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    Home > Biochemistry News > Biotechnology News > Centrifugal separation method

    Centrifugal separation method

    • Last Update: 2020-10-26
    • Source: Internet
    • Author: User
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    (i) differential centrifugation method
    it uses different particles in the centrifugal force field to settle the difference, under the same centrifugal conditions, the sethering speed is different, through increasing the relative centrifugal force, so that a non-uniform mixture of the size of the liquid, different shapes of particles sub-precipitation. The operation process is generally after centrifugation with the dumping method to separate the upper liquid and precipitation, and then the upper liquid plus high speed centrifugation, separate the second part of the precipitation, so that the high speed, step by step to separate the required substances.
    resolution of differential centrifugation is not high, the precipitation coefficient in the same order of magnitude of various particles are not easy to separate, often used in other separation means before the extraction of crude products.
    (ii) rate zone with centrifugation method . The rate zone centrifugation method is the loading of density gradient mediums (e.g. sucrose, glycel, KBr, CsCl, etc.) in the
    centrifuge tube
    before centrifugation, and the sample to be separated is laid out at the top of the gradient fluid, at the bottom of the centrifugal tube, or in the middle of the gradient layer, together with the gradient fluid. After centrifugation, the media density at the near rotation axis (X1) is the smallest and the media farthest from the rotating axis (X2) is the densest, but the maximum media density must be less than the minimum density of particles in the sample, i.e. ρP>ρm.
    This method is based on the separation of particles in the gradient fluid settlement velocity is different, so that particles with different semen velocity in different density gradient layers divided into a series of zones, to achieve the purpose of separation from each other. Gradient fluid acts as a medium and
    stabilizer
    during centrifugation and after centrifugation, avoiding layered particle remix due to mechanical vibration.
    Because ρP>ρm S>0 is known, the centrifugal time of the centrifugal method should be strictly controlled, both to allow sufficient time for various particles to form a zone in the medium gradient, but also to control before any particle reaches precipitation. If the centrifuge time is too long, all samples can reach the bottom of the centrifuge tube;
    this method is an incomplete subsiding, the subsidon is greatly affected by the size of the substance itself, generally applied in cases of different sizes of matter and the same density. Common gradient fluids are Ficoll, Percoll and sucrose.
    (iii) iso-density centrifugation method . The isodyscing method is the density gradient of the pre-arranged medium before centrifugation, which contains the density of all particles in the separated sample, the sample to be separated is laid on top of the gradient liquid or mixed with the gradient fluid first, after centrifugation begins, when the gradient fluid gradually forms the density gradient of the bottom thick and the top of the tube, at the same time the original evenly distributed particles are also redistred.
    When the density of the bottom medium of the tube is greater than the density of the particles, i.e. the particles float up at ρm>ρP, and at ρP>ρm at the top of the bend, the particles sink, and the final particle enters its own density position, which is ρP-ρm, at which point dx/dt zero particles no longer move, particles form a pure component of the zone, and the density of the sample particles, but not the size of the particles and other parameters, so as long as the speed, temperature does not change, then extended centrifugal time can not change the position of these particles.
    this method is generally applied when the size of the substance is similar and the density difference is large. The commonly used gradient fluid is CsCl.
    (iv) the preparation of the gradient solution . 1, the selection principle of gradient material as an ideal gradient material should have the following points:
    (1) and the separated biological material does not react that is completely inert, and easy to separate from the separated biological particles.
    (2) can achieve the required density range, and in the required density range, the viscosity is low, the penetration pressure is low, the ion strength and pH change is small.
    (3) does not corrode centrifugation equipment.
    (4) is easy to purify, cheap or easy to recycle.
    (5) concentration is easy to determine, e.g. with a retrout rate.
    (6) for the super-speed centrifugal analysis work, its physical properties, thermodynamic properties should be known. These conditions are ideal, and there is almost no gradient material that fully meets each performance.
    following are several gradient materials that basically conform to the above principles:
    (1) sugars: sucrose, glycelin, polysucrose (Ficoll), right-hand glyceride, glycogen.
    (2) inorder salts: CsCl (chlorpyride), RbCl (chlorinated ), NaCl, KBr, etc.
    (3)
    organic
    iodide: triiodine benzoyl glucose (matrizamide, etc.).
    (4) silicone sol: such as Percoll.
    (5)
    protein
    : e.g.
    serum
    albumin.
    (6) heavy water.
    (7) non-water-soluble organic matter: such as fluorocarbons.
    2, the application range of gradient materials
    (1) sucrose: water solubility, stable properties, high permeability pressure, its highest density of up to 1.33g/ml, and because of the low price is easy to prepare, is now commonly used in the laboratory cytors, viruses, RNA separation gradient materials, but due to large permeation pressure, should not be used for cell separation.
    (2) polysucrose: the commodity name Ficoll, often used Ficoll-400 is relative to the molecular weight of 400,000, Ficoll penetration pressure is low, but its viscosity is particularly high, for this reason often mixed with pan-shadow glucosamine to reduce viscosity. It is mainly used to isolate various cells including blood cells, fibroblasts, tumor cells, mouse liver cells, etc.
    (3) cesium chloride: is an ionized medium, water solubility, up to a maximum density of 1.91g/ml. Because it is heavy metal salt, the gradient formed at centrifugation has a good resolution and is widely used for the separation of
    DNA
    , granules, viruses and lipoproteins, but at a more expensive price.
    (4) halides: KBr and NaCl can be used for lipid
    protein separation
    , KI and NaI can be used for RNA separation at a resolution higher than radon salts. NaCl gradients can also be used to isolate lipoproteins, and NaI gradients can isolate natural or denatured DNA.
    (5) Percoll: Is a commodity name, it is a SiO2 colloidal bread layer of polyethylene pyridoxine (PVP), low penetration pressure, it has a small impact on biological materials, and particle stability, cooling and freezing and thawing conditions are stable, its viscosity is high, and unstable at acidic pH and high ion strength. It can be used for the separation of cells, cells, and viruses.
    .
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