Centrifuge is one of the most commonly used methods to separate proteins, enzymes, nucleic acids and cell sub components, and is also a commonly used method of separation, purification or clarification in biochemical laboratories.In particular, ultrafast freeze centrifugation has become a common technical method in the research of biomacromolecule laboratories.
Centrifuge technology is to use the strong centrifugal force generated when the object rotates at high speed to make the suspended particles placed in the rotating body settle or float, so that some particles can achieve the purpose of concentration or separation from other particles.The suspended particles here often refer to cells, organelles, viruses and biological macromolecules that are made into suspended state.When the centrifuge rotor rotates at high speed, when the density of suspended particles is greater than the density of surrounding medium, the particles move away from the axis direction and sedimentation occurs;If the particle density is lower than the density of the surrounding medium, the particle moves towards the axis and floats.There are many types of commonly used centrifugesLow speed centrifugeThe maximum speed of shall not exceed 6000rpm,High speed centrifugeBelow 25000 rpm,UltracentrifugeThe maximum speed of is more than 30000 rpm.
Technical Introduction
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Centrifuge separation is to makeCentrifugal accelerationexceedGravitational accelerationIt is a method to accelerate the precipitation and removal of impurities in the liquid medicine by increasing the sedimentation speed.Its principle is to use the density difference of the mixture to separate the feed liquid, which is more suitable for separating suspensions containing fine particles or flocs that are difficult to settle and filter.[1]Centrifuge is a kind of instrument and equipment that uses the high-speed rotation of motor to generate centrifugal force field to separate and extract different particles mixed in suspension or immiscible liquids with different densities in emulsion according to the differences in density, shape, size and other aspects between different substances. It is widely used in biology, medicine, chemical industry and other fields.The technology of separating, purifying and extracting samples with a centrifuge is called centrifugal separation technology.Common centrifugation technologies include precipitation centrifugation, differential centrifugationDensity gradient centrifugation, analytical ultracentrifugation, centrifugal elutriation, zone centrifugation and continuous flow centrifugation, among which precipitation centrifugation, differential centrifugation and density gradient centrifugation are commonly used in the laboratory.Other technologies need specialCentrifugeOr turn around.[2]
Differential centrifugation
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Differential centrifugation is based on the difference of sedimentation speed (i.e. sedimentation coefficient) caused by different particle size and density. It can increase the centrifugal speed by grading or conduct high-speed and low-speed centrifugation alternately, so that the particle samples (or macromolecules) with different mass can be settled from the mixed liquid to the bottom of the tube in batches, so as to achieve the purpose of separation.This method is applicable to the separation of components with large difference in sedimentation coefficient in mixed samples. More accurately, it is the separation of mixed samples with difference in sedimentation coefficient of 1 to several orders of magnitude. The greater the difference, the better the separation effect.
Differential centrifugationGenerally, the rotor with fixed angle is adopted, and the heaviest particles will all sink to the bottom of the pipe through centrifugal sedimentation at a lower speed.Continue to precipitate the supernatant at a higher speed to obtain the second heavy particle sample.The sample particles with different weights can be obtained by gradually increasing the centrifugal speed to achieve the purpose of separation.However, the above is only an ideal state. Generally, the precipitation obtained in each step is not uniform, and light particles are usually mixed.This is because the particles of various weights are evenly distributed in the solution before centrifugation. When centrifugation starts, all particles move to the bottom of the tube according to their own settling speed, so the light particles close to the bottom of the tube will also sink to the bottom of the tube and mix into the heavy particles.Generally, in order to obtain a more pure particle sample, it is also necessary to precipitateHeavy overhang, precipitate again at the same speed.After repeated several times, particles with basically uniform size can be obtained.But generally in practical use, differential centrifugation is not usually used for fine separation.It is only used for the separation of two kinds of particles whose s values differ by one order of magnitude or more, and the precipitation cannot achieve complete recovery.
