple two-armed or digital scales. If the weights of the sample tubes are different, counter-balances must be prepared by filling similar tubes with water. Unbalanced rotors are subject to extremely high forces during rotation. Even a small asymmetry of the weights around the axis of rotation can result in the breakage of the rotor shaft at high angular speeds. In such cases, the rotor may also damage the whole device and even cause serious personal injuries.


Figure 1.15. A, Bench-top Eppendorf centrifuge with samples arranged symmetrically around the axis of the rotor. B, Semi-preparative, refrigerated centrifuge with a rotor for Eppendorf tubes. C, Preparative, refrigerated centrifuge with a rotor for the handling of volumes up to 6 litres. D, Semi-preparative rotor with lid. E, Preparative rotor with lid.

Centrifuge tubes

Figure 1.16. Centrifuge tubes.

The rotation speed of centrifuges is often specified as the number of revolutions per minute (RPM). However, as the force applied to the sample depends not only on the actual RPM value but also on the radius of the rotor, the relative centrifugal force (RCF) is more informative about a particular experiment. This defines acceleration according to the mass of particles floating in the sample. Therefore, RCF values are given as relative acceleration values (the centrifugal acceleration compared to g, ~9.8 m/s2, the acceleration due to gravity on the surface of Earth). Thus, if the same sample is spun at equal RPM values in two centrifuges with different rotor geometries (different rotor radii), the results will be different. However, if equal RCF values are applied, sedimentation forces will be identical. Therefore, RPM values are only informative when specified together with the rotor type or radius.

Simple centrifuges can provide maximal accelerations around 104g. Ultracentrifuges (Figure 1.17) are capable of operating at maximal accelerations in the range of 105-106g. To reach high angular speeds, ultracentrifuges generate vacuum around the rotor to decrease aerodynamic drag. Ultracentrifuges can be used for preparative (e.g. removal of cell debris from a lysate prior to the isolation of recombinant proteins) or analytical purposes (e.g. investigation of the interactions or the oligomerisation of different biomolecules). (The reader may remember that the first evidence of the semi-conservative replication of DNA also came from density gradient ultracentrifugation experiments.)


Figure 1.17. A, Micro-ultracentrifuge (RPMmax = 100000 min-1, RCFmax = 300000 g). B, Rotors compatible with the micro-ultracentrifuge. C, Semi-preparative ultracentrifuge (RPMmax = 70000 min-1, RCFmax = 350000 g). D, Rotors compatible with the semi-preparative ultracentrifuge.