Definition of Ultracentrifuge
Ultracentrifuge is an advanced and sophisticated centrifuge that runs at a high rate and is able to separate smaller molecules that can’t be separated from traditional centrifuges.
Rotor speeds inside an ultracentrifuges can vary between 60,000 and 150,000 rpm. Ultracentrifuges tend to be used in laboratories that are more well-equipped to carry out more sophisticated operations. They are bigger in size and operate samples in groups or as continuous flow systems. The majority of ultracentrifuges are refrigerated in order to reduce the heat caused by the increased speed.
Principle of Ultracentrifuge
Ultracentrifuges work using the same principles like all centrifuges. The process of working an ultracentrifuge relies on the sedimentation principle. It stipulates that the more dense particles are more likely to settle in comparison to the less dense particles in gravity. However, the process of sedimentation for particles in gravity would require more time, which is the reason an additional force is employed to assist in the process of sedimentation. In an ultracentrifuge, the specimen is rotated around an axis. This results in a force perpendicular to the axis, known as centrifugal force, which is a force that affects various particles in the sample.
Larger molecules move more quickly, while smaller molecules move more slowly. In the same way, larger molecules are moved towards the outer edges of the tubes , whereas smaller molecules are rotated toward the central part inside the tube. When the process is complete when the larger heavier and dense molecules settle into pellets that are placed at on the inside in the tube. The smaller particles are either supernatant suspended in it or floating at the top of the tube.
Types of Ultracentrifuge
Based on the purpose and the purpose, ultracentrifuges can be of two kinds:
1. Analytical ultracentrifuge (AUC)
Analytical centrifuges, as their name suggests, are ultracentrifuges used to analyze various particles inside the specimen. An analytical ultracentrifugation (AUC) is a flexible and reliable method to perform qualitative analysis of macromolecules in solution. Ultracentrifuges are equipped with detection systems that monitor the spin and movement of particles in real time to calculate the coefficient of sedimentation, which helps in the study of particles based on their shape dimensions, size, and mass.
Analytical ultracentrifugation used to determine of the molecular mass of a macromolecule could be done using the sedimentation velocity method or the sedimentation equilibrium approach. The properties of macromolecules’ hydrodynamics can be described through the sedimentation coefficients. The coefficients can be derived by the speed at which the concentration boundary for a specific biomolecules shifts in gravity fields. The sedimentation coefficient may be used to determine changes in the dimensions and shapes of macromolecules in response to changing experimental conditions.
The three different optical system are offered to the ultracentrifuge that is analytical (absorbance as well as interference and fluorescence) which allow the precise and specific monitoring of sedimentation in real-time. One of of detection systems utilized in ultracentrifuges can be that of the Schlieren optical system, which observes the particles inside the centrifuge in real time to determine their location and motion. Based on the location the sedimentation coefficients can be calculated, which allows the measurement of the characteristics of various molecules. Analytical ultracentrifuge is often used to determine the nature of the properties of biomolecules such as protein and nucleic acid.
2. Preparative ultracentrifuge
Preparative ultracentrifuges are centrifuges which are used to isolate and separate of particles within an experiment by means of centrifugation. When a run is prepared for an ultracentrifuge, the contents in the tubes are analysed following the centrifugation process in contrast to the centrifuge for analysis which analyzes at the time of the centrifugation.
Preparative ultracentrifuges are able to be used for a variety of centrifugation techniques such as density gradient centrifugation differential centrifugation and isopycnic centrifugation. The particles of the sample are separated based on their density or the size. In centrifugation using a density gradient and isopycnic centrifugation, particles in a sample are separated based on their density. Different particles that are present in a sample are separated by forming bands that are separated into distinct levels, where they have a density that of each particles is the same as what the volume of media.
In differential centrifugation however particles are separated through applying different speeds to the rotors. Larger particles settle at slower speeds, while smaller particles require greater speeds to be separated. Since particles are separated based on size and density Ultracentrifuges that are preparative can be utilized to determine the size and density of various particles.
Instrumentation/ Parts of Ultracentrifuge
Ultracentrifuges have various components and parts that perform various tasks. Rotors are an essential element of an ultracentrifuge. Ultracentrifuges utilize all three types of rotors: vertical rotors and swinging bucket rotors Fixed-angle rotors, and. The rotor that swings bucket is the most frequently used rotor for ultracentrifuges because it provides the greatest concentration of particles. This is due to the direction of the centrifugal force in vertical rotors is aligned with the location that the tube is in.
This drive acts as the motor that spins the rotor which is holding cells or tubes that hold molecules in a solution or suspensions of particles. In addition to the interchangeable electric drive and rotors it comes with analytical rotors which can hold as many as four cells temperature control devices that have flexibility and control from 0deg up to 40deg cells with thicknesses that exceed 10 times the sensitivities with wedge-shaped quartz windows that allow multiple cells to be utilized at the same time.
The temperature system permits the control of temperature within the unit since heat is produced frequently in the operation of an ultracentrifuges operating at high speed. An analytical ultracentrifuge comes with two-dimensional comparators, printer desk calculators interfering, absorption, and interference optical systems. These optical systems are essential to analyze in real-time molecules. A gradient-forming device hand refractometers as well as a recording spectrophotometer that has a the fraction collector and flow cell could also be found in a pre-preparative ultracentrifuge.
