Centrifugal technology principle and types

The centrifugal method is one of the most commonly used protein, enzyme, nucleic acids and cell separation, biochemical laboratory method is commonly used in separation, purification or clarification. Especially the ultracentrifuge method has become a widely used technique of molecular biological research in the laboratory.
Centrifuge (centrifuge) is a device for carrying out centrifugal technology. Many different types of centrifuges, in accordance with the purpose of use, can be divided into two categories, namely the preparative centrifuge and analysis type centrifuge. The former is mainly used for the separation of biological materials, each separation capacity of the sample is relatively large, and the latter is mainly used to study the pure macromolecules, including the nature of certain particles such as ribosomal material, sample size for each analysis is very small, according to the material to be tested in centrifugal field behavior (continuous monitoring optical system available in the centrifuge), can infer its purity, shape and relative molecular mass properties. These two types of centrifuge due to different purposes, the main structure are also different.
Centrifugal principle
The centrifugal sample into a centrifuge rotor tubes, centrifuge drive, liquid samples with centrifugal pipe of uniform circular motion, and then produces an outward centrifugal force. Because of the different particle mass, density, size and shape vary from one another, in centrifugal field with a fixed size of sedimentation velocity is not the same, so we can get the separation between.
The centrifugal force and the relative centrifugal force
Produce an outward centrifugal force of solid particles in solution to do circular motion, which is defined as:
F = m ω 2
Type in:
F is the centrifugal force strength; m is the effective mass settling particles;
Angular velocity ω for centrifugal rotor rotation, the unit is rad/s
R centrifugal radius (CM), namely the rotor center shaft to the settlement of the distance between particles.
Obviously, the centrifugal force increases with the speed and grain quality, while decreases with decreasing the centrifugal radius. At present, the centrifugal force is usually to relative centrifugal force Fcf, namely the size of the centrifugal force of the F relative to the earth's gravity (G) how many times, the unit is g, the calculation formula is as follows:
Fcf = 1.119 x 105 (H) 2R × G
As can be seen, at the same speed, the difference of F, Fcf will be of great difference, the actual application of general average. In the centrifugal experimental report, Fcf, average R, centrifugal time t and liquid medium conditions should be expressed, because they are and settling velocity of sample is directly linked to. Obviously Fcf is a just and centrifuge related parameters, and has no direct relationship with the sample.
Sedimentation velocity and coefficient
A particle to settlement, it must be the solution to volume replacement out below it, it only when the particle mass is more than by quality replacement of liquid can be by centrifugal means, or otherwise, during centrifugation particles will float upwards, not sinking. When the particles in motion, regardless of the direction, it must be through the solvent molecules, the friction is always with the particle movement in the opposite direction.
Movement velocity and particle friction force is proportional to the size, shape, and dielectric properties and by the particles::
Mp and M s respectively for particle mass and volume of solvent quality type.
In the formula of Mp and M s are difficult to determine, in order to establish relations between the molecular size and sedimentation coefficient, introducing the new concept of the sedimentation coefficient. The sedimentation coefficient is defined as the ratio of settling velocity and centrifugal force centrifugal field or unit of the settling velocity of particles, it is calculated using the Svedberg unit, 1S = 1 × 10-13 s. For example, a sedimentation coefficient of RNase A is 1.85 × 10-13 s, can be written 1.85S.
In recent years, in biochemistry, molecular biology and biological engineering books and literature, for some large molecular compounds, when the detailed structure and the molecular weight is not very clear, often with a sedimentation coefficient of this concept to describe the size of them. Such as ribosomal RNA (rRNA) 30 s and 50s subunits, where s is the sedimentation coefficient, now more for the classification of large biological molecules, particularly nucleic acids.
Type centrifugal technology
Maximum speed method
(1) moving boundary ultracentrifugation
With several components of the samples were centrifuged at high enough in centrifugal field, each particle attains its maximum settling velocity, then the sample from the beginning. Centrifugal tube upper gradually formed transparent supernatant, a series of concentration at the interface and forms corresponding to the sample components, interface moving relative to each component is characteristic of.
