What is centrifugation 1
"Sample preparation in the biochemical laboratory"
The centrifuge is indispensable in the biochemical laboratory - regardless of whether you are working with cells, proteins or DNA - at some point there will come a time when unwanted components have to be separated. In addition to other techniques such as filtration or chromatography, centrifugation is an established and easy-to-use technique.
The basics of centrifugation
During centrifugation, the solid particles present in a mixture with a liquid medium are separated in a centrifugal force field due to their different sedimentation speeds. The sedimentation depends on the size, shape and density of the components. Larger particles sediment faster than smaller ones and particles with higher density sediment faster than those with lower density. In addition, the density and viscosity of the medium have an influence on the sedimentation speed. In media with high viscosity, particles sediment more slowly than in media with lower viscosity. This allows a mixture of different particles with different sizes and densities to be separated by centrifugation.
The Swedish chemist and Nobel laureate in chemistry in 1926, The (Theodor) Svedberg (1884-1971), was the first to mathematically grasp these relationships and use his formula known as the Svedberg equation to calculate the sedimentation speed of particles.
An important parameter for biochemistry is the sedimentation coefficient, which results from the ratio of sedimentation speed and centrifugal acceleration. This quotient reflects a characteristic molecular property that can be determined experimentally. It will be in Svedberg units (S.) for which the following applies: 1 S. = 1013 Seconds. For many biochemical particles they are S.Values are known, some have even become part of their scientific name, such as for the prokaryotic ribosome 30S.-RNA.
Construction and equipment of a typical centrifuge from the laboratory
A centrifuge consists of the rotor, which is driven by a motor to the desired revolutions. It is equipped with centrifuge tubes, either centrifuge tubes or, for larger sample quantities, with centrifuge beakers made of special, thick-walled glasses or plastics such as PTFE, PP or HDPE, which hold the sample. They have conical or rounded bases.
The design of the rotors depends on the sample volume and area of application. Tabletop centrifuges are used to separate sample quantities in the µl and ml range; larger centrifuges can hold up to several hundred milliliters of sample volume.
The relative centrifugal acceleration G varies from a few 103G up to several 106G, where 1 G corresponds to the acceleration due to gravity. For high speeds, ultracentrifuges are used, which are operated in a vacuum in order to avoid air resistance and the resulting frictional heat.
Centrifuges also differ in the type of rotor
Centrifuges often have a fixed-angle rotor in which the centrifuge vessels are centrifuged at a fixed angle of around 30 ° to the rotor axis. With a vertical rotor, the sample tubes are vertical, i.e. perpendicular to the rotor axis. This type of rotor has the advantage of very short sedimentation distances and centrifugation times, which is why it is particularly suitable for areas with a high sample throughput.
A variant of this is the so-called NVC rotor, where NVC stands for Near Vertical Centrifugation. The centrifuge tubes are at a small angle to the rotor axis; angles between 7.5 ° and 9 ° are common, which should facilitate sampling.
In the swing-bucket rotor, the sample vessels swing horizontally in the direction of the gravitational field during centrifugation. This type of rotor is particularly popular for separating large volumes.
Different centrifugation methods for different questions
With differential, isopycnic and zone centrifugation there are three different techniques that use different physical parameters for the separation.
Differential centrifugation uses differences in the sedimentation speed
By means of differential centrifugation, the different cell components of a cell extract are separated by their different sedimentation speeds. It is always the first step when starting sample preparation in the laboratory. Here, the sample is sequentially at different G- Numbers centrifuged and the supernatant used in each case in the next centrifugation step. A typical scheme for separating a cell extract looks like this:
- In the first centrifugation step, 1000 G Whole cells and cell nuclei are centrifuged off and are found as a precipitate on the bottom of the centrifuge beaker or tube as a so-called pellet.
- The supernatant will be at 2000 in the next step G centrifuged, whereby cell components such as mitochondria, peroxisomes and lysosomes are separated as a pellet.
- At 80,000 G microsomes and small vesicles are separated from the rest, while large complexes, such as ribosomes, are only separated at an acceleration of 150,000 G
Differential centrifugation is therefore a very simple technique for obtaining different cell fractions.
Isopycnic and zone centrifugation use density gradients
Zone centrifugation is suitable for the separation of particles with similar sedimentation velocities but different sizes. Here the sample is applied to a discontinuous density gradient, for example a sucrose gradient. This consists of layers of increasing concentration, typically between 18 and 30%, and thus increasing densities. The density gradient is created by carefully layering the differently concentrated solutions, with the sucrose solution with the highest density at the bottom of the centrifuge beaker or tube.
The sample is then centrifuged at low speeds for a defined period of time. Due to the different densities, the particles migrate with different sedimentation speeds and thereby separate into discrete bands. At the end, these can be carefully sucked off with a Pasteur pipette.
It is important to stop the centrifugation after a certain time, otherwise all the particles would eventually migrate to the bottom of the centrifuge vessel.
Isopycnic centrifugation, also known as sedimentation equilibrium centrifugation, is suitable for particles of similar size but different density. A density gradient is also used in this technique, but it is not layered beforehand, but rather forms itself as a continuous gradient during centrifugation. Centrifugation takes place at high GNumbers and is time-consuming, as a stable gradient sometimes only develops after 24 to 48 hours. With this method, the particles migrate in the gradient up to exactly that point where the density of the medium corresponds to their own. Since the sedimentation speed is zero in this so-called isopycnic zone, the particles no longer migrate, so that the centrifugation time does not have to be considered as meticulously as with zone centrifugation.
The particles contained in the individual layers are usually not clearly recognizable in a continuous gradient, which is why they are not sucked off from above with a pipette, but a hole is made in the bottom of the centrifuge and the content is collected in fractions. A typical medium for a continuous gradient are cesium chloride solutions (CsCl), with the aid of which, for example, nucleic acids can be separated.
The analytical ultracentrifugation helps with special questions
The analytical ultracentrifuge is not an everyday centrifuge in the laboratory. It is a combination of an ultracentrifuge and an optical detector, either a spectrophotometer or a refractometer. Instead of sample tubes, UV-permeable cuvettes with windows made of quartz or sapphire are used here. At the beginning and during the centrifugation, the absorption of the solution is measured repeatedly, from which the concentrations of the sedimenting particles can be determined. For example, the exact sedimentation coefficients of molecules can be determined from the measured values.
Another important area includes the study of molecular complexes, since individual molecules and molecules associated with complexes differ in their sedimentation velocities.
This technique is used, for example, to study protein-protein or protein-DNA interactions.
In addition to the various other techniques for sample preparation and separation, centrifugation is still one of the most important and widely used techniques in the biochemical laboratory.
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