What Is an Ultracentrifuge?

A centrifuge is a device that separates liquid phase heterogeneous systems by centrifugal force. According to the different sedimentation coefficients, masses, and densities of materials, the method of applying strong centrifugal force to separate, concentrate and purify materials is called centrifugation. Generally speaking, a centrifuge with a speed above 30 000 r / min is called ultracentrifugation. Centrifugation technology, especially ultracentrifugation technology, is an indispensable means in molecular biology, biochemical research and industrial production.

Chinese name: Ultracentrifuge

Foreign name: ultracentrifuge, supercentrifuge, UCF

Introduction of Ultracentrifuge

In 1924, T.Svedberg and Rinde developed the world's first turbo ultracentrifuge, which has developed rapidly and has been widely used in various fields of scientific research and production. The maximum speed of the centrifuge rotor has reached 150,000 r / min (Beckman Optima MAX-XP, 2010 data), and the maximum centrifugal force is 1019000 X g (Beckman Optima MAX-XP, 2010 data). In addition, usually above 30,000 rpm is called ultracentrifugation. The business card above is not universal, but I can't change it there, so write here. (Butterfly only changed this paragraph, the rest will be discussed)

Centrifuges have many advantages as a means. For example, ultracentrifugation can be performed at low temperatures, protecting the activity of biological macromolecules. The preparative centrifuge has a large load, which can separate and purify several grams of samples at a time, which is much larger than the samples on chromatography and electrophoresis. Analytical centrifuges can measure not only the molecular weight of a substance, but also the purity, conformation, and sedimentation coefficient of a substance. Therefore, centrifugation technology plays an important role in biological research, and it is the most convenient and effective tool for separating and purifying cells, proteins, nucleic acids, enzymes and virus isolation.

Ultracentrifuge principle

When the suspension containing fine particles stands still, the suspended particles gradually sink due to the effect of the gravity field. The heavier the particles, the faster they sink, whereas the particles with a density lower than the liquid will float up. Particles move down in the gravitational field

Ultracentrifuge The speed of movement is related to the size, shape and density of the particles, and also to the strength of the gravity field and the viscosity of the liquid. Particles the size of red blood cells, several micrometers in diameter, can be observed to settle under normal gravity.

In addition, when the substance settles in the medium, it is accompanied by a diffusion phenomenon. Diffusion is unconditional absolute. Diffusion is inversely proportional to the mass of the substance, and the smaller the particles, the more severe the diffusion. The settlement is relative and conditional. It can only be moved by external force. The sedimentation is directly proportional to the weight of the object. The larger the particles, the faster the sedimentation. For particles smaller than a few microns, such as viruses or proteins, they are in a colloidal or semi-colloidal state in solution, and it is impossible to observe the sedimentation process using only gravity. Because the smaller the particles, the slower the sedimentation, and the more serious the diffusion phenomenon. Therefore, a strong centrifugal force needs to be used to force the particles to overcome the diffusion

Benchtop ultracentrifuge Generates settlement motion.

Centrifugation is to use the strong centrifugal force generated by the high-speed rotation of the centrifuge rotor to accelerate the sedimentation rate of particles in the liquid and separate the different sedimentation coefficients and buoyant density substances in the sample. The magnitude of the centrifugal force (F) depends on the angular velocity (, r / min) of the centrifugal rotor and the distance (r, cm) of the material particles from the mandrel. Their relationship is: F = 2R ^[1]

For convenience, F is often expressed as a relative centrifugal force, which is a multiple of gravity. That is, dividing the F value by the acceleration of gravity g (approximately equal to 9.8m / s2.) To obtain how many times the centrifugal force is gravity, called how many g. For example, the average radius of the centrifuge rotor is 6cm. When the rotation speed is 60 000 r / min, the centrifugal force is 240 000 × g, which means that the centrifugal force acting on the centrifuged substance at this time is 240,000 times the daily gravity.

