Shanghai Xinzhuang Laboratory is divided into a preparative centrifuge and an analytical centrifuge by a centrifuge. The preparative centrifuge is mainly used to separate various biological materials. The sample volume of each separation is relatively large, and the analytical centrifuge generally has The optical system is mainly used to study the physical and chemical properties of pure biological macromolecules and particles. According to the behavior of the substance to be tested in the centrifugal field (continuous monitoring by the optical system in the centrifuge), the purity, shape and molecular weight of the substance can be inferred. . Analytical centrifuges are all ultracentrifuges.
1. Preparative centrifuges can be divided into three categories:
(1) Ordinary centrifuge: the maximum speed is about 6000 rpm, the maximum relative centrifugal force is nearly 6000×g, and the capacity is several tens of milliliters to several liters. The separation form is solid-liquid sedimentation separation, the rotor has angular and external pendulum type, and its rotation speed cannot be strictly controlled. Usually, without a freezing system, it is operated at room temperature to collect large particles that are easy to settle, such as red blood cells, yeast cells, and the like. The centrifuge is driven by an AC commutator motor. The carbon brush of the motor is easy to wear. The speed is regulated by a voltage regulator. The starting current is large and the speed is not uniform. The general rotor is placed on a hard steel shaft. It is extremely important to accurately balance the tube and contents, otherwise the centrifuge will be damaged.
(2) High-speed refrigerated centrifuge: the maximum speed is 20,000 ~ 25000 rpm (r / min), the maximum relative centrifugal force is 8900 × g, the maximum capacity can reach 3 liters, the separation form is also solid-liquid sedimentation separation, the rotor is equipped with various angles Head, slewing rotor, zone rotor, vertical rotor and large capacity continuous flow rotor, generally have a refrigeration system to eliminate the heat generated by the friction between the high speed rotating rotor and the air, the temperature of the centrifuge chamber Can be adjusted and maintained at 0 ~ 40C, speed, temperature and time can be strictly and accurately controlled, and has a pointer or digital display, usually used for microbial cells, cell debris, large organelles, ammonium sulfate precipitation and immunoprecipitate Isolation and purification work, but can not effectively settle the virus, small organelles (such as nucleoprotein bodies) or individual molecules.
(3) Ultracentrifuge: The rotation speed can reach 50,000 to 80,000 rpm, and the relative centrifugal force can reach up to 510,000×g. The most famous manufacturers include Beckman in the United States and Hitachi in Japan. The centrifugal capacity is from tens of milliliters to 2 liters. The form is differential sedimentation separation and density gradient zone separation, and the centrifuge tube balance allows an error of less than 0.1 gram. The advent of ultracentrifuges has enabled a new expansion in the field of biological sciences. It enables the separation of subcellular organelles observed in electron microscopy in the past, as well as the isolation of viruses, nucleic acids, proteins and polysaccharides.
The ultracentrifuge consists of four parts: drive and speed control, temperature control, vacuum system and rotor. The driving device of the ultracentrifuge is driven by a water-cooled or air-cooled motor through a precision gear box or belt, or directly driven by a variable frequency induction motor, and controlled by a microcomputer. Since the diameter of the drive shaft is thin, the thin shaft is rotated. It can have a certain elastic bending to adapt to the slight imbalance of the rotor without causing vibration or shaft damage. In addition to the speed control system, there is an overspeed protection system to prevent the rotation speed from exceeding the maximum specified speed of the rotor. This causes a tear or explosion of the rotor. For this purpose, the centrifugal chamber is sealed with an armor plate that can withstand such an explosion.
The temperature control is to directly and continuously monitor the temperature of the centrifugal chamber by the infrared radiation sensor installed under the rotor to ensure more accurate and sensitive temperature regulation. This infrared temperature control is more sensitive than the thermocouple control device of the high-speed centrifuge. more acurrate.
The ultracentrifuge is equipped with a vacuum system, which is the main difference between it and the high speed centrifuge. When the speed of the centrifuge is below 2000 rpm, the friction between the air and the rotating rotor produces only a small amount of heat. When the speed exceeds 20000 rpm, the heat generated by the friction increases remarkably. When the speed is above 40,000 rpm, the heat generated by the friction is generated. This becomes a serious problem. For this reason, the centrifugal chamber is sealed and vacuumed by a vacuum pump system in which the mechanical pump and the diffusion pump are operated in series, the temperature change is easily controlled, and the friction force is small, so that the required ultra-high rotational speed can be achieved.
2. Turn your head:
(1) Angle rotor: The angle rotor refers to the rotor with a certain inclination angle of the centrifuge tube and the rotating shaft. It is made of a complete piece of metal with 4 to 12 mechanical holes for the centrifuge tube, ie the centrifuge tube, the angle between the central axis of the hole and the axis of rotation is between 20 and 40 degrees, angle The larger the settlement, the stronger the separation and the better the separation effect. The advantage of this type of rotor is that it has a large capacity, a low center of gravity, a balanced operation, and a long service life. When the particles settle, they first collide with the centrifugal tube in the direction of centrifugal force, and then slide along the tube wall to the bottom of the tube, so the tube Particle deposition occurs on one side. This phenomenon is called “wall effectâ€. The wall effect easily disturbs the convection caused by sudden shifting of the settled particles and affects the separation effect.
