Ultracentrifuge Definition, Principle, Types, Uses

What is Ultracentrifuge?

• An ultracentrifuge is an exceptional piece of laboratory equipment designed to separate particles and molecules based on their density. It is a highly sophisticated and advanced version of a centrifuge, capable of achieving extremely high rotational speeds that traditional centrifuges cannot match. By harnessing these incredible speeds, an ultracentrifuge can effectively separate smaller molecules and particles that would otherwise remain mixed when subjected to lower centrifugal forces.
• The rotors in an ultracentrifuge are designed to spin at astonishing rates, ranging from 60,000 to 150,000 revolutions per minute (rpm). This rapid rotation generates immense centrifugal forces, enabling the precise separation of components based on their density gradients. The ability to reach such high speeds is a defining characteristic of the ultracentrifuge and sets it apart from other types of centrifuges.
• Ultracentrifuges are primarily utilized in well-equipped laboratories where more advanced scientific operations are conducted. Due to their larger size and complex mechanisms, they are commonly found in specialized research facilities or institutions that require sophisticated separation techniques. These powerful instruments are employed in various scientific fields, including biochemistry, molecular biology, and virology, among others.
• The operation of an ultracentrifuge can be performed in different modes. It can work in batches, where a fixed number of samples are processed simultaneously, or it can function as a continuous flow system, where samples are continuously fed into the centrifuge. The choice of operation mode depends on the specific experimental requirements and the nature of the samples being analyzed.
• Given the substantial heat generated by the high speeds of an ultracentrifuge, most of these devices are equipped with refrigeration systems. These refrigeration systems help to maintain a controlled temperature within the centrifuge, preventing excessive heat buildup that could potentially affect the samples or the integrity of the equipment itself. By managing the temperature, scientists can ensure optimal conditions for the separation process and minimize any potential heat-related issues.
• In summary, an ultracentrifuge is a cutting-edge centrifuge that operates at an incredibly high speed, facilitating the separation of smaller molecules and particles that are otherwise challenging to separate using traditional centrifugation methods. Its ability to achieve speeds ranging from 60,000 to 150,000 rpm makes it an indispensable tool in advanced laboratory settings. By incorporating refrigeration systems, ultracentrifuges effectively manage the heat generated during operation, enabling precise and controlled separations.

Definition of Ultracentrifuge

An ultracentrifuge is an advanced centrifuge that operates at extremely high speeds to separate smaller molecules and particles based on their density.

Principle of Ultracentrifuge

The principle underlying the operation of an ultracentrifuge is based on the same fundamental principle as other centrifuges. It utilizes the concept of sedimentation, where particles of varying densities settle at different rates when subjected to a gravitational force. However, in the case of an ultracentrifuge, an additional force is applied to expedite the sedimentation process.

When an ultracentrifuge is in operation, the sample is subjected to high-speed rotation around an axis. This rotation generates a perpendicular force known as the centrifugal force, which acts on the particles within the sample. As a result, larger molecules experience a greater centrifugal force and move faster, while smaller molecules are influenced to a lesser extent and move more slowly.

Simultaneously, the denser molecules within the sample are driven outward, towards the periphery of the tubes, while the less dense molecules tend to migrate towards the center of the tube. This differential movement allows for the separation of the particles based on their size and density.

Once the centrifugation process is complete, the larger and denser particles have settled down, forming compacted pellets at the bottom of the tube. In contrast, the smaller and less dense particles either remain suspended in the supernatant or float on the surface, depending on their buoyancy.

By employing this principle of sedimentation under the influence of centrifugal force, the ultracentrifuge enables the precise separation and isolation of particles based on their size, density, and sedimentation properties. This technique is particularly valuable in various scientific applications, ranging from studying the behavior of macromolecules to purifying biomolecules for research or medical purposes.

Types of Ultracentrifuge

Based on the purpose and the purpose, ultracentrifuges can be of two kinds:

1. Analytical ultracentrifuge (AUC)

• The analytical ultracentrifuge (AUC) is a specialized type of ultracentrifuge used for the analysis of various particles present in a sample. AUC, specifically analytical ultracentrifugation, is a versatile and robust method employed for the quantitative analysis of macromolecules in solution.
• Analytical ultracentrifuges are equipped with detection systems that monitor the spinning and position of particles in real-time. These detection systems enable the determination of the sedimentation coefficient, which aids in the analysis of particles based on their shape, size, and masses.
• For the determination of the relative molecular mass of a macromolecule, analytical ultracentrifugation can be performed using two main approaches: sedimentation velocity and sedimentation equilibrium. Sedimentation velocity measures the rate at which a concentration boundary of the biomolecules moves in the gravitational field, providing information about their hydrodynamic properties. Sedimentation equilibrium, on the other hand, establishes an equilibrium between sedimentation and diffusion to determine the distribution of the macromolecules’ molecular masses.
• The sedimentation coefficient obtained from AUC experiments can be used to characterize changes in the size and shape of macromolecules under different experimental conditions. It serves as a valuable parameter for studying the properties and behavior of biomolecules.
• Analytical ultracentrifuges offer various optical systems for precise and selective observation of sedimentation in real-time. These systems include absorbance, interference, and fluorescence techniques. One example of a detection system used in the ultracentrifuge is the Schlieren optical system, which observes the particles’ position and movement during centrifugation.
• The analytical ultracentrifuge is commonly employed in the determination of properties of biomolecules such as proteins and nucleic acids. It enables researchers to gain insights into their size, shape, molecular mass, and other important characteristics, contributing to a deeper understanding of their structure and function.
• In summary, the analytical ultracentrifuge (AUC) is an instrumental tool used for the quantitative analysis of particles, particularly macromolecules, in a sample. Its sophisticated detection systems and sedimentation analysis methods facilitate the characterization of biomolecules based on their size, shape, and hydrodynamic properties. AUC is widely utilized in the study of biomolecules like proteins and nucleic acids, providing valuable information for various scientific and research applications.

2. Preparative ultracentrifuge

• Preparative ultracentrifuges are specialized centrifuges primarily used for the isolation and separation of particles within a sample through the process of centrifugation.
• In contrast to analytical centrifuges, where analysis is conducted during the centrifugation process, preparative ultracentrifuges involve analyzing the contents of the tubes after the centrifugation period has concluded.
• These ultracentrifuges can be operated for various types of centrifugation processes, including density gradient centrifugation, differential centrifugation, and isopycnic centrifugation.
• In density gradient centrifugation and isopycnic centrifugation, the particles within a sample are separated based on their density. Different particles present in the sample are isolated into distinct bands at specific levels where the density of the particle matches that of the surrounding medium.
• On the other hand, in differential centrifugation, particles are separated by subjecting them to different speeds of the rotors. Larger particles settle down at lower speeds, while smaller particles require higher speeds to achieve separation.
• Due to the separation of particles based on density and size, preparative ultracentrifuges are capable of determining the density and size of different particles within a sample.
• These instruments find applications in various fields, such as biochemistry, molecular biology, and biotechnology, where the isolation and separation of specific particles are crucial for research, diagnostics, or production purposes.
• In summary, preparative ultracentrifuges are specialized centrifuges used for the isolation and separation of particles within a sample. They operate through different types of centrifugation processes, enabling the separation of particles based on their density or size. These instruments are valuable for determining the density and size of various particles and find applications in diverse scientific and industrial settings.

Instrumentation/ Parts of Ultracentrifuge

• Ultracentrifuges are complex instruments composed of various parts and components, each serving a specific function in the centrifugation process.
• One essential component of an ultracentrifuge is the rotor. Ultracentrifuges utilize different types of rotors, including vertical rotors, swinging bucket rotors, and fixed-angle rotors. The swinging bucket rotor is the most commonly used type in ultracentrifuges as it yields the highest concentration of particles. This is because the direction of centrifugal force in the vertical rotors aligns with the position of the tubes, allowing particles to settle more efficiently.
• The drive is another crucial part of an ultracentrifuge. It is the power unit responsible for spinning the rotor, which holds cells or tubes containing the solution of molecules or suspended particles. The drive provides the necessary rotational force to achieve the high speeds required for ultracentrifugation.
• Ultracentrifuges may also include additional features and components to enhance their functionality. For example, analytical rotors accommodating multiple cells or tubes may be available, allowing for simultaneous analysis of different samples. Temperature control devices are often incorporated to regulate the system’s temperature, as heat generation is common during high-speed operation. Wedge quartz windows are utilized to enable the use of multiple cells or tubes simultaneously, and cells with different thicknesses provide a range of sensitivity for analysis.
• Analytical ultracentrifuges may feature a two-dimensional comparator with a printing desk calculator, as well as interference and absorption optical systems. These optical systems are essential for real-time analysis of molecules, providing valuable information during the centrifugation process.
• In the case of preparative ultracentrifuges, additional components may be present. These can include a gradient-forming device for density gradient centrifugation, hand refractometers for monitoring refractive index changes, and a recording spectrophotometer with a flow cell and fraction collector for collecting and analyzing fractions of separated particles.
• Overall, the instrumentation of an ultracentrifuge consists of rotors, drives, temperature control systems, analytical and optical components, and other specialized devices depending on the specific type and purpose of the ultracentrifuge. These various parts work in harmony to enable efficient and precise separation and analysis of particles within a sample.

