Type of centrifugal technique
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 velocity, at which time the sample begins to separate. The upper layer of the centrifuge tube gradually forms a clear supernatant and forms a series of concentration interfaces corresponding to the components of the sample, the movement of the interface being characteristic with respect to each component.
Although the purification separation of the components may not be achieved by this method, the sedimentation velocity of each component can be determined by monitoring the movement of the interface. In order to achieve separation between components, the centrifugation process must be stopped after the desired sample has settled. The deposited sample is resuspended in a new solvent and centrifuged at a lower rate to cause large particles of contaminants to settle, while the purified sample remains in solution and is centrifuged repeatedly to obtain a pure sample. The method is called differential sedimentation centrifugation, which is very useful for separation between cell components. It is also possible to achieve separation between different components by gradually increasing the rotational speed, as shown in Figure 2-36.
Figure 2-36 Schematic diagram of differential separation of cell homogenate
(a) cell homogenate; (b) cell debris; (c) mitochondria, peroxisomes, lysosomes; (d) microsomes, ribosomes; (e) cytosol; (soluble proteins and small molecule organisms) )
(2) Moving zone with ultracentrifugation Differential centrifugation Before the centrifugation, the components are evenly distributed throughout the solution, so the separation is generally not ideal, and the mobile zone ultracentrifugation is a density gradient (Dens heart Gradient) centrifugal technique. The density of the solution in the centrifuge tube is different before the centrifugation (the density increases from top to bottom). The maximum density in the gradient medium should be less than the minimum density of the sample material. The characteristic is that the separation of the material depends on the mass of the sample material particles. That is, the sedimentation coefficient of the sample material, rather than the density of the sample material, is suitable for separating substances of similar density and size and shape, which is a non-equilibrium separation method. When the sample material is gently spread on the liquid level of the density gradient medium, the centrifuge is started, and under the action of the centrifugal force, a zone of different substances is formed after a certain time. When the centrifugation is continued, each zone will reach the bottom of the pipe one by one, so the centrifugation is stopped before the zone with the fastest settlement reaches the bottom of the pipe, and each zone is collected. The most commonly used compounds for preparing density gradients are sucrose, glycerin, cesium chloride and barium sulphate.
Equal density method Equal density centrifugation is also called sedimentation balance method. The so-called equal density means that the density of the sample is equal to the density of the medium, and is actually carried out in a gradient density medium. The feature of this technique is that the sedimentation separation is independent of the size and shape of the sample material and depends on the density of the sample material. This method is very similar to the PH gradient isoelectric focusing method in electrophoresis. During centrifugation, the particles settle or float upward depending on their density until they move to the same solvent gradient as their own density. The result is dependent on the sample. The difference in material density forms a zone in the gradient solvent.
In the experimental method, a method of preparing a density gradient solution in advance may be used. Generally, two kinds of stock solutions are prepared, and their concentrations determine the limit of the gradient solution finally formed. The discontinuous gradient solution can be formed by stepwise reduction of the density, gradually adding liquid from the bottom of the centrifuge tube to the upper portion, or a continuous gradient density can be produced by a gradient mixer. Stock solutions are typically prepared using two densities of sucrose solution or two densium ruthenium chloride solutions. The sample is typically spread on the surface of the solution and then centrifuged.
An equilibrium density gradient method can also be used in the experimental method. In this centrifugation method, the gradient of the medium is not pre-formulated, but is gradually formed due to the centrifugal force during the centrifugation. The sample material and the concentrated salt solution of barium chloride are well mixed. After the start of centrifugation, the barium salt forms a continuously increasing density gradient from the centrifuge tube to the bottom of the centrifuge tube due to the centrifugal force. The different component substances in the biological sample settle or float in the process of centrifugation to find the solution density gradient zone with the same density as the self, and the different substances finally reach the corresponding zone, thereby achieving separation. This method relies on the formation of a low molecular weight strontium salt density gradient under the action of a centrifugal field, which typically requires long periods of centrifugation (2 to 3 days). Obviously, the density of the sample material should be between the maximum and minimum density in the media gradient, otherwise the sample will sink to the bottom of the centrifuge tube or float to the top of the solution.
Regardless of the mode of operation, the sample components in each zone are ultimately collected separately. Generally, the following two methods can be used:
(1) Puncture method This is a convenient and ideal partial collection method. A metal hollow needle is used to pierce the tube from the bottom of the centrifuge tube. The components in the different zones are separately discharged from the needle tube from bottom to top, and then collected by a part of the collector.
(2) Substitute method A plug with a collecting conduit is added to the centrifuge tube. The plug is simultaneously filled with an infusion catheter inserted into the bottom of the centrifuge tube, and a high-density centrifugal medium is injected from the infusion tube. Its density is higher than the maximum density formed in the centrifuge tube. As the replacement liquid is continuously injected, the solution in the centrifuge tube gradually rises and continuously flows out of the collection conduit, and then is collected separately by a part of the collector.
Centrifuge types Generally, centrifuges can be classified into three types: general centrifuges, high-speed centrifuges, and ultracentrifuges depending on the rotor speed.
(1) General centrifuge Generally speaking, the maximum speed does not exceed 6000r/min. It is a common centrifuge; such as the domestic 80-1 type, LXJ-II type. Centrifugal speed and relative centrifugal force are measured, as shown in Figure 2-37.
Ordinary centrifuge rotors operate at room temperature, the temperature inside the rotor chamber is generally uncontrollable, and the rotor has a fixed angle and suspension suspension format.
The solid precipitate formed during centrifugation is called a platen, and the liquid phase is called the supernatant. The two phases were separated by a pour method.
