Centrifugation
By applying a larger effective gravitational force to the mixture, like a centrifuge does, the separation of the particles is accelerated.
The most common application is the separation of solid from highly concentrated suspensions, which is used in the treatment of sewage sludges for dewatering where less consistent sediment is produced.
[3] The centrifugation method has a wide variety of industrial and laboratorial applications; not only is this process used to separate two miscible substances, but also to analyze the hydrodynamic properties of macromolecules.
In the chemical and food industries, special centrifuges can process a continuous stream of particle turning into separated liquid like plasma.
[5] In a liquid suspension, many particles or cells will gradually fall to the bottom of the container due to gravity; however, the amount of time taken for such separations is not feasible.
, where g represents the relative centrifugal force (RCF) and r the radius from the center of the rotor to a point in the sample.
[6] However, depending on the centrifuge model used, the respective angle of the rotor and the radius may vary, thus the formula gets modified.
is a constant; r is the radius, expressed in centimetres, between the axis of rotation and a point in the sample; and rpm is the speed in revolutions per minute.
Microcentrifuges are specially designed table-top models with light, small-volume rotors capable of very fast acceleration up to approximately 17,000 rpm.
The microcentrifuge is normally used in research laboratories where small samples of biological molecules, cells, or nuclei are required to be subjected to high RCF for relatively short time intervals.
[10] Low-speed centrifuges are used to harvest chemical precipitates, intact cells (animal, plant and some microorganisms), nuclei, chloroplasts, large mitochondria and the larger plasma-membrane fragments.
[9] These machines have maximum rotor speeds of less than 10 000 rpm and vary from small, bench-top to large, floor-standing centrifuges.
[11] High-speed centrifuges are typically used to harvest microorganisms, viruses, mitochondria, lysosomes, peroxisomes and intact tubular Golgi membranes.
[9] High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres.
The rotors may come with different adapters to hold various sizes of test tubes, bottles, or microtiter plates.
[9] Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles and, additionally, whilst microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
Analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation.
It is a commonly used biomolecular analysis technique used to evaluate sample purity, to characterize the assembly and disassembly mechanisms of biomolecular complexes, to determine subunit stoichiometries, to identify and characterize macromolecular conformational changes, and to calculate equilibrium constants and thermodynamic parameters for self-associating and hetero-associating systems.
[13] Analytical ultracentrifuges incorporate a scanning visible/ultraviolet light-based optical detection system for real-time monitoring of the sample’s progress during a spin.
[6] The most widely used application of this technique is to produce crude subcellular fractions from a tissue homogenate such as that from rat liver.
This is more likely to occur when working with a solution that has a layer of suspension floating on a dense liquid, which in fact have little to no density gradient.
[17] A centrifuge can be used to isolate small quantities of solids retained in suspension from liquids, such as in the separation of chalk powder from water.
In such situations, the aqueous discharge is obtained at the opposite outlet from which solids with a specific gravity greater than one are the target substances for separation.
[23] By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large-grained sols in terms of their gravitational sedimentation.
This gave hemoglobin a resulting weight of approximately 16,000 dalton (Da) but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present).
[24][25] How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea that proteins are macromolecules rather than colloids.
[26] In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the ultracentrifuge was created to apply the theory of sedimentation-diffusion.
[24] The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.
[24] Further application of centrifugation showed that under different conditions the large homogeneous particles could be broken down into discrete subunits.
[17] This method was also used in Meselson and Stahl's famous experiment in which they proved that DNA replication is semi-conservative by using different isotopes of nitrogen.