Procedure/ Steps of Ultracentrifuge
The procedure for operating an ultracentrifuge can differ based on whether it’s an analytical ultracentrifuge or a preparative. In general, here are the steps that must be taken when performing an analysis-based ultracentrifugation
- Smaller sample sizes (20-120 millimeters) are collected in cells for analysis and placed in the ultracentrifuge.
- The ultracentrifuge is operated to create a centrifugal force that results in a movement of biomolecules that are randomly distributed throughout the solvent, radiating towards the outside of the rotation.
- The distance of particles from their center are measured using the Schlieren optical system.
- An illustration is created by comparing the concentration of the solute in relation to the squared distance to the central point of rotation using the molecular mass calculated.
The following is the procedure to follow for the operation of an ultracentrifuge preparative:
A. Density gradient centrifugation
- In the process of making a preparative ultracentrifuge with a density gradient it is necessary for a density gradient to be made. To do this the process, a thin layer of sucrose with less concentration is placed over the top layer of sucrose that is more concentrated, that creates a sucrose density gradient. Some ultracentrifuges are equipped with devices for forming gradients that create the gradient themselves.
- The sample is then placed inside the centrifuge tube, with the gradient and after that, the tubes will be placed on those racks that are part of the rotors that are in the ultracentrifuges.
- The temperature and timing are set by the ultracentrifuge before the procedure is initiated.
- The lid is shut and the process is begun.
- The particles move through their gradients until they arrive at a point at which their density is comparable to with the denseness of medium around them.
- The fragments are then removed and separated, giving particles that are isolated.
B. Differential centrifugation
- In differential centrifugation samples are homogenized within the buffer that it is in.
- The sample is placed inside the centrifuge tube that is then operated by an exact centrifugal force over an exact amount of time and at a specific temperature.
- After this process the pellet will have formed in the middle of the tube. This is then separated from supernatant.
- The supernatant is then added to an unopened centrifuge tube in which it is centrifuged at a different speed for a specific period of duration and at a certain temperature.
- The supernatant is removed from pellets that have formed.
- This process continues until the elements are separate from one the other.
- The particles are identified through the testing of indicators specific to the particular particles.
Uses of Ultracentrifuge
Due to their intrinsic differences, analytical and preparative ultracentrifugation are used for different purposes:
- determination of the purity (including the presence of aggregates) and oligomeric state of macromolecules, by recording sedimentation velocity data
- determination of the average molecular mass of solutes in their native state
- Study of changes in the molecular mass of supramolecular complexes,
- using either sedimentation velocity, sedimentation equilibrium (or both)
- the detection of conformation and conformational changes
- subcellular fractionation
- affinity purification of membrane vesicles
- separation of DNA components
- colloid separation
- virus purification
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:
- 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:
- 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.
- 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 and Limitations of Ultracentrifugation
From the invention of the ultracentrifuge’s first prototype during the 1920s by Svedberg until today the advances in science that have resulted from the use of ultracentrifugation to biology materials science, chemistry and other fields, are numerous. Through its most obvious applications ultracentrifugation expanded the boundaries of research in biology down to the subcellular level in that it allowed the isolation of tiny particles like organelles subcellular membranes, the ribonucleic acid. With the introduction of analytical ultracentrifugation, science moved one step further toward understanding the submicroscopic world. It also gave researchers the ability to better understand the size, shape, and the structure. However, ultracentrifugation is not without its own drawbacks, just like every other technique used in labs. It is a good example:
- A low yield of samples – In the process of preparative ultracentrifugation samples need to be washed several times after spinning, in order to ensure there is no cross-contamination of the different fractions. The samples for preparative centrifugation are typically smaller in terms of size (e.g. tissues, cellular) or the volume (e.g. cell suspensions, blood). Every wash step the sample goes through to, it loses material. Consequently following an ultracentrifugation process the yield may be quite low.
- Ultracentrifugation remains a lengthy processthat can take many hours to separate all the elements of a mixture.
- Ultracentrifuges are expensive equipment that require ongoing maintenance. This is why ultracentrifuges can be rarely found in labs However, they are usually only one unit for each department or university.
Ultracentrifuges are prone to malfunction, that is why they must be operated with complete precautions. The most important precautions include:
- When operating an ultracentrifuge, the manual of the manufacturer should be adhered to.
- The rotors need to be handled with care and be regularly checked to look for indications of corrosion or cracking.
- As you insert the tubes into the rotors, the size of the sample within the tubes should be the same.
- The quantity of tubes that are added to an ultracentrifuge must be regulated to ensure that they are in balance. If there isn’t enough sample loads accessible, distilled water needs to be added to adjust.
- The rotor’s speed must not exceed the value assigned to it.
- The chamber’s lid must always be closed during the process.
- To avoid damage to rotors, the manufacturers’ guidelines of rotor maintenance and management must be observed.
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.
- 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.
- 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.
- 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.
- 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.
- Regular maintenance. This should be done by certified operators to ensure the proper long-term function of the ultracentrifuge.