Centrifugation is a process that involves spinning a mixture at a high speed to separate its contents based on density. It is used at different speeds for various separation needs. While low-speed centrifugation assists in whole-cell pelleting, isolating subcellular biomolecules requires higher speed centrifugation up to and exceeding 100,000 rotations per minute (RPM). This method is called ultracentrifugation. It allows particles at different densities to sediment at different rates based on their physical properties while keeping the mixture's temperature under control. Ultracentrifugation is essential in biomedical applications for its ability to purify proteins, nucleic acids, organelles and viruses. It can also be used to study the structures of these molecules.

Principles of Ultracentrifugation

Similarly to a standard centrifuge, an ultracentrifuge follows the principle of sedimentation, suggesting that high-speed spinning causes particles in a solution to experience a force that repels them from the axis of rotation. This force, known as the centrifugal force, is proportional to the molecular weight of the particle, the radius of the rotor and the square of the angular velocity (i.e., how fast the particle rotates around the axis).

The liquid also exerts opposing forces on the particles, such as friction and buoyant forces. These forces have a significant impact on the sedimentation order. For particles denser than the fluid, the centrifugal force easily exceeds the buoyant force, allowing rapid sedimentation towards the bottom edge of the tube. On the other hand, less dense particles may remain suspended or float towards the surface, while no sedimentation occurs if the particle and the fluid have the same density. Eventually, large and dense particles rapidly aggregate at the bottom, while the smaller and lighter ones remain suspended at different levels inside the liquid.

This principle was studied by the Swedish chemist Theodor Svedberg, who won the Nobel Prize for his work on ultracentrifugation. He also invented the sedimentation coefficient, or Svalbard Unit, to measure particle size based on its sedimentation velocity.¹

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Ultracentrifugation vs High-speed Centrifugation

It is important to note that while ultracentrifuges and high-speed centrifuges both use centrifugal forces instead of gravity-based sedimentation, they should not be confused. High-speed centrifuges operate between 15,000 – 30,000 rpm, while ultracentrifuges can exceed 100,000 rpm. Although high-speed centrifuges can handle larger sample volumes, the extreme speed employed in ultracentrifugation makes it more efficient at separating small particles like viruses than high-speed centrifugation.

Types of Ultracentrifugation

Preparative and analytical are the two main ultracentrifuge types, each serving a distinct purpose.

Analytical ultracentrifugation (AUC) is used to analyze the physical properties of the particles in a liquid mixture. Analytical ultracentrifuges have detection devices that monitor particle spinning velocity and location in real-time through absorbance or interference optics. Analytical ultracentrifugation features two modes of analysis. Sedimentation velocity leverages high-speed spinning to infer particle size and shape from the sedimentation rates of particles, revealing sedimentation coefficients, the heterogeneity of the sample, and molecular interactions, such as protein-protein binding. In contrast, the sedimentation equilibrium mode operates at a lower spinning speed to generate an equilibrium state where sedimentation and diffusion balance each other. Thus, the molar masses of analytes and equilibrium constants of interacting analytes can be calculated.

Preparative ultracentrifugation is solely used for isolating and purifying desired macromolecules from their mixture. Although they do not feature the real-time detection capacities of AUCs, they allow precise separation of particles based on size and density, making them available for analysis after centrifugation. Thanks to their ability to process large sample volumes and compatibility with robust separation methods, preparative ultracentrifuges are essential in purifying viruses, nucleic acids and proteins for downstream vaccine development or gene therapy applications.

Ultracentrifugation Techniques and Methods

Preparative ultracentrifugation can be conducted in three different modes to separate particles.

