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Different Types of Centrifugation Techniques

    In this different types of centrifugation techniques post we have briefly explained about definition, revolutions per minute (RPM), relative centrifugal force (RCF), principle, rotors, types, and ultracentrifuge.

    Centrifugation is a separation or concentration process for materials suspended in a liquid medium. The effect of gravity on particles in suspension serves as the theoretical foundation for this technique. Gravity causes particles of different masses to settle at different rates in a tube.

    The centrifugal force is proportional to the rotor’s rotation rate. The centrifuge is made up of a rotor that is enclosed in a refrigerated chamber and powered by an electric motor.

    Different Types of Centrifugation Techniques

    Centrifugation is a separation technique in which the centrifugal force/acceleration causes denser molecules to move to the periphery and less dense particles to move to the centre.

    The centrifugation process is based on the perpendicular force created when a sample is rotated around a fixed point. The rate of centrifugation is determined by the particle size and density in the solution.

    Revolutions Per Minute (RPM)

    In terms of centrifugation, revolutions per minute (RPM) is simply a measurement of how quickly the centrifuge rotor completes a full rotation in one minute. It basically tells us how fast the rotor is spinning.

    Centrifuges have a speed range that they can achieve, which varies depending on the centrifuge. A low-speed centrifuge may spin at as little as 300 RPM, whereas a high-speed centrifuge may spin at up to 15000 RPM.

    Ultracentrifuges, the most powerful type of centrifuge, are also available; they can spin at speeds in excess