Although the use of this method may not be able to achieve component isolation, but can move through the monitoring interface to determine the settling velocity of each component. In order to realize the separation between the components, must be in the required sample settlement after centrifugal process. Sediment samples resuspension to new solvents, and with low speed centrifugation of the pollutants in the particle sedimentation, while the samples in the solution to be purified, after repeatedly centrifugation to obtain pure samples, this method is called the differential sedimentation centrifugal method, it is very useful for cell separation between components. Separation can also through the method of speed increasing realization among different components,
Cell homogenates differential separation
(a) cell homogenate; (b) cell debris; (c) mitochondria, peroxisomes, lysosomes; (d) microsomal, ribosome; (E) cell liquid; (soluble protein and small molecular biology)
(2) moving zone ultracentrifugation
Differential centrifugation before centrifugation were evenly distributed over the entire solution, therefore the separation is generally not ideal, while the moving zone ultracentrifugation in a density gradient centrifugation technique (Dens Gradient), at different centrifugal before centrifuge tube solution density (from top to bottom density increases), the minimum density of maximum density should be less than the sample gradient medium, its characteristic is the separation depends on the material in the sample material grain quality. The sedimentation coefficient of sample, rather than depending on the sample density of the material, which is suitable for the separation of similar density and different size and shape of the material, which belongs to a method for the separation of non equilibrium. When the sample material gently on centrifuge in liquid density gradient medium, starting, under the action of centrifugal force, after a certain period of time will form a different material zones. When the centrifugal each zone will go to the bottom of the pipe, so, in the settlement of the fastest zone reaches the bottom of pipe to stop prior to centrifugation, and each zone division collection. The most commonly used preparation density gradient of sucrose, glycerol, CSCL compounds and cesium sulfate etc..
Density method
Density gradient centrifugation method is also called the sedimentation equilibrium method. The so-called density refers to the density equal to sample density and medium, is actually in the density gradient medium. The technology is characterized by subsidence separation and sample size and shape, and depends on the sample density. PH gradients isoelectric focusing method this method is very similar to the electrophoresis, the centrifugal, particles according to their density in different settlement or float upwards, until moving to the same solvent gradient and its density so far, the result is still substance density gradient solution in a zone forming agent.
The experimental method can be used to pre preparation method of density gradient solution, the first general prepared two kinds of liquid reserves, limit concentration determines their final form gradient solution. Through stepwise reduced density, from the bottom of the centrifuge tube to the upper gradually add fluid to form discontinuous gradient solution, can also produce a continuous density gradient by a gradient mixer. Cesium chloride solution of sucrose solution liquid reserves generally use two kinds of density or two density to prepare. Samples are generally spread on the solution surface, then start centrifugal.
The experimental method can also be used in equilibrium density gradient method, the centrifugal method, medium gradient is not prepared in advance, but in the centrifugal process, due to the centrifugal force and gradually formed. Concentrated salt solution sample material and CSCL are fully mixed evenly, centrifugal began, effect of cesium salt due to the centrifugal force, the centrifugal nozzle density gradient from the bottom to the centrifugal tube to form a continuous increasing. In biological samples with different components during centrifugation sedimentation or float in search of solution density gradient zone and its density with different material, reach the corresponding zone, so as to realize the separation. The formation of cesium salt density gradient of this method relies on centrifugal field under the action of low molecular weight, centrifugal generally require long time (2~3 days). Obviously, sample density should be between the maximum and minimum density, medium gradient between otherwise, samples will sink to the bottom of the centrifuge tube or float to the top of solution.
Either way, will eventually be collected separately in the sample group of each zone is divided, in general can be realized by the following two methods:
(1) puncture
This is a convenient and ideal part of the collection method. With a metal hollow needle from the centrifuge tube bottom prick pipe band, set different area divided from bottom to top successively from the needle tube were out, and then use the fraction collector collected separately.
(2) substitution method
In the centrifugal nozzle with a collecting conduit plug, plug and is provided with an infusion catheter insertion centrifugal pipe from the bottom, the transfusion tube into the centrifugal medium high density. The density is higher than the maximum density formed in centrifugal tube. When replacing the liquid is continuously injected solution centrifuge tube gradually rise, and continuously from the collection pipe out, then part of the collection were collected.
Centrifuge type
The commonly used centrifuge according to the rotor speed of different size can be divided into ordinary centrifuge, high-speed centrifuge and ultracentrifuge three.
(1) general centrifuge
In general, the maximum speed of not more than 6000r / min belonging to the ordinary centrifuge; such as made of 80 - 1, LXJ - Ⅱ etc.. Measure centrifuge speed and relative centrifugal force, as shown in Figure 2 - 37.
Ordinary centrifuge rotor operating at room temperature, room temperature is generally unable to control the rotor, a fixed angle and rotor suspended format.
Solid sediment layer formed when called centrifugal plate, the liquid portion is called the supernatant. Separation of two-phase by pouring method.
(2) high speed centrifuge
Speed can reach 25000r / min for high speed centrifuge; speed over 25000r / min above for ultracentrifuge. High speed centrifuge with cooling device, so the rotor can control indoor temperature. Rotor indoor temperature control in 4 ℃.. The main purpose of large capacity continuous flow centrifuge is from a number of cultures (5~500 L) collected in yeast and bacteria. Another kind is the low capacity refrigerated centrifuge, type many, the maximum capacity of up to 3L. This kind of centrifuge with angle type internal can transform and swing out rotor, they used for collecting microorganisms, cell debris, cells, organelles, ammonium sulfate precipitate, immune precipitate the enzyme extract etc.. Special attention is, every time before centrifugation, should according to how much head models and samples, setting the sample height.