Summery

• Rotors:
  • Vertical rotors
  • Swinging bucket rotors (most commonly used)
  • Fixed-angle rotors
• Drive:
  • Power unit that spins the rotor
  • Generates the necessary rotational force
• Additional Features and Components:
  • Analytical rotors accommodating multiple cells
  • Temperature control devices (0° to 40° range) to regulate system temperature
  • Cells with varying thicknesses for sensitivity range
  • Wedge quartz windows enabling the use of multiple cells simultaneously
• Optical Systems (Analytical Ultracentrifuge):
  • Interference and absorption optical systems
  • Two-dimensional comparator with a printing desk calculator
  • Real-time analysis of molecules
• Additional Components (Preparative Ultracentrifuge):
  • Gradient-forming device for density gradient centrifugation
  • Hand refractometers for monitoring refractive index changes
  • Recording spectrophotometer with flow cell and fraction collector for analysis and collection of separated particles

Procedure/ Steps of Ultracentrifuge

Here are the steps involved in the procedure of operating an ultracentrifuge, specifically for analytical ultracentrifugation:

1. Sample Preparation:
   • Take small sample sizes (20-120 mm3) of the solution containing biomolecules.
   • Transfer the sample into analytical cells designed for the ultracentrifuge.
2. Loading the Sample:
   • Place the analytical cells containing the sample into the ultracentrifuge rotor.
   • Ensure proper balance and secure positioning of the rotor to avoid imbalance during operation.
3. Centrifugation:
   • Start the ultracentrifuge, initiating the rotation of the rotor at the desired speed.
   • The centrifugal force causes the biomolecules within the sample to migrate radially outwards from the center of rotation.
4. Optical Detection:
   • Utilize the Schlieren optical system or other suitable optical systems available in the ultracentrifuge.
   • The optical system observes the position and movement of the molecules during centrifugation.
5. Data Collection:
   • Record the solute concentration versus the squared radial distance from the center of rotation.
   • This data is typically collected over time during the centrifugation process.
6. Analysis:
   • Plot a graph using the collected data, with solute concentration on the y-axis and squared radial distance on the x-axis.
   • Analyze the graph to determine the molecular mass of the biomolecules present in the sample.

It’s important to note that the specific steps and techniques may vary depending on the type of ultracentrifuge, the objectives of the experiment, and the specific protocols followed by the laboratory or research facility. The procedure outlined above provides a general overview of the steps involved in performing analytical ultracentrifugation.

The following is the procedure to follow for the operation of an ultracentrifuge preparative:

A. Density gradient centrifugation

1. Density Gradient Preparation:
   • Prepare a sucrose density gradient by layering a less concentrated sucrose solution over a more concentrated sucrose solution.
   • Alternatively, if the ultracentrifuge has a gradient-forming device, it can create the gradient automatically.
2. Sample Placement:
   • Carefully place the sample to be separated on top of the density gradient in the centrifuge tube.
   • Ensure the sample is layered on the gradient without disturbing the gradient layers.
3. Rotor Preparation:
   • Position the centrifuge tubes with the samples in the racks of the ultracentrifuge rotor.
   • Ensure proper balance and alignment of the rotor to avoid any imbalance during centrifugation.
4. Temperature and Time Setting:
   • Set the desired temperature and centrifugation time on the ultracentrifuge control panel.
   • The temperature control ensures stability and control during the centrifugation process.
5. Closing and Starting the Process:
   • Close the lid of the ultracentrifuge securely to maintain the internal environment during centrifugation.
   • Start the centrifugation process according to the specified speed and time parameters.
6. Gradient Separation:
   • As the centrifuge spins, particles within the sample travel through the density gradient.
   • Each particle will eventually reach a point where its density matches that of the surrounding medium within the gradient.
7. Fraction Collection:
   • Carefully remove the fractions from the centrifuge tubes, taking care to isolate each fraction.
   • The fractions collected will contain particles separated based on their density within the gradient.
8. Particle Isolation:
   • Separate the particles of interest obtained in each fraction, typically by transferring them to separate containers or tubes.
   • These isolated particles can be further analyzed or utilized for specific applications.