Differential centrifugation technologyIt is widely used, especially for the separation, crude extraction and concentration of bioactive substances, such as animal and plant viruses, various subcellular components (nuclei, chloroplasts, mitochondria, etc.), and biological macromolecules such as nucleic acids and proteins.[2]
Gradient centrifugation
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Density gradient centrifugationIt is a separation method that enables the sample to be separated to carry out centrifugal sedimentation or sedimentation balance in the density gradient medium, and finally distribute it to some specific positions in the gradient to form different zones, also known as zone centrifugation.Density gradient centrifugationIt can be separated not only according to the weight and sedimentation coefficient of sample particles, but also according to the density, shape and other characteristics of sample particles.The density gradient centrifugation only uses one speed in the whole centrifugation process, and there is no need to change the experimental parameters in the midway, while the differential centrifugation requires speed adjustment, resuspension and repeated centrifugation.Density gradient centrifugation is suitable for separating samples with different densities, while differential centrifugation is suitable for separating components with different sedimentation coefficients in mixed samples.
Density gradient centrifugationIts advantages are: good separation effect, which can obtain relatively pure sample particles at one time;It has a wide range of applicationsDifferential centrifugationSimilarly, the particles with different sedimentation coefficients can be separated, and the particles with a certain difference in buoyancy density can be separated;The particles will be suspended in the corresponding position to form a zone, without forming sediment and being squeezed, so it can maximize thebiological activity；The sample processing capacity is large, and multiple samples can be processed at the same time;It is insensitive to temperature changes and disturbances caused by acceleration and deceleration.Density gradient centrifugationThe disadvantages are: long centrifugation time, need to prepare density gradient medium solution, and high skill requirements for operators.The density gradient centrifugation method usually adopts bucket type horizontal rotor, zone rotor and near vertical rotor.
Before centrifugation, the centrifuge tube is filled with separation medium (such as sucrose, glycerin, etc.) to form continuous or discontinuous density gradient medium, and then the sample is added for centrifugation, which can be divided into:
Velocity zone
Before centrifugation,Centrifugal tubeFirst, sucrose, glycerin, CsCl, Percoll and other density gradient media are loaded into the gradient liquid. The samples to be separated are placed on the top of the gradient liquid, at the bottom of the centrifuge tube or in the middle of the gradient layer. Centrifuge with the gradient liquid, and use the different settling speed or floating speed of each particle in the gradient liquid to make the particles with different settling speed in the gradient layer of different density, so as to achieve the purpose of separating each other.This method can be used to separate various biological samples such as cells, viruses, chromosomes, lipoproteins, DNA and RNA.
Prefabricated gradient
It is required to prepare density gradient media with thick bottom and thin top in advance before centrifugation. Common media include sucrose, CsCl, Cs2SO4, etc. The sample to be separated is generally laid on the gradient liquid top. If it needs to be carried in the middle of gradient liquid or at the bottom of the tube, the density of sample liquid needs to be adjusted.After centrifugation, the sample particles with different densities reach the gradient layer with the same density as their own, that is, they reach the position of equal density and are separated.
Autogenous gradient
Some density media will form their own gradient after centrifugation, such as Percoll, which can rapidly form gradient, CsCl, Cs2SO4 and triiodoformGlucosamineStable gradient can also be produced after long time centrifugation.needCentrifugal separationAfter centrifugation, the gradient medium will gradually form a density gradient of thick at the bottom of the tube and thin at the top of the tube due to the centrifugal force. At the same time, it can drive the original mixed sample particles to redistribute and reach the gradient layer with the same density as its own, that is, reach the position of equal density to obtain separation.[2]
Settlement equilibrium
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The separation is carried out according to the difference of buoyancy density of the separated material. The initial density of the medium used is about equal to the density of the separated material. The medium forms a density gradient during the centrifugal process, and the separated material settles or floats to the medium area with the same density to stay and form a zone.[3]
Centrifugal tool
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CentrifugeCentrifuge is a device that implements centrifugal technology.There are many types of centrifuges. According to the purpose of use, there are two types, namelyPreparation centrifugeandAnalytical centrifuge。The former is mainly used to separate biological materials, and the volume of samples separated each time is relatively large, while the latter is mainly used to study pure productsMacromolecular substances, including the properties of some particles such as ribosomes. The sample volume for each analysis is very small. According to the behavior of the substance to be measured in the centrifugal field (which can be continuously monitored by the optical system in the centrifuge), its purity, shape, relative molecular weight and other properties can be inferred.The main structures of the two types of centrifuges are different due to their different uses.Centrifuges commonly used can be divided intoOrdinary centrifuge、High speed centrifugeandUltracentrifugeThree types.