3) ultra high speed centrifuge
Common and high speed centrifuge is mainly used for the separation and preparation of biological macromolecules and subcellular components; ultracentrifuge with centrifugal force more than 500000 x g, can make the subcellular fractionation, and can be used for virus isolation, can also be used for the determination of protein, nucleic acid molecular weight etc..
According to different functions, and can be divided into preparative ultracentrifuge and analytical ultracentrifuge.
Because of high speed will produce a lot of heat, so the centrifuge with freezing device, in order to reduce indoor temperature of rotor. At the same time. The rotor is operating under vacuum, can reduce the friction. Analysis of centrifugal sedimentation process, must be monitored for solid particles. Therefore, ultracentrifuge with an optical system, the optical path and centrifugal tube is vertical, and through the solution in the centrifugal pipe, simultaneous determination of optical density or light transmittance, can also speed and moving boundary settlement detection settling particles. These contribute to the determination of sample analysis.
The preparative ultracentrifuge:
Mainly by the drive and speed control, temperature control, vacuum system and the rotor is composed of four parts.
Drive and speed control: drive most ultracentrifuge is by water or air cooled motor through a gear or belt transmission, or the direct use of variable frequency motor connected to the rotor shaft. Because the drive shaft diameter is only 0.476cm, so, during rotation of the fine shaft bending degree of elasticity, so as to adapt to the head slightly unbalanced, not to cause vibration or rotation of uranium injury. Controller using rheostat and with screw speed to rotor speed. In addition to the speed control system, there is an over speed protection system, in order to prevent the speed exceeds the specified maximum speed when the head caused by rotor tearing or explosion. For this purpose, a centrifugal cavity always use armor can withstand the explosive closed.
Temperature control: the temperature control is the placement of infrared radiation sensor in turn below the direct and continuous monitoring of rotor temperature, to ensure more accurate temperature control more sensitive.
Vacuum system: when the speed of more than 4000 R / min, the friction between the air and the rotating shaft of a serious problem. In order to eliminate the heat source. The ultracentrifuge generally add vacuum system. The centrifugal cavity sealing, vacuum pump system and the two series of vacuum. Mechanical vacuum pump first working pump and general laboratory of the same, it can be evacuated to 13.33~666Pa. Once the centrifuge chamber pressure decreased to below 33.33Pa, the water diffusion pump began to work. Using the two pump, the vacuum degree to achieve and maintain in 0.13~0.26Pa. In the case of friction reduction, the speed can be increased to the required speed.
Head: preparative ultracentrifuge the head have various. Generally can be divided into two categories: angle rotor and swing out rotor. Angle between rotor cavity and the axis of rotation angle in 20~45 degrees. The advantages of this kind of rotor is a large capacity, high speed. Another swing out rotor is composed of a head suspension with 6 free bucket (centrifugal tube). When the rotor is static, the handle vertical suspension; when the head under the action of centrifugal force and speed up to 200 ~ 800 R / min. The bucket is swing to the horizontal position.
The rotor is mainly to density gradient sedimentation method and design. Its main advantage is the gradient material can be placed in a vertical centrifugal tube, and the centrifugal tube to maintain the level of. Appears across the centrifugal pipe band in a horizontal position settlement to centrifuge tube in different regions of the sample, rather than the angular rotor as an angle. Therefore, when removed from the head of the centrifuge tube. Not like in angle rotor as, re positioning the settlement components. This kind of rotor's weaknesses are: the time required for the forming zone between the moment is long, the analytical ultracentrifuge analytical ultracentrifuge using a special design of the rotor and the detection system, so as to continuously monitor the material in the process of settlement of a centrifugal field.
Analytical ultracentrifuge head is oval, the rotor by a natural shaft coupled to a drive device of high speed. A rotating turret in a frozen and vacuum rubber. There are 2~6 mounted centrifugal cup chamber head, a centrifugal cup is fan-shaped, down light. An optical system with a centrifuge, can subsidence by UV absorption and refractive index change monitoring centrifugal cup in the centrifugal substances during. At a predetermined time can shoot photos, settlement of the material cup material deposition process, lens formed between heavy particles and light particle interface like a refraction, created a "peak" in the photographic plate detection system for settlement, continuous, interface ahead, so the peak also mobile. From the peak shift speed can be obtained index substance sedimentation velocity.
Analytical ultracentrifuge is mainly used for the determination of relative molecular mass of biological macromolecules, estimated class purity and detection of biological macromolecular conformational changes etc..