B. Differential centrifugation

1. Sample Preparation:
   • Homogenize the sample in a suitable buffer medium to ensure a uniform distribution of particles.
2. Centrifuge Tube Preparation:
   • Transfer the homogenized sample into a centrifuge tube, taking care not to introduce any air bubbles.
3. Centrifugation:
   • Operate the centrifuge at a specific centrifugal force, predetermined based on the size and density of the particles of interest.
   • Set the desired centrifugation time and temperature.
4. Pellet Formation:
   • During centrifugation, particles in the sample sediment based on their size and density.
   • At the end of the centrifugation cycle, a pellet forms at the bottom of the centrifuge tube.
5. Supernatant Removal:
   • Carefully decant or aspirate the supernatant (liquid above the pellet) into a new centrifuge tube.
   • Take caution not to disturb or transfer any of the pellet.
6. Repeat Centrifugation:
   • Place the new centrifuge tube containing the collected supernatant in the centrifuge.
   • Centrifuge the supernatant at a different centrifugal force, time, and temperature than the previous step.
7. Pellet Separation:
   • Following centrifugation, a new pellet will form at the bottom of the tube.
   • Separate the supernatant from the pellet as done in the previous step.
8. Repeat and Iterate:
   • Repeat the process of centrifugation and separation, gradually increasing the centrifugal force or adjusting other parameters.
   • Continue the steps until the desired separation and isolation of particles is achieved.
9. Particle Identification:
   • Once all particles have been separated, identify and characterize them using specific indicators or tests unique to the particles of interest.
   • Analyze the isolated particles to determine their composition, size, density, or other relevant characteristics.

Uses of Ultracentrifuge

1. Organelle Fractionation: Preparative ultracentrifuges are used in biology to separate and isolate specific cell organelles such as mitochondria, ribosomes, and viruses from complex mixtures.
2. Nucleic Acid Separation: Density gradient centrifugation using ultracentrifuges is employed to separate nucleic acids like DNA and RNA. Cesium salt gradients are commonly used for this purpose.
3. Characterization of Macromolecules: Analytical ultracentrifugation enables the detection and characterization of macromolecular conformational changes. It helps in understanding how pH, temperature, and other environmental factors influence the structure and behavior of macromolecules.
4. Determination of Molecular Properties: Analytical ultracentrifuges aid in determining the stoichiometry, molecular masses, and size of various macromolecules. This information is crucial for understanding their functions and interactions.
5. Study of Biomolecular Complexes: Ultracentrifugation techniques allow researchers to investigate the assembly and disassembly of biomolecular complexes. This helps in understanding their formation, stability, and dynamics.
6. Density Determination: Ultracentrifuges are utilized to determine the density of macromolecules. This information aids in characterizing the composition and purity of biological samples.
7. Purification of Biological Extracts: Ultracentrifugation is employed to purify crude biological extracts by separating different components based on their size, density, or other properties. This enables the isolation of specific biomolecules for further analysis or downstream applications.

Centrifugation Versus Ultracentrifugation

Ultracentrifugation as a substitute for centrifugation (and the reverse) is a sign of the fundamental differences between these two techniques. The fundamental differences between centrifugation as well as ultracentrifugation comprise:

1. The spinning speed which, in turn, increases the force exerted by centrifugal force onto the specimens. The rotor in an ultracentrifuge may spin at a speed of 1,000 000 x G unlike the majority of benchtop centrifuges that are restricted to 65 000 x G (Biocompare 2019, 2019a, and 2019b). This leads to another fundamental differentiator:
2. Vacuum and refrigeration systems, that are required in ultracentrifuges. Due to the extremely high speed of spinning ultracentrifuges are equipped with refrigeration and vacuum systems to prevent damage to the device or sample caused by frictional force or overheating. In centrifuges that are benchtop both of these methods are not required, and are only available on the most basic centrifuges such as microcentrifuges that are small with no refrigeration system, showing none.
3. Type of pellet, that results from fractionation of samples: since ultracentrifuges are able to achieve higher speed of spinning, the type of suspended matter (pellet) produced by either one or the other is different and ultracentrifugation allows for the separation of smaller particles that benchtop centrifugation. In labs in biology, subcellular fractionation to separate cytosolic content (such as the entire cytosol chloroplasts or mitochondria) from the cell nuclei could be accomplished by centrifuges that are benchtop. However, in order to separate smaller components, such as small vesicles or ribosomes larger centrifugal forces that can only be achieved by ultracentrifuges (Momen-Heravi 2017, and Ohlendieck and Harding Ohlendieck & Harding, 2017,).

Advantages of Ultracentrifugation

• Subcellular Level Research: Ultracentrifugation enables the isolation and study of tiny particles such as organelles, subcellular membranes, and ribonucleic acid (RNA), expanding the boundaries of research in biology to the subcellular level.
• Size, Shape, and Structure Analysis: Analytical ultracentrifugation provides valuable insights into the size, shape, and structure of macromolecules, allowing researchers to better understand their properties and functions.
• Advances in Various Fields: Ultracentrifugation has contributed to advancements in fields like biology, materials science, chemistry, and others, leading to significant discoveries and breakthroughs in scientific research.