Technical Principles
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Put the sample into the centrifuge tube of the centrifuge rotating head. When the centrifuge is driven, the sample solution will follow the centrifuge tubeUniform circular motionTherefore, an outward centrifugal force is generated.Because the mass, density, size and shape of different particles are different from each other, the settling speed in the same centrifugal field with a fixed size is also different, so that they can be separated from each other.
centrifugal force
When the solid particles in the solution move in a circle, an outward centrifugal force is generated, which is defined as:
F = mω2r
Where:
F is the strength of centrifugal force;M is the effective mass of settling particles;
ω is the angular velocity of centrifugal rotor rotation, and its unit is rad/s;
R is the centrifugal radius (cm), that is, the distance between the central axis of the rotor and the settling particles.
Obviously, the centrifugal force increases with the increase of rotating speed and particle mass, and decreases with the decrease of centrifugal radius.Centrifugal force is usuallyRelative centrifugal forceFcf refers to the number of times the centrifugal force F is relative to the gravity (G), and the unit is g. Its calculation formula is as follows:
Fcf = 1.119×105(h)2r×g
It can be seen that at the same speed, due to the difference of f, the difference of Fcf will be large, and the average value is generally taken in practical application.In the report of centrifugation experiment, conditions such as Fcf, r average, centrifugation time t and liquid medium should be expressed, because they are directly related to the sedimentation rate of the sample.Obviously, Fcf is a parameter only related to the centrifuge, but not directly related to the sample.
Speed and coefficient
To settle a particle, it must replace the solution with the same volume below it. This can only be achieved by centrifugation when the particle mass is greater than the mass of the replaced liquid. Otherwise, the particle will float upward rather than sink during centrifugation.When a particle is moving, it will pass through the solvent molecule regardless of its direction, and the friction force generated is always opposite to the direction of particle movement.
The size of friction force is proportional to the movement speed of particles, and is affected by the size, shape and medium properties of particles: where:
F is the friction coefficient of particles in the accelerator, which is related to the size, shape and medium properties of particles;
V is the settling velocity of particles.
Due to the existence of centrifugal force, particles will accelerate until the friction is equal to the centrifugal force.In this case, the net force on the particles is zero, and the particles will move at the maximum speed.
Where Mp and Ms are respectively the mass of particles and the mass of solvent of equal volume.
Mp and Ms in the above formula are difficult to determine. In order to establish the relationship between molecular size and settlement coefficient, a new concept of settlement coefficient is introduced.The sedimentation coefficient is defined as the ratio of sedimentation velocity to centrifugal force or the sedimentation velocity of particles in unit centrifugal field, which is calculated in svedberg unit, 1S=1 × 10-13 s.For example,Ribonuclease AThe settlement coefficient of is 1.85 × 10-13 s, which can be recorded as 1.85S.
In recent years, for some macromolecular compounds, when their detailed structure and molecular weight are not very clear, the concept of sedimentation coefficient is often used to describe their size in biochemical, molecular biology, bioengineering and other books and literatures.asRibosomal RNA(rRNA) has 30 s subunit and 50 s subunit, where s is the sedimentation coefficient. Now it is mostly used for the classification of biological macromolecules, especially nucleic acids.[3]
type
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Maximum speed method
(1) Moving interface ultracentrifugation
When a sample containing several components is centrifuged in a sufficiently high centrifugal field, each particle reaches its maximum settling speed, and then the sample begins to separate.The upper layer of the centrifuge tube gradually forms transparent supernatant, and forms a series of concentration interfaces corresponding to each component of the sample. The movement of the interface is characteristic of each component.
