What Is Ultracentrifugation? [A Primer]

Ultracentrifugation is an advanced technique used in laboratories to separate and analyze complex mixtures of biological molecules or particles based on their size, shape, density or molecular structure. By spinning samples at extremely high speeds, this method enables scientists and researchers to isolate different components, including cells, proteins and viruses. Ultracentrifugation is a pivotal process in scientific research, helping to advance studies in drug development, disease research, biotechnology and more.

This article will provide an in-depth look at ultracentrifugation, including how it works, its practical applications, how to care for your ultracentrifuge and more.

What Is Ultracentrifugation?

As described in our introduction, ultracentrifugation is a highly specialized laboratory technique that uses high centrifugal forces and intense rotational speeds to separate small particles based on their size, shape and density. Ultracentrifuges — the instruments that perform this process — can spin samples at speeds between 80,000 and 100,000 rotations per minute (RPM), generating centrifugal forces of up to 800,000 x g. This immense force separates biological particles as small as macromolecules such as proteins and nucleic acids, which would be challenging or otherwise impossible to isolate with traditional centrifugation methods.

Ultracentrifugation relies on the principle of sedimentation, wherein particles move through a medium — typically a liquid — under centrifugal force. The rate at which a particle settles (sediments) is primarily determined by its sedimentation coefficient, or s-value, which is influenced by its size, shape and density relative to the surrounding medium. Denser or larger particles sediment faster, while smaller or lighter particles take longer to settle or may remain suspended in the solution.

The ultracentrifugation process is as follows:

1. An operator prepares samples in sealed tubes or containers balanced with precision to ensure stable operation at high speeds. It’s common during sample preparation to use a gradient medium to further enhance particle separation based on their physical properties.
2. Once the operator has loaded the sample into the ultracentrifuge, the machine accelerates the rotor to the designated speed, generating the required centrifugal force. The intense rotational speed forces particles to move outward from the center of rotation, driving denser components to migrate to the bottom of the tube, while less dense components remain near the top.
3. During ultracentrifugation, the particles separate according to their respective sedimentation rates, creating distinctive layers or bands within the tubes. These layers can then be individually extracted for further analysis, purification or use in downstream applications.

Ultracentrifugation typically operates at controlled, low temperatures to prevent degradation of sensitive biological materials, such as proteins or nucleic acids, ensuring sample integrity throughout the procedure. Many ultracentrifuges are also equipped with a vacuum system to minimize air resistance and friction, allowing for more efficient acceleration and reducing the risk of rotor damage during high-speed operation.

Ultracentrifugation is a vital technique across various fields of life sciences, including molecular biology, biochemistry and virology. It’s frequently employed for applications such as protein purification, isolation of organelles, viral particle separation and DNA/RNA fractionation. Its ability to precisely separate subcellular components make it indispensable in both basic research and industrial settings, such as pharmaceutical manufacturing and biotechnology, where product purity and sample integrity are paramount.

Centrifugation vs. Ultracentrifugation

Though both ultracentrifugation and centrifugation separate components within a mixture using centrifugal force, they differ significantly in terms of the forces they generate and the types of particles they’re intended to isolate, making each method suited for different applications.

Centrifuges operate at relatively low speeds, typically up to 30,000 RPM. These speeds produce centrifugal forces of up to 110,500 x g, which is sufficient for separating larger particles such as cells, cell debris or even large organelles such as mitochondria. The process involves spinning a sample in a centrifuge, where denser components move outward, forming a pellet at the bottom of the tube, while lighter components remain in the liquid phase.

In contrast, ultracentrifugation operates at much higher speeds, generating extreme centrifugal forces that allow for the separation of much smaller particles, such as viruses, ribosomes, proteins and nucleic acids. Ultracentrifugation relies on similar principles as standard centrifugation but offers a higher level of precision. It also requires more complex sample preparation, often utilizing gradient mediums such as sucrose or cesium chloride to further enhance particle separation.

While centrifugation is effective for more routine applications involving larger components, ultracentrifugation provides the speed and precision needed for separating much smaller, more delicate particles.

