Centrifugation is a technique, method or process 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, which is applied by a team called centrifugal.
Definition
By centrifugation, the components of a fluid sample can be separated and analyzed. These components are different classes of molecules or particles. As particles, various cell fragments are referred, among others, to organelles of cells, even in many cell types.Theodor Schweder is considered one of the major pioneers of research in centrifugation. The Nobel Prize in 1926, determined that molecules or particles, along with their sizes, have different coefficients of sedimentation. S "S" comes from Swedar, in honor of his actions.
Therefore, the particles have specific sedimentation rates. This means that not all behave in the same way under the action of centrifugal force, expressed in revolutions per minute (rpm), or as a function of the rotor's radius (relative centrifugal force).
S and the factors that determine it, for example, are the characteristics of molecules or particles; properties of the medium; the technique or method of centrifugation; and the type of centrifuge used among other aspects.
Centrifugation is classified according to the usefulness of the same. in preparation, when limited to the separation of the components of the sample; And in analytics, when it wants to analyze isolated molecule or particle. On the other hand, it can also be classified according to the conditions of the process.
Centrifugation is essential for the advancement of scientific knowledge among its various types. Used in research centers, it has facilitated the understanding of complex biochemical and biological processes among many others.
Basis of Centrifugation
The centrifugation process is based on the fact that the molecules or particles that make up a sample in solution will move around while rotating in a device called a centrifuge. This causes the particles to separate from the surrounding medium while sedimenting at different speeds.
The process is based exclusively on the principle of sedimentation. According to this, particles that have a higher density, while the rest of the components of the substance or medium remain suspended. Why? Because molecules or particles have their own shape, size, mass, volume and density. Therefore, not all of them manage sediment in the same way, which translates into a different sedimentation coefficient S; and therefore, at a different sedimentation rate.
These are the properties that allow the molecules or particles to be separated by centrifugal force at a certain centrifugal speed.
Centrifugal force
Centrifugal force will be affected by several factors that will determine sedimentation: those inherent to the molecules or particles; In the characteristics of the environment they are found; and factors related to the centrifuge where the centrifugation process is carried out.
The mass, specific volume, and flotation factor of the molecules or particles are the factors that affect sedimentation. Regarding the surrounding environment, the mass of the solvent displaced, the density of the medium, the resistance to advance and the coefficient of friction are important.
As in the centrifuge, the most important factors affecting the sedimentation process are rotor type, angular velocity, centrifugal force and, consequently, centrifugal velocity.
Types of centrifuges
There are several types of centrifuges by which the sample can be subjected to different centrifugation speeds.
Whereas in so-called supercentrifugadoras, a greater speed range near 25,000 g can be achieved. And in ultracentrifuges, the speed is much higher, reaching 100,000 g.
According to other criteria, there are microcentrifuges or table centrifuges, which are specialized to process centrifugation in a small volume of sample, reaching the range of 12,000 to 15,000 g.
There are high-capacity centrifuges that allow the centrifugation of large and high-speed samples, such as ultracentrifuges.
In general, a number of factors must be controlled to prevent the rotor and sampler from overheating. For this, ultracentrifuges have been created, with special conditions of vacuum or refrigeration, among others.
Types of rotors
One of the determining elements is the type of rotor, the device that rotates and where the tubes are placed. There are different types of rotors. The main ones include rotators, fixed angle rotors and vertical rotors.
In tilt rotors, when placing the tubes in the devices of this type of rotor and when turning, 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.
Type of centrifugation
The types of centrifugation vary according to the purpose of its application and the conditions in which the process is carried out. These conditions may vary depending on the type of sample and the nature of what you want to isolate and/or analyze.
There is a first classification criterion depending on the purpose or purpose of its attainment: preliminary centrifugation and analytical centrifugation.
Preparation centrifuge
This name is obtained when centrifuge is mainly used to separate molecules or particles, particles, cell fragments or cells for later use or analysis. The amount of sample typically used for this purpose is relatively large.