Therefore, the speed r / min and the centrifugal force g are not directly proportional to each other, but also related to the radius. At the same speed, the radius is doubled, and the centrifugal force (g value) is also doubled. The relationship between the speed (r / min) and the centrifugal force (g value) can be converted using the following formula:

RCF = F centrifugal force / F gravity = m2r / mg = 2r / g = (2r / r × rpm) 2r / g (note that the units are converted into standard units uniformly)

Where: r is the radius (cm), and g is the centrifugal force (g value) ^[2]

Centrifugation method of ultracentrifuge

Overview of Ultracentrifuge

The basic principle of separating biological macromolecules and subcellular substances by centrifugation is to form different zones according to their different sedimentation speeds in liquid media or their density and stay in different positions in liquid media to separate them. One apart. The former is the sedimentation rate method. The maximum density of the liquid medium is smaller than the density of the smallest particles in the sample. The high speed and short time are used for centrifugation. The latter is the sedimentation balance method. The maximum density of the liquid medium is Greater than the density of the smallest particles in the sample, choose a lower speed and longer time for centrifugation. There are several forms of actual operation:

Ultracentrifuge

This method is to choose different centrifugation speeds to separate different components. For example, first settle large particles and supernatant with low speed, settle medium particles with high-speed centrifugal supernatant, and settle small particles with ultracentrifuge. The method of increasing the centrifugal force to separate the upper fluid was used to separate particles of different sizes.

This method is relatively simple and convenient. The separated components sink to the bottom of the tube, so the desired components can be concentrated quickly and the volume can be reduced. However, there is a contradiction between recovery rate and purity. To increase the recovery rate, it is bound to increase the speed or centrifugation time, so that particles of similar size will sink to the bottom of the tube. Therefore, this method is mostly used for crude purification or preliminary concentration of samples.

() Ultracentrifuge speed zone (or speed gradient) centrifugation

In this method, the sample is placed on a continuous density gradient liquid. By centrifugation, large particles settle quickly and small particles settle slowly. Over time, the same particles are at the same depth

A band is formed, so the various components are separated. It is suitable for separating substances with the same density and different sizes. For example, the density of different protein components is similar, but the molecular weights are different. It can be easily separated by this method. However, it is not easy to separate substances with different densities and similar sizes.

Sucrose gradient is a commonly used medium. Different concentrations of sucrose solutions (5% -60%) were used to prepare gradients for gradient centrifugation. Then collect the components in different zones separately.

Ultracentrifuge sedimentation balance centrifugation

This is the static method. A liquid gradient is formed in the centrifuge tube. During centrifugation, each component sinks at different speeds, but eventually stays in the same density as itself, forming a narrow equilibrium band and maintaining relative stability. In order for all components of the sample to reach their equilibrium position, long centrifugation is required. This method is suitable for separating similar-sized but different-density substances, such as nucleic acid. Cesium chloride gradient is a medium commonly used for equilibrium centrifugation. The resolution is very high, and components with a density difference of 0.05 can be distinguished. However, the centrifugation time requires dozens to tens of hours, and the price is expensive. A polysucrose solution under the trade name "Percoll" has been used in place of the cesium chloride gradient. The medium can quickly form a gradient, which can greatly reduce the centrifugation time to 1-2h and still have a good separation effect. And there are matching marker beads (Density marker beads) to determine the density of the sample.

Ultracentrifuge centrifuge classification: speed, capacity and use

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According to the speed, capacity, and use, it can be roughly divided into analytical ultracentrifuge, double-speed ultracentrifuge, high-speed refrigerated centrifuge, large-capacity refrigerated low-temperature centrifuge, general low-speed centrifuge and desktop (high-speed, low-speed) centrifuge Machine and so on.

Ultracentrifuge Analytical Ultracentrifuge

The maximum speed is above 60,000r / min, and it is equipped with a suspension drive. The speed selection is very fine, and the speed control and temperature control precision are high. It is equipped with two-hole to six-hole analysis rotors made of aluminum alloy or titanium alloy. The latest Beckman Optima MAX-XP desktop ultracentrifuge currently has a maximum speed of 1,500,000 rpm (2,500 rpm), and it runs very quietly (noise is only 47dBa), it is a more advanced analysis model. Equipped with a perfect cylindrical lens optical system, interference light system and ultraviolet absorption scanning optical system, and can use specially prepared data processing microcomputer to automatically calculate physical parameters such as sedimentation coefficient and molecular weight. Older models include MSE-75 (discontinued production) dual-purpose ultracentrifuges for preparation and analysis.

