During the experiment, due to the differences in various properties of the samples, only by selecting the correct centrifugation method can the expected separation and purification results be obtained. The commonly used centrifugation methods include differential centrifugation and density gradient centrifugation. The density gradient centrifugation method can be further divided into rate zone centrifugation and equal density gradient centrifugation. In this issue, we will introduce the centrifugal method of differential centrifugation in detail.

Differential centrifugation method of centrifugation

Differential velocity centrifugation method, also known as centrifugal force differential separation method.

Principle of differential centrifugation method

By utilizing the differences in sedimentation coefficients of each component in the sample, different particles are subjected to different centrifugal forces. After multiple centrifugations, the centrifugal speed gradually increases, and different particles are sequentially settled to achieve centrifugal separation.

The principle of differential centrifugation can be explained by the expression of centrifugal force: F=ma, where a=ω 2R.

Due to the small mass of small particles, a large centrifugal force is required for separation. To meet the requirements of high centrifugal force, it is necessary to increase its rotational speed in order to separate. Separating particles of different sizes using different centrifugal forces is a dynamic separation method, especially for particles with significant differences in settling velocity.

If there are three different particle sizes in the separated sample, namely large, medium, and small, and different centrifugal forces are applied to them, the larger particles will settle first due to their larger mass; Separate the supernatant and perform a second centrifugation at a higher speed to separate the intermediate particles; Take the supernatant again with greater centrifugal force and higher rotation speed, and then settle the small particles to achieve separation of different particles, as shown in Figure 1.

Figure 1 Differential centrifugation

Due to the presence of some secondary particles in the precipitate of each particle, in order to purify a certain particle, it is necessary to dilute the precipitate of that particle and then centrifuge it for precipitation, in order to ultimately prepare particles of ideal purity.

Advantages and disadvantages of differential centrifugation method

Differential centrifugation is mainly used to separate particles with significant differences in diameter and density. Its advantages include short separation time, high repeatability, large sample processing capacity, and can be used for initial separation of a large number of samples.

However, when using differential centrifugation to separate complex samples, or when high purity requirements are needed for separation, multiple centrifugations may be required, making the operation complicated; Secondly, due to multiple cleaning, dissolution, and re precipitation of the precipitate, it is easy to cause intermediate losses, which can lead to poor centrifugal resolution; In addition, in actual separation, due to convection, diffusion during centrifugation, and pollution during sediment collection, some components with similar sedimentation coefficients cannot be separated and purified. Due to the limitations of differential centrifugation in terms of sample purity and recovery rate, it is mainly used for preliminary separation and purification of a large number of samples, in preparation for further detection or experimentation.

For example, we can perform multiple centrifugation and precipitation/supernatant collection steps on animal liver tissue homogenates with different relative centrifugal forces and times to obtain preliminary separation, enrichment, and precipitation of sample nuclei, mitochondria, lysosomes, ribosomes, etc., which can be used for other detection or fine purification experiments in the next step. The specific processing procedure is as follows:

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