Although this method may not be able to achieve the purification and separation of components, the sedimentation rate of each component can be measured by monitoring the movement of the interface.To achieve the separation of components, the centrifugation process must be stopped after the required sample sedimentation.The deposited sample is then suspended in a new solvent, and centrifuged at a lower speed to precipitate large particles of pollutants, while the purified sample is left in the solution. After repeated centrifugation, a pure sample can be obtained. This method is called differential sedimentation centrifugation, which is very useful for the separation of cell components.The separation of different components can also be achieved by gradually increasing the rotating speed.
(2) Moving zone ultracentrifugation
The components are evenly distributed in the whole solution before differential centrifugation, so the separation is generally not ideal. The moving zone ultracentrifugation is a kind of density gradient centrifugation technology. Before centrifugation, the density of solutions in the centrifuge tube is different (the density increases from top to bottom), and the maximum density in the gradient medium should be less than the minimum density of the sample material,Its characteristic is that the separation of substances depends on the quality of the sample material particles, that is, the sedimentation coefficient of the sample material, rather than the density of the sample material, so it is suitable for separating substances with similar density but different size and shape, which belongs to a non-equilibrium separation method.When the sample material is gently laid on the liquid surface of the density gradient medium, start the centrifuge, and under the action of centrifugal force, different material zones will be formed after a certain time.When centrifugation continues, each zone will reach the pipe bottom one by one. Therefore, stop centrifugation before the zone with the fastest sedimentation reaches the pipe bottom, and collect each zone separately.The most commonly used compounds for preparing density gradient are sucrose, glycerin, cesium chloride and cesium sulfate.
Isodensity method
Isodensity centrifugation is also calledSettlement balance method。so-calledIsodensityIt means that the density of the sample is equal to the density of the medium, which is actually carried out in the gradient density medium.The characteristic of this technology is that the sedimentation separation is independent of the size and shape of the sample material, but depends on the density of the sample material.This method is very similar to the PH gradient isoelectric focusing method in electrophoresis. During centrifugation, particles will settle or float upward according to their density until they move to the solvent gradient with the same density as their own. The result is that zones will be formed in the gradient solvent according to the density of the sample substance.
In the experimental method, the density gradient solution can be prepared in advance. Generally, two stock solutions are prepared first, and their concentration determines the limit of the final gradient solution.The discontinuous gradient solution can be formed by gradually adding liquid from the bottom to the top of the centrifuge tube by reducing the density step by step, or byGradient mixerProduces a continuous gradient density.The stock solution is generally prepared with sucrose solution of two densities or cesium chloride solution of two densities.The sample is generally spread on the surface of the solution, and then centrifugation is started.
The balance density gradient method can also be used in the experimental method. In this centrifugal method, the gradient of the medium is not pre prepared, but gradually formed due to the centrifugal force during the centrifugal process.The sample material and the concentrated salt solution of cesium chloride are fully mixed evenly. After centrifugation, the cesium salt, due to the centrifugal forceCentrifugal tubeA continuous increasing density gradient is formed from the nozzle to the bottom of the centrifuge tube.In the process of centrifugation, different components of biological samples settle or float up to find a solution density gradient zone with the same density as their own, and different substances eventually reach the corresponding zone to achieve separation.This method depends on the formation of low molecular weight cesium salt density gradient under the action of centrifugal field, which generally requires a long time of centrifugation (2-3 days).Obviously, the density of the sample material should be between the maximum and minimum density in the medium gradient, otherwise, the sample will sink to the bottom of the centrifuge tube or float to the top of the solution.
Implementation method
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Puncture method
This is a convenient and ideal partial collection method.A hollow metal needle is inserted into the centrifuge tube from the bottom of the tube, and the components in different zones flow out from the needle tube successively from bottom to top, and then collected separately with some collectors.
Substitution method
Add a plug with a collecting catheter at the centrifugal pipe mouth, and a transfusion catheter is simultaneously installed on the plug to insert into the bottom of the centrifugal pipe, and inject high-density centrifugal medium from the transfusion pipe. Its density is higher than the maximum density formed in the centrifugal pipe.When the replacement solution is continuously injected, the solution in the centrifuge tube gradually rises and flows out of the collection tube, and then is collected separately with some collectors.