(2) Horizontal rotor: This rotor consists of 4 or 6 freely movable buckets (centrifugal sleeves) that are suspended. When the rotor is stationary, the bucket is suspended vertically. When the rotor speed reaches 200 to 800 revolutions per minute, the bucket swings to the horizontal position. This rotor is most suitable for density gradient zone centrifugation. The advantage is that the gradient material can be placed. In a vertical centrifuge tube, the sample strip separated during centrifugation is perpendicular to the longitudinal axis of the centrifuge tube, unlike the interface of the sample sediment in the angled rotor head at an angle to the centrifuge tube, which facilitates centrifugation after the end of centrifugation. The layers were taken out of each sample strip that had been separated. The disadvantage is that the sedimentation distance of the particles is long and the time required for centrifugation is also long.
(3) Zone turret: The zone turret has no centrifuge tube, and is mainly composed of a rotor barrel and a retractable top cover. The rotor barrel is provided with a cross-shaped baffle device to divide the barrel into four or more sectors. The chamber has a conduit therein, and the gradient or sample liquid is pumped from the inlet tube in the center of the rotor, through which the tubes are distributed around the rotor, and the separator in the rotor maintains the stability of the sample strip and the gradient medium. The sedimentation of the settled sample particles in the zone rotor is different from that of the angle and the external pendulum rotor. Under the radial scattering centrifugal force, the sedimentation distance of the particles is constant, so the "wall effect" of the zone rotor is It is very small, can avoid the disorder of zone and sedimentation particles, has good separation effect, and has the advantages of high rotation speed, large capacity, easy recovery gradient and no influence on resolution, making ultracentrifugation possible for preparation and industrial production. The disadvantage of the zone rotor is that the sample and the medium are in direct contact with the rotor, which has high corrosion resistance requirements and complicated operation.
(4) Vertical rotor: The centrifuge tube is placed vertically, the sedimentation distance of the sample particles is the shortest, and the time required for centrifugation is also short. It is suitable for centrifugation in the density gradient zone. After the centrifugation, the liquid level and the sample zone should be turned 90 degrees. Therefore, the speed is slower.
(5) Continuous flow rotor: It can be used for the concentration and separation of a large number of culture fluids or extracts. The rotor is similar to the zone rotor. It consists of a rotor barrel and a rotor cover and an auxiliary device with inlet and outlet. The inlet continuously flows into the rotor, and under the action of centrifugal force, the suspended particles settle on the rotor barrel wall, and the supernatant flows out from the outlet.
3. Centrifuge tube:
The centrifuge tube is mainly made of plastic and stainless steel. The commonly used materials for the plastic centrifuge tube are polyethylene (PE), polycarbonate (PC), polypropylene (PP), etc., wherein the PP tube has good performance. Plastic centrifuge tubes have the advantage of being transparent (or translucent) with low hardness and can be removed by puncture. The disadvantage is that it is easy to deform, has poor corrosion resistance to organic solvents, and has a short service life.
The stainless steel tube has high strength, no deformation, heat resistance, frost resistance and chemical resistance. However, it should also avoid contact with highly corrosive chemicals such as strong acids and alkalis.
The plastic centrifuge tube has a tube cover, and the tube cover must be tightly closed before being centrifuged, and the liquid is inverted without leakage. The cap has three functions:
1 Prevent sample leakage. This is especially important when used with radioactive or highly corrosive samples. 2 Prevent the sample from volatilizing. 3 Support the centrifuge tube to prevent deformation of the centrifuge tube.
4. Analytical centrifuge:
The analytical centrifuge uses a specially designed rotor and optical inspection system to continuously monitor the sedimentation of the material in a centrifugal field. Thereby determining its physical properties.
The turret of the analytical ultracentrifuge is elliptical to avoid stress concentration at the holes. The rotor is coupled to a high speed drive through a flexible shaft that rotates in a freezer and vacuum chamber with 2 to 6 chambers containing centrifuge cups. The centrifuge cup is fan shaped quartz. The light can be up and down. The centrifuge is equipped with an optical system. During the whole centrifugation, the material settled in the centrifuge cup can be monitored by ultraviolet absorption or refractive index change, and a photograph of the sedimentation material can be taken during a predetermined period. When analyzing the sedimentation of the material in the centrifuge cup, the interface formed between the heavy particles and the light particles acts like a refractive lens, resulting in a "peak" on the photographic substrate of the detection system. Pushing forward, so the peak also moves, and the speed at which the peak moves can be calculated as the settling velocity of the sample particles.
Analytical ultracentrifuge and its main feature is that it can get some important information in a short time with a small amount of sample, can determine the existence of biomacromolecules, its approximate content, calculate the sedimentation coefficient of biomacromolecules, combined with interface diffusion, Estimating the size of the molecule, detecting the heterogeneity of the molecule and the proportion of each component in the mixture, determining the molecular weight of the biomacromolecule, and detecting the conformational change of the biomacromolecule.
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