Analytical centrifugation
Analytical centrifugation is performed for the purpose of measuring or analyzing physical properties, such as the sedimentation coefficient and the molecular mass of sedimentary particles.
Depending on the purpose centrifugal can be done by applying various standardized conditions; As is the case with, for example, one of the techniques of ultracentrifugal analytical, which allows to analyze the separation of molecules or particles, even sedimentation occurs.
In some specific cases, the use of quartz centrifuge tubes may be required. Thus, they allow the passage of visible and ultraviolet light, as the molecules are observed and analyzed with an optical system during the centrifugation process.
In short, there are other classification criteria based on the characteristics or conditions in which the centrifugation process is performed. These are: differential centrifugation, zonal or band centrifugation and isocyanic or equilibrium sedimentation centrifugation.
Differential centrifugation
This type of centrifugation consists of a sample for centrifugation, usually with an angular rotor, for a specified time and speed. It is based on the separation of particles by their difference in sedimentation velocity, which is directly related to their sizes. With greater size and greater S, sediment in the bottom of the tube; Whereas those who are younger will remain suspended.
The suspended separation of the precipitate is important in this type of centrifugation. The suspended particles should be disintegrated or removed from the tube, so that the pellet or pellet may be suspended in another solvent for further purification; That is, it is centrifuged again.
Such a technique is not useful for separating molecules. Instead, it can be used to separate, for example, cell organelles, from cells, among other particles.
Zonal or Band Centrifugation
Zonal or band centrifugation serves the separation of components of the sample based on the difference in S when one crosses a medium along a density gradient; For example ficol or sucrose.
The sample is placed over the shield of the test tube. Thereafter, it proceeds to centrifuge at high speed and the separation into individual bands arranged with the medium (as if it were a gelatin in several layers). Particles with a low value of S are at the beginning of the medium, while those with a large or high S are directed down the tube.
With this process, components found in different sedimentation bands can be separated. It is important to control the timing well to prevent all molecules or particles in the sample at the bottom of the tube.
Isocyanic centrifugation and other types
• There are many other types of centrifugation, such as isocyanic. It specializes in isolating macromolecules even though they are of the same type. DNA fits very well into these types of macromolecules, as it presents variations in the sequence and amount of its nitrogenous bases; And so, sediment at different speeds.
• There is also ultracentrifugation, through which the sedimentation characteristics of biomolecules are studied, a process that can be monitored by means of ultraviolet light, for example.
It has been useful in the knowledge of subcellular structures, or organisms. This has allowed advances in molecular biology and the development of polymers.
Applications
There are myriad areas of daily work in which different types of centrifugation are used. They serve in healthcare, in biomedical laboratories, in the pharmaceutical industry, among other fields. However, its importance can be summarized in two words: distinct and characteristic.
Separates particles
In chemistry, different centrifugation techniques have been extremely important for several reasons. This allows the separation of two molecules or miscible particles. Helps to eliminate impurities, substances or unwanted particles in a sample; For example, a sample in which only proteins should be preserved.
In a biological sample, such as blood, plasma can be separated from the cellular component by centrifugation. This contributes to the realization of a variety of biochemical or immunological tests in plasma or serum, as well as for routine or developmental studies.
Even centrifugation allows the separation of different cell types. From a blood sample, for example, red blood cells can be isolated from leukocytes or white blood cells, and also from platelets.
The same utility can be achieved with centrifugation in any of the biological fluids: urine, cerebrospinal fluid, amniotic fluid, among many others. In this way many types of analysis can be done.
As a characterization technique
It has also allowed the study or analysis of the characteristics or hydrodynamic properties of many molecules; Mainly complex molecules or macromolecules.
As well as many macromolecules such as nucleic acids. This has facilitated the characterization of subtypes of the same molecule as RNA, among many other applications.
Author says
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