Limitations of Ultracentrifugation

• Low Sample Yield: Preparative ultracentrifugation often involves multiple washing steps to prevent cross-contamination between different fractions. These wash steps can lead to a loss of material, resulting in a low yield of samples after the ultracentrifugation process.
• Lengthy Process: Ultracentrifugation is a time-consuming process that can take many hours to separate all the components of a mixture. The extended duration can limit the efficiency and productivity of experiments or analyses.
• Expensive Equipment and Maintenance: Ultracentrifuges are expensive pieces of equipment that require ongoing maintenance and care. This cost factor makes ultracentrifuges less accessible, and they are typically found in specialized labs or research facilities.
• Limited Availability: Due to their cost and maintenance requirements, ultracentrifuges may be limited in availability within labs. Often, there is only one unit per department or university, making access to the equipment more restricted.

Precautions

1. Follow Manufacturer’s Manual: Always refer to the manufacturer’s manual and follow the recommended guidelines and procedures for operating the ultracentrifuge. This ensures proper usage and helps prevent any potential risks.
2. Handle Rotors with Care: Handle the rotors carefully and inspect them regularly for signs of corrosion, damage, or cracking. Damaged rotors can compromise the safety and performance of the ultracentrifuge.
3. Maintain Equal Sample Volume: When adding tubes into the rotor, ensure that the volume of the sample in each tube is equal. Imbalanced samples can cause uneven forces during centrifugation, leading to potential damage to the equipment or inaccurate results.
4. Balance Tube Load: It is important to balance the number of tubes added to the ultracentrifuge rotor. If there is an insufficient sample load, balance the rotor by adding distilled water to the empty tubes according to the manufacturer’s guidelines.
5. Adhere to Recommended Speeds: Do not exceed the assigned rotor speed limits specified by the manufacturer. Operating the ultracentrifuge at excessive speeds can pose a safety risk and potentially damage the equipment.
6. Keep the Chamber Closed: Ensure that the lid of the ultracentrifuge chamber remains closed during operation. Opening the lid while the rotor is spinning can lead to accidents, such as sample leakage or rotor imbalance.
7. Follow Rotor Care Guidelines: To prevent damage to the rotors, adhere to the manufacturer’s instructions for rotor care and management. This may include guidelines for cleaning, storage, and maintenance of the rotor components.

The Ultracentrifuge: How to Use and How to Care

Modern ultracentrifuges are heavy, sturdy equipment that requires certain know-how for proper usage and care.

1. Rotor balance. As in all centrifuges, sample spinning requires a proper balance of the weight inside the rotor. Given the extremely high spinning speed inside the ultracentrifuge’s rotor, the impact of subtle imbalances may be shockingly strong. Modern ultracentrifuges have some buffer capacity, to absorb slight weight imbalances, and when there is too much imbalance, an automatic system shuts off the device. Moreover, in all ultracentrifuges, the rotor is encapsulated in a strong heavy metallic cage, to avoid vibrations and projections that could damage the sample and endanger the operator. Yet, it is of vital importance that the ultracentrifuge is properly loaded, according to the manufacturer’s instructions.
2. Sample position in rotor. All rotor positions must be filled. Even when there are only a few tubes, the rest of the positions must be occupied with blank samples of equivalent weight. To avoid both rotor and sample damage, it is important to set the ultracentrifuge to slow acceleration and deceleration modes. This is especially important in density gradients, as the sudden stop of the spinning may affect the separation of the gradient layers (Ohlendieck & Harding, 2017). Ultracentrifuges are expensive devices, which are required to accurately separate particles in solution. To ensure the proper function of the ultracentrifuge, care measures must be undertaken regularly. Apart from safety, proper loading of the rotor avoids excessive vibration, which can cause damage to the device.
3. Centrifuge cleaning. Maintenance and cleaning of the rotor must be done with non-abrasive detergents to avoid corrosion. Rotor cleaning is especially important to ensure that there are no remnants of the samples that were centrifuged, and therefore, should always be performed after spinning.
4. Storage. Whenever the device is not used, or simply for overnight storage, rotors must be kept in a dry room, properly cleaned, and left to dry in an inverted position, to avoid the accumulation of water in the sample cells.
5. Regular maintenance. This should be done by certified operators to ensure the proper long-term function of the ultracentrifuge.

FAQ