Common centrifugation methods used in the laboratory

(1) Differential centrifugation, also known as stepwise centrifugation, is a method in which particles with different sedimentation rates are separated step by step by using varying centrifugal speeds and centrifugation times. During operation, a suspension containing two types of particles is first centrifuged at a moderate speed to allow the larger particles to settle down. The supernatant is then carefully decanted into another centrifuge tube, after which the centrifugal force is increased and the sample is centrifuged for a specified duration to separate the smaller particles. This process is repeated several times until the desired degree of separation is achieved. Differential centrifugation is primarily used for separating particles that differ significantly in size and density. The advantages of differential centrifugation include: simplicity of operation—after centrifugation, the supernatant can be easily separated from the pellet by decanting; the use of large-capacity angular rotors is possible; short separation times and high reproducibility; and large sample throughput. However, the disadvantages include: limited resolution and poor separation efficiency—particles with sedimentation coefficients within the same order of magnitude are difficult to separate, making it impossible to obtain pure particles in a single run; severe wall effects, especially when particles are large or the concentration is high, leading to precipitation on one side of the centrifuge tube; particle compression—excessive centrifugal force or prolonged centrifugation time can cause particles to deform, aggregate, and lose activity.

Figure 1: Schematic Diagram of the Differential Centrifugation Principle

(2) Density Gradient Centrifugation (also known as Zonal Centrifugation): This method is further divided into rate-zonal centrifugation and isopycnic zonal centrifugation. In this technique, a sample is subjected to centrifugation in a medium containing a specific inert density gradient, allowing particles to reach certain defined positions within the gradient under a given centrifugal force, thereby achieving separation into distinct zones. The advantages of this method include: excellent resolution, superior separation efficiency, and the ability to obtain relatively pure particles in a single run; it has a broad applicability, capable of separating particles with both large and small sedimentation coefficients as well as particles with varying buoyant densities; particles remain undistorted and retain their activity, and the formation of zones is protected from mixing caused by convection. The disadvantages are: longer centrifugation times; the need to prepare a density gradient; and stringent operational requirements that can be challenging to master.
① Rate-zonal centrifugation: This method relies on the differing sedimentation rates of particles under centrifugal force. After centrifugation, particles with different sedimentation rates settle into distinct density gradient layers, forming several separate sample bands that are thereby separated from one another. Commonly used separation media in clinical practice include Ficoll, Percoll, and sucrose. To isolate mononuclear cells from venous blood, the first separation medium divides the mononuclear cells (lymphocytes and monocytes) in the blood into a single layer and simultaneously extracts them. In contrast, the Percoll separation medium separates lymphocytes and monocytes into two distinct gradient layers, allowing for their separate extraction. Although the latter method offers superior separation efficiency, it is more cumbersome to perform.

Figure 2: Schematic Diagram of Rate-Zone Centrifugation

② Density-Zone Centrifugation: When particles of different densities are subjected to a centrifugal force field, they either sediment downward or rise upward, continuously migrating along the density gradient until they reach a point where their densities become exactly equal—this point is known as the isopycnic point. At this point, the particles form a distinct band, hence the technique is called density-zone centrifugation. The effectiveness of density-zone centrifugation in achieving separation depends on the difference in buoyant densities among the particles: the greater the density difference, the better the separation effect. This method is independent of particle size and shape; however, the latter two factors do influence the rate and time required to reach equilibrium, as well as the width of the resulting band.

Figure 3: Schematic Diagram of Density-Zone Centrifugation

(3) Analytical Ultracentrifugation
① Working Principle of an Analytical Ultracentrifuge: The analytical ultracentrifuge consists primarily of an elliptical rotor, a vacuum system, and an optical system. The rotor is connected to a high-speed drive mechanism via a flexible shaft, which allows the rotor to establish its own axis of rotation during spinning. The rotor spins within a cryogenically cooled vacuum chamber and houses two chambers: an analysis chamber and a balancing chamber. The analysis chamber typically has a capacity of 1 ml and is arranged in a fan-like configuration within the rotor; its operating principle is similar to that of a conventional horizontal rotor. The analysis chamber is equipped with quartz windows on both its upper and lower surfaces. The optical system installed in the centrifuge ensures that the substances undergoing sedimentation within the chamber can be continuously observed throughout the entire centrifugation process. Sedimentation can be monitored either by the absorption of ultraviolet light (e.g., for proteins and DNA) or by differences in refractive index. As the substances sediment in the analysis chamber, the interface formed between heavy and light particles acts like a refracting lens, producing a “peak” on the photographic plate of the detection system. Since sedimentation continues unabated, the interface advances forward, causing the “peak” to shift as well. By measuring the speed at which this peak moves, it is possible to determine the rate of substance sedimentation.

Figure 4: Schematic Diagram of the Analytical Ultracentrifuge System

② Application scope of analytical ultracentrifugation: Determination of the relative molecular weights of biological macromolecules. Among the methods used for determining relative molecular weights, the sedimentation velocity method is the most widely applied; by recording the sedimentation process photographically, it is possible to calculate the sedimentation coefficient of the particles. Estimation of the purity of biological macromolecules. Analytical ultracentrifuges have been extensively employed in studying the purity of DNA preparations, viruses, and proteins. Detection of conformational changes in biological macromolecules. Such changes can be confirmed by examining differences in the sedimentation velocities of samples.