The Pros and Cons of Ultracentrifugation

Ultracentrifugation is a cornerstone of laboratory research and has proven indispensable across various scientific fields due to the following:

• Precision: Ultracentrifugation provides exceptional resolution, enabling the separation of particles based on subtle differences in size, shape and density — a critical factor for researchers who need to isolate subcellular components that cannot be effectively separated using standard centrifugation.
• Versatility: Ultracentrifuges are versatile tools capable of supporting a wide range of research and industrial applications. From molecular biology and virology to pharmaceutical development and genomics, ultracentrifugation plays an essential role in everything from basic biological research to therapeutic development. Its ability to separate biomolecules of different sizes and densities makes it essential for characterizing macromolecules, studying protein interactions and preparing viral vectors for vaccines.
• Sample Purity: Ultracentrifugation is able to achieve high levels of sample purity through the use of gradient mediums, which enable researchers to carefully layer samples by density and ensure biomolecular fractions remain separate. This level of purity is crucial for downstream applications where contamination could interfere with experimental results or product efficacy.
• Control: Modern ultracentrifuges offer sophisticated control options, including programmable speeds, temperature regulation and vacuum systems that minimize friction and sample degradation. These features ensure that sensitive biological samples are maintained in optimal conditions throughout the separation process.

But ultracentrifugation is not without its challenges. It demands meticulous sample preparation and technical expertise and can be a time-intensive process, especially when compared to standard centrifugation.

Ultracentrifuges are also sophisticated pieces of equipment whose high-speed operation generates significant mechanical stress. As a result, they require careful calibration and rotor inspections to ensure proper performance. Even minor issues, such as rotor imbalance or improper vacuum seals can affect the performance of an ultracentrifuge and compromise the integrity of an experiment, making routine maintenance essential.

Ultracentrifugation Techniques

There are two primary types of ultracentrifuges — analytical ultracentrifuges and preparative ultracentrifuges — each of which serve different purposes.

Analytical ultracentrifuges (AUCs) are used to study the properties of macromolecules in a solution rather than merely separate their components. These instruments allow for the real-time monitoring of sedimentation behavior, providing insights into molecular weight, shape, size and interactions between biomolecules such as proteins, nucleic acids and lipids. AUCs are equipped with optical detection systems — typically either absorbance or interference options — which enable researchers to track sedimentation profiles as the sample is spun at high speeds. This continuous observation provides valuable information about molecular interactions, aggregation states and conformational changes of biomolecules.

By comparison, preparative ultracentrifuges are used primarily for separating and isolating different components within a mixture based on their size, density and shape. Preparative ultracentrifugation focuses on the physical separation of particles, allowing for the collection of purified fractions for downstream experiments or applications. Preparative centrifuges do not feature real-time monitoring systems like AUCs but can accommodate larger sample volumes. These instruments are also equipped with different types of rotors, such as fixed-angle, swinging-bucket and vertical rotors, to optimize the separation process for specific types of particles.

In addition to two types of ultracentrifuges, there are also multiple ultracentrifugation techniques, each with their own set of applications. These include:

• Differential Ultracentrifugation: One of the most basic techniques, differential ultracentrifugation separates particles based on their size and density by applying sequential centrifugation at progressively higher speeds. After each spin, the pellet (the denser particles) and the supernatant (the lighter particles) are separated. This method is commonly used to isolate organelles from homogenized cell samples, to separate larger cellular debris from smaller macromolecules and to purify specific subcellular components for biochemical analysis.
• Sedimentation Velocity Ultracentrifugation: Sedimentation velocity ultracentrifugation (SV-AUC) is an analytical technique that measures the rate at which particles move through a solvent under centrifugal force. The speed at which a particle sediments depends on its size, shape and density, as well as the viscosity of the solvent. This technique provides insights into the size distribution of particles in a solution and is commonly used to determine molecular weights and conformational changes, to analyze protein complexes and to study molecular heterogeneity and aggregation states.
• Sedimentation Equilibrium Ultracentrifugation: Sedimentation equilibrium ultracentrifugation (SE-AUC) is another analytical technique, but, unlike SV-AUC, it measures the equilibrium state where centrifugal force and diffusion balance out. SE-AUC is used to determine the molecular weight of macromolecules without requiring prior knowledge of their shape. This method is often used to determine molecular weight, study reversible interactions and complex formation and investigate protein-ligand and protein-protein interactions in biophysical studies.
• Density Gradient Ultracentrifugation: A powerful technique that separates particles based on their buoyant density, density gradient ultracentrifugation involves spinning samples in a gradient medium that forms layers of increasing density. Particles migrate to the point in the gradient where their density matches that of the surrounding medium, creating distinct bands that can be isolated.There are two main types of density gradient ultracentrifugation: isopycnic (equilibrium) centrifugation and rate-zonal (velocity) centrifugation.
  • Isopycnic centrifugation separates particles based on their buoyant density alone. Samples are spun until the particles reach the point where their density matches that of the gradient medium. This technique is widely used for purifying nucleic acids, isolating viruses and fractionating organelles and membrane vesicles in cellular studies. Since particles migrate to their equilibrium positions regardless of size, this method ensures high purity and is ideal for tasks requiring precise separation based on density differences.
  • Rate-zonal centrifugation separates particles based on their size and shape as they move through a gradient of decreasing density. In this technique, the sample is layered on top of a pre-formed gradient, and centrifugation is applied for a set period. Larger particles sediment more quickly than smaller ones, allowing for separation by size; however, operators must take care to stop the centrifuge before the particles reach the bottom of the tube. Rate-zonal centrifugation is commonly used for protein and virus purification, studying macromolecular complexes and subcellular fractionation.