Differential ultracentrifugation

Differential ultracentrifugation involves varying the rotational speed to settle particles over multiple rounds. First, the ultracentrifuge is spun slowly to sediment the large particles. After collecting the pellet, the supernatant is spun faster to settle smaller particles. This method is beneficial for organelle isolation.²

Density gradient centrifugation

Density gradient centrifugation isolates particles at separate bands using a special medium, such as a sucrose gradient or cesium chloride. This medium generates a density gradient within the tube. Thus, different particles settle at distinct points where their density matches the density within the medium. Density gradient centrifugation offers high-resolution purification, separating particles with similar sizes and densities. Therefore, it is a powerful tool for generating high-purity viral vectors by isolating them from contaminated ones.³

Application of Ultracentrifugation in Life Sciences

Ultracentrifugation plays a vital role in life sciences by separating macromolecules, organelles, viruses and nanoparticles, which are difficult to isolate using traditional centrifugation.

Protein purification by ultracentrifugation

Ultracentrifugation is suitable for separating individual proteins and large protein complexes without the risk of denaturation. Rate-zonal centrifugation is often preferred for size-based separation of enzymes and protein biomarkers.⁴

Virus purification using ultracentrifuges

Ultracentrifuges are significant components of viral vector manufacturing protocols to eliminate residual DNA, host cell proteins and replicating viruses from the vector product. Density gradient ultracentrifugation with iodixanol or cesium chloride helps achieve high purity and yield.⁵

DNA/RNA fractionation with ultracentrifuges

DNA molecules with varying forms, such as circular, linear or supercoiled, can be segregated using isopycnic centrifugation. Ultracentrifugation is widely used for DNA or RNA isolation during plasmid DNA/RNA preparation.

Nanoparticle separation

Ultracentrifugation is integral in nanoparticle characterization. By applying high centrifugal forces, researchers decipher nanoparticle diameter and weight, which helps them optimize these particles for small molecule drug discovery, delivery and biomaterial applications.⁶

Key Components of an Ultracentrifuge System

Rotors

The rotor is the central component responsible for holding and spinning the tubes to generate centrifugal forces. Different rotor types are:

1. Swinging Bucket Rotors: Used for horizontal spinning of the tubes, ideal for forming well-defined bands in gradient separations
2. Vertical Rotors: Tubes are held in a vertical orientation parallel to the axis of rotation, which supports higher speed limits and enables rapid separation, especially in isopycnic centrifugation.
3. Fixed-angle rotors hold the tubes at 14-40 degrees to the vertical axis while spinning and are ideal for rapid pelleting.

Detection systems

Analytical ultracentrifuges comprise detection systems, such as interference and absorption optical systems, as well as control panels for real-time monitoring and analysis of particles as they sediment.

Ultracentrifuge and heating system

Temperature maintenance is vital when spinning samples at extreme speeds. Cooling mechanisms, such as an interior refrigeration system, keep the environment at a desired temperature range to prevent the denaturation of temperature-sensitive biomolecules like enzymes.

High-speed centrifuge vacuum systems

Ultracentrifuge rotors are placed in a vacuum chamber to minimize air friction that may cause heat buildup. This allows the ultracentrifuge to reach and exceed 100,000 rpm without heat generation and mechanical wear. Vacuum systems enhance rotor longevity and ensure stable and reproducible separation.

References

1. Svedberg T. The Ultra-Centrifuge and the Study of High-Molecular Compounds. 1937.
2. Hu J, Liu Y, Du Y, Peng X, Liu Z. Cellular organelles as drug carriers for disease treatment. J Control Release 2023;363:114-135.
3. Sternisha SM, Wilson AD, Bouda E, Bhattacharya A, VerHeul R. Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation. Eur Biophys J 2023;52(4):387-392
4. Rikkert LG, Engelaer M, Hau CM, Terstappen LW, Nieuwland R, Coumans FA. Rate zonal centrifugation can partially separate platelets from platelet‐derived vesicles. Res Pract Thromb and Haemost 2020;4(6):1053-1059.
5. Janc M, Zevnik K, Dolinar A, Jakomin T, Štalekar M, Bačnik K, et al. In-depth comparison of adeno-associated virus containing fractions after CsCl ultracentrifugation gradient separation. Viruses 2024;16(8):1235.
6. Xu X, Cölfen H. Ultracentrifugation techniques for the ordering of nanoparticles. Nanomaterials 2021;11(2):333.

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