(2) High-speed centrifuges can reach 25000r/min for high-speed centrifuges; those with rotational speeds above 25000r/min are ultracentrifuges. High speed centrifuges typically have a refrigeration unit so that the temperature in the rotor chamber can be controlled. The temperature in the rotor chamber is generally controlled at 4 ° C. . The main use of large-capacity continuous-flow centrifuges is to collect yeast and bacteria from a large number of cultures (5~500 L). The other type is a low-capacity freezing centrifuge with many models and a maximum capacity of 3L. These centrifuges have a variety of internally convertible angle and flattening rotors, which are used to collect microorganisms, cell debris, cells, large organelles, ammonium sulfate precipitates, crude extracts of immunoprecipitase enzymes, and the like. It is important to note that the sample height should be set according to the type of rotor and the number of samples before each centrifugation.
(3) Ultra-high-speed centrifuges General and high-speed centrifuges are mainly used to separate and prepare biological macromolecular substances and subcellular components; ultracentrifuges have a centrifugal force of more than 500,000 × g, which can separate the subcellular organelles and can be used to separate viruses. It can be used to determine the relative molecular mass of proteins, nucleic acids, and the like.
Depending on the function, it can be divided into a preparative ultracentrifuge and an analytical ultracentrifuge.
Since the high speed generates a large amount of heat, the centrifuge is equipped with a freezer to reduce the temperature inside the rotor chamber. Simultaneously. The rotor is operated under vacuum to reduce friction. For analytical centrifugation, the solid phase particle sedimentation process must be monitored. To this end, the ultracentrifuge is equipped with an optical system that is perpendicular to the centrifuge tube and passes through the solution in the centrifuge tube to simultaneously measure the optical density or light transmittance. It can also detect the sedimentation velocity and moving interface of the settled particles. These measurements help to analyze the condition of the sample.
1 preparative ultracentrifuge:
It mainly consists of four parts: drive and speed control, temperature control, vacuum system and rotor.
Drive and speed control: The drive of most ultracentrifuges consists of a water-cooled or air-cooled motor that is shifted by a precision gearbox or belt, or directly connected to a rotor shaft with a variable frequency motor. Since the diameter of the drive shaft is only 0.476 cm, the fine shaft can be flexed flexibly during rotation to accommodate the slight imbalance of the rotor without causing vibration or uranium damage. The varistor and the controller with the rotator are used to select the speed of the rotor. In addition to the speed control system, there is an overspeed protection system to prevent the rotor from tearing or exploding caused by the speed exceeding the maximum specified speed of the rotor. For this purpose, the centrifugal chamber is always sealed with an armor plate that can withstand such an explosion.
Temperature control: The temperature control is to directly and continuously monitor the temperature of the rotor by the infrared radiation sensor placed under the rotor to ensure more accurate and sensitive temperature regulation.
Vacuum system: When the speed exceeds 4000 r/min, the frictional heat between the air and the rotating shaft becomes a serious problem. In order to eliminate this heat source. Ultracentrifuges generally add a vacuum system. The centrifuge chamber was sealed and evacuated by two vacuum pump systems operating in series. The first working pump is the same as the mechanical vacuum pump in the general laboratory, which can be evacuated to 13.33~666Pa. Once the pressure in the centrifugal chamber is reduced below 33.33 Pa, the water-cooled diffusion pump also starts to work. With these two pumps, the vacuum can be achieved and maintained at 0.13~0.26Pa. In the case of reduced friction, the speed is likely to rise to the required number of revolutions.
Rotor: There are various types of rotors used in preparative ultracentrifuges. Generally can be divided into two categories: angle rotor and 甩 flat rotor. The angle between the corner of the angled rotor and the axis of rotation is between 20 and 45 degrees. The advantage of this type of rotor is that it has a large capacity and a high speed. Another type of flat-type rotor consists of six freely movable buckets (centrifugal sleeves) suspended from a rotating head. When the rotor is stationary, these hangers are vertically suspended; when the rotor reaches the speed of 200~800 r/min under the action of centrifugal force. The bucket is flattened to a horizontal position.
This rotor is mainly designed for density gradient sedimentation. The main advantage is that the gradient material can be placed in a centrifuge tube that is kept vertical, while the tube remains level during centrifugation. Samples that settled in different locations in the horizontal position in the horizontal position exhibited a strip across the centrifuge tube, rather than being angled as in an angled rotor. Therefore, when removing the centrifuge tube from the rotor. The settling component is not repositioned as in the corner rotor. The disadvantages of this type of rotor are: the long time between the formation of the zone, and the 2 analytical ultracentrifuge analytical ultracentrifuge uses a specially designed rotor and detection system to continuously monitor the substance in a centrifugal field. Settling process.
The turret of an analytical ultracentrifuge is elliptical and the turret is coupled to a high speed drive through an innate shaft. The rotor rotates in a frozen and vacuum gel. There are 2~6 chambers with centrifuge cups on the rotor. The cups are fan-shaped and can be transmitted up and down. The centrifuge is equipped with an optical system that monitors the settled material in the centrifuge cup by UV absorption or refractive index changes throughout the centrifugation. At a predetermined time, a photograph of the sedimentation material can be taken, and during the sedimentation of the material in the cup, the interface formed between the heavy particles and the light particles acts like a refractive lens, and a "peak" is generated on the photographic substrate of the detection system. As the sedimentation continues, the interface advances before it is asked, so the peak also moves. An index of the rate of material settling can be obtained from the speed at which the peak moves.
Analytical ultracentrifuges are mainly used for the determination of relative molecular mass of biological macromolecules, estimating the purity of the sample compartment and detecting changes in the conformation of biological macromolecules.
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