How to Care for an Ultracentrifuge

Proper use and maintenance of your ultracentrifuge is vital to ensuring reliable performance, sample integrity and the longevity of the equipment. Following the best practices listed below reduces the risk of equipment damage, safety hazards and compromised experiments:

Proper Equipment Use

• Sample Preparation and Loading: Before running an ultracentrifuge, verify that samples are properly sealed in tubes specifically designed to withstand high speeds. Depending on the ultracentrifugation technique, you may need to prepare gradient solutions and layer them carefully to avoid mixing. It’s equally important to balance each sample tube against another tube with equal volume and mass to prevent motor imbalances during operation. Even a small imbalance can cause mechanical strain or equipment damage, so use an accurate balance to verify tube weights before placing them in the rotor.
• Choosing the Right Rotor: Different types of rotors are designed for specific separation tasks. For example, swinging-bucket rotors are ideal for rate-zonal centrifugation, while fixed-angle rotors are well-suited for differential ultracentrifugation. If you’re unsure which rotor to select for a particular ultracentrifugation technique, consult the manufacturer’s instructions for use (IFUs) and your protocol requirements. Be sure to check that the rotor is properly secured before starting the ultracentrifuge.
• Setting Parameters: Before starting any experiment, consult your protocol to determine the appropriate centrifugal force (often expressed in xg) and use the ultracentrifuge’s settings to convert that to the necessary RPM for your specific rotor. If your experiment requires temperature control, be sure to set the temperature according to the sample’s needs and ensure the ultracentrifuge’s refrigeration system is working properly.
• Safety Precautions: Double-check to confirm that the rotor lid is securely fastened and that the chamber is properly sealed. Ultracentrifuges generate extreme force, and any failure in the rotor or an imbalance in the tubes can lead to serious accidents.

Maintenance and Ongoing Care

• Regular Cleaning: Spills, leaks or other residual materials inside the rotor chamber can damage the equipment or lead to contamination between runs, so be sure to clean both the rotor and chamber after each use with a non-abrasive, mild detergent and a soft cloth. Immediate cleaning is especially crucial for gradient separation protocols that use dense mediums such as cesium chloride, as these substances can crystallize and cause damage.
• Rotor Care: The rotor is a high-stress component and requires regular inspections for signs of wear, such as scratches, cracks or corrosion — all of which can compromise its structural integrity. Most rotors come with a lifespan measured in the number of ultracentrifugation runs or hours of use, so maintain detailed records of rotor use. Never exceed the number of runs listed in the manufacturer’s IFUs and replace the rotor if you detect any damage. When not in use, store rotors in a dry, temperature-controlled environment to prevent degradation or corrosion.
• Lubrication and Sealing: Regularly inspect the ultracentrifuge’s lid and rotor seals, ensuring that the O-ring and gaskets are in good condition. Replace parts when they begin to appear worn and properly lubricate moving parts, such as the rotor lid lock and hinges, to maintain smooth operation and prevent mechanical wear.
• Vacuum System Maintenance: Most ultracentrifuges are equipped with vacuum systems to reduce air resistance during high-speed spins. Regularly check this pump for performance issues and confirm the seals are functioning correctly. If this vacuum system is compromised, it can reduce the efficiency of the ultracentrifugation process or cause overheating, putting sensitive samples at risk.
• Calibration and Preventative Maintenance: Routine calibration ensures that the RPM and g-force settings remain accurate, which is critical for reproducible results. Schedule professional calibration and equipment servicing at least once per year, following the manufacturer’s IFUs.

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