Ultracentrifuge preparation type ultracentrifuge

This model has been developed into the fifth generation since it was commercialized in 1955. The maximum speed of the production overspeed off-machine can reach 83,000r / min, the maximum relative centrifugal force field (R · C · F) below S15,000 × g, the life of the driving device has reached 20 billion revolutions, and it is equipped with various speeds and speeds. Capacity aluminum alloy, titanium alloy angle rotor, horizontal rotor, vertical rotor, zone rotor and other special rotors. Each rotor is equipped with hundreds of metal or plastic centrifuge tubes on multiple sides. Ancillary equipment can optionally be equipped with optical devices for analysis, a density gradient forming-collecting instrument, a special sample-adding sampler for zone rotor operation, a density gradient pump, an integrator, and a pipe cutter. The new model has the advantages of high degree of automation (built-in microprocessor control machine), long life, low noise, no need for water cooling and low failure rate. ^[4]

High Speed Refrigerated Centrifuge

High-speed refrigerated centrifuges generally rotate in atmospheric air and have high-power drive motors and refrigeration compressors. The rated speed is between 16,000 and 21,000 r / min, and the maximum linear speed transferred does not exceed 28 Om / s (that is, below the transonic zone, the air disturbance is small and easy to control). This centrifuge is equipped with a variety of rotors made of aluminum alloy, including angle rotors, horizontal rotors, vertical tube rotors, continuous flow rotors, zone rotors, and cell elutriation rotors. The maximum centrifugal acceleration is below 55,000xg. ^[5]

Ultracentrifuge centrifuge operation steps and maintenance precautions

Ultracentrifuge operation steps

1. Turn on the power and open the centrifuge lid.

2. Assemble the centrifuge tube as required.

3. Install the centrifugal rotor as required.

4. Close the centrifuge lid.

5. Enter the centrifuge data and program the centrifuge.

6. Vacuum down and start running the program.

7. After the program is finished, go to vacuum.

8. Open the centrifuge lid and remove the rotor.

9. Remove the centrifuge tube and analyze.

Selection of ultracentrifuge centrifuge tubes

Glass centrifuge tubes must not be used on high and ultracentrifuges.
PA tube: stable chemical properties, translucent, resistant to high temperature disinfection.
PP tube: stable chemical properties, translucent, resistant to high temperature disinfection. ? PC tube: good transparency, high hardness, can withstand high temperature disinfection. But it is not resistant to strong acids and alkalis and some organic solvents. It is mainly used for centrifugation above 50,000 (rev / min). ? CN tube: The texture is soft and transparent, but it is not resistant to strong acids and alkalis and certain organic solvents, and cannot be autoclaved. Suitable for density gradient centrifugation such as sucrose and glycerin. Be transparent and facilitate collection.
Check the table: 10,000 (rev / min) RCF = 6000 and substitute it into the formula F = 1 × 6000 = 6 (kg)
50,000 (rev / min) RCF = 150000 Substitute into the formula F = 1 × 150000 = 150 (kg)
If the cap of the centrifuge tube is poorly sealed, the liquid cannot be filled (for high-speed centrifugation and using an angle head) to prevent overflow.
Consequences of spillage: contamination of the rotor and centrifugal cavity, affecting the normal operation of the sensor

Precautions for Ultracentrifuge Maintenance

1. If the lid of the centrifuge tube is poorly sealed, the liquid cannot be filled (for high-speed centrifugation and using an angle head) to prevent overflow. The spillage contaminates the rotor and centrifugal cavity and loses its balance, affecting the normal operation of the sensor. 2. During ultracentrifugation, the liquid must be filled with the centrifuge tube, and the vacuum should be evacuated when it is separated.
3. When using the angle head, do not forget to cover the rotating head cover. If it is not covered, the eddy current resistance and frictional temperature rise in the centrifugal cavity, which is equivalent to adding extra burden to the centrifuge motor and refrigerator, affecting the centrifuge. Service life. ^[6]

Precautions for Ultracentrifuge Maintenance

1. Clean the centrifuge cavity and rotor, and dry the condensation water in the cavity.
2. After use, open the door until the room temperature returns to normal. ^[7]