The centrifugation It is a technique, method or procedure that mechanically or physically separates molecules or particles with different densities and which are also present in a liquid medium. Its cornerstone is the application of centrifugal force, applied by equipment called a centrifuge..
By centrifugation, the components of a fluid sample can be separated and analyzed. Among these components are the different classes of molecules or particles. As particles, reference is made to different cell fragments, to the organelles of cells, even to various types of cells, among others..
Theodor Svedger is considered one of the leading pioneers in centrifuge research. Nobel Prize in 1926, determined that molecules or particles with their own sizes have different sedimentation coefficients S. The "S" comes from Svedger, in honor of his works.
The particles, therefore, have characteristic sedimentation rates. This means that not all of them behave in the same way under the action of a centrifugal force expressed in revolutions per minute (rpm), or as a function of the radius of the rotor (relative centrifugal force, g).
Among the factors that determine S and its velocity are, for example, the characteristics of the molecules or particles; the properties of the medium; the technique or method of centrifugation; and the type of centrifuge used, among other aspects.
Centrifugation is classified according to its usefulness. In preparative, when it is limited to the separation of the components of the sample; and in analytics, when it also seeks to analyze the separated molecule or particle. On the other hand, it can also be classified based on the process conditions.
Centrifugation in its different types has been essential for the advancement of scientific knowledge. Used in research centers, it has facilitated the understanding of complex biochemical and biological processes, among many others..
Article index
The centrifugation process is based on the fact that the molecules or particles that make up a sample in solution, will rotate when rotating in a device called a centrifuge. This causes the separation of the particles from the environment that surrounds them as they settle at different speeds..
The process is specifically based on the theory of sedimentation. According to this, the particles that have a higher density will settle, while the rest of the substances or components of the medium will remain suspended..
Why? Because molecules or particles have their own sizes, shapes, masses, volumes and densities. Therefore, not all of them manage to sediment in the same way, which translates into a different sedimentation coefficient S; and consequently at a different sedimentation rate.
These properties are what allow the molecules or particles to be separated by centrifugal force at a given centrifugation speed..
The centrifugal force will be influenced by several factors that will determine the sedimentation: those inherent to the molecules or particles; to the characteristics of the environment in which they are found; and factors related to the centrifuges where the centrifugation procedure is carried out.
In relation to the molecules or particles, the mass, the specific volume and the flotation factor of the same are influencing factors in sedimentation..
Regarding the environment that surrounds them, the mass of the displaced solvent, the density of the medium, the resistance to advance and the coefficient of friction are important..
Regarding the centrifuge, the most important factors influencing the sedimentation process are the type of rotor, the angular velocity, the centrifugal force and consequently the centrifugal speed..
There are several kinds of centrifuges by which the sample can be subjected to different speeds of centrifugation..
Depending on the maximum speed they reach, expressed in centrifugal acceleration (Relative centrifugal force g), can be classified simply as centrifuges, having a maximum speed of approximately 3,000 g.
While in the so-called supercentrifuges, it can reach a greater range of speeds close to 25,000 g. And in the ultracentrifuges, the speed is much higher, reaching 100,000 g.
According to other criteria, there are microcentrifuges or tabletop centrifuges, which are special to perform the centrifugation process with a small sample volume, reach a range of 12,000 to 15,000 g.
High-capacity centrifuges are available that allow higher-speed, larger volumes of samples to be centrifuged such as ultracentrifuges..
In general, several factors must be controlled to protect the rotor and sample from overheating. For this, ultracentrifuges have been created with special vacuum or refrigeration conditions, among others..
One of the determining elements is the type of rotor, a device that rotates and where the tubes are placed. There are different types of rotors. Among the main ones are swingarm rotors, fixed angle rotors and vertical rotors.
In tilting rotors, when placing the tubes in the devices of this type of rotor and when rotating, the tubes will acquire an arrangement perpendicular to the axis of rotation..
In fixed angle rotors, the samples will be located inside a solid structure; as seen in the image and in many centrifuges.
And in the vertical rotors in some ultracentrifuges, the tubes will rotate parallel to the axis of rotation..
The types of centrifugation vary according to the purpose of their application and the conditions in which the process is carried out. These conditions may be different depending on the type of sample and the nature of what you want to separate and / or analyze..
There is a first classification criterion based on the objective or purpose of its performance: preparative centrifugation and analytical centrifugation..
It receives this name when centrifugation is used mainly to isolate or separate molecules, particles, cell fragments or cells, for their later use or analysis. The amount of sample that is generally used for this purpose is relatively large.
Analytical centrifugation is carried out in order to measure or analyze the physical properties, such as the sedimentation coefficient and the molecular mass of the sedimented particles..
Centrifugation based on this objective can be performed by applying different standardized conditions; as is the case, for example, of one of the analytical ultracentrifugation techniques, which allows to analyze the molecules or particles that are separated, even as sedimentation is carried out.
In some specific cases, the use of quartz centrifuge tubes may be required. Thus, they allow the passage of visible and ultraviolet light, since during the centrifugation process the molecules are observed and analyzed with an optical system..
Precisely, there are other classification criteria depending on the characteristics or conditions in which the centrifugation process is carried out. These are: differential centrifugation, zone or band centrifugation, and isopycnic or sedimentation equilibrium centrifugation..
This type of centrifugation consists of subjecting a sample to centrifugation, generally with an angle rotor, for a specific time and speed..
It is based on the separation of particles due to their difference in sedimentation speed, which is directly related to their sizes. Those that are larger and larger S, settle at the bottom of the tube; while those that are smaller, will remain suspended.
Suspended separation of the precipitate is vital in this type of centrifugation. Suspended particles must be decanted or removed from the tube, so that the sediment or pellet can be suspended in another solvent for subsequent purification; that is, it is centrifuged again.
This type of technique is not useful for separating molecules. Instead, it can be used to separate, for example, cellular organelles, cells, among other particles.
The zonal or band centrifugation performs the separation of the components of the sample based on the difference of S when passing through a medium with a preformed density gradient; like Ficoll, or sucrose, for example.
The sample is placed on top of the gradient of the test tube. Next, it is centrifuged at high speed and the separation takes place in different bands arranged along the middle (as if it were a gelatin with multiple layers)..
The particles with a lower value of S remain at the beginning of the medium, while those that are larger or have a higher S, go towards the bottom of the tube.
With this procedure, the components found in the different sedimentation bands can be separated. It is important to control the time well to avoid that all the molecules or particles of the sample settle to the bottom of the tube..
-There are many other types of centrifugation, such as isopycnic. This specializes in separating macromolecules, even if they are of the same type. DNA fits very well in this type of macromolecules, since it presents variations in the sequences and quantity of its nitrogenous bases; and therefore sediment at different speeds.
-There is also ultracentrifugation, by which the sedimentation characteristics of biomolecules are studied, a process that can be monitored using ultraviolet light, for example..
It has been useful in understanding subcellular structures, or organelles. Likewise, it has allowed advances in molecular biology and in the development of polymers..
There are innumerable areas of daily life in which the different types of centrifugation are used. They are used for the health service, in bioanalytical laboratories, in the pharmaceutical industry, among other areas. However, its importance can be summed up in two words: separate and characterize.
In chemistry, different centrifugation techniques have been extremely important for many reasons..
It allows to separate two miscible molecules or particles. Helps remove unwanted impurities, substances or particles in a sample; for example, a sample in which you only want to preserve the proteins.
In a biological sample, such as blood, plasma can be separated from the cellular component by centrifugation. This contributes to the performance of different types of biochemical or immunological tests on plasma or serum, as well as for routine or special studies..
Even centrifugation allows the different types of cells to be separated. From a blood sample, for example, red blood cells can be separated from leukocytes or white blood cells, and also from platelets.
The same utility can be obtained with centrifugation in any of the biological fluids: urine, cerebrospinal fluid, amniotic fluid, among many others. In this way, a wide variety of analyzes can be carried out..
It has also made it possible to study or analyze the characteristics or hydrodynamic properties of many molecules; mainly of complex molecules or macromolecules.
As well as numerous macromolecules such as nucleic acids. It has even made it easier to characterize details of the subtypes of the same molecule such as RNA, among many other applications..
-Thanks to the different centrifugation techniques, advances have been made in the exact knowledge of complex biological processes such as infectious disease and metabolism, among others..
-By means of centrifugation, many ultrastructural and functional aspects of molecules and biomolecules have been elucidated. Among such biomolecules may be mentioned the proteins insulin and hemoglobin; and on the other hand, nucleic acids (DNA and RNA).
-With the support of centrifugation, the knowledge and understanding of many of the life-sustaining processes has expanded. One of them is the Krebs cycle.
In this same area of utility, it has influenced the knowledge of the molecules that make up the respiratory chain. Thus, giving light to the understanding of the complex process of oxidative phosphorylation, or true cellular respiration, among many other processes..
-Finally, it has contributed to the study of various processes such as infectious disease, by allowing the analysis of the route followed by the DNA injected by a phage (bacteria virus) and the proteins that the host cell can synthesize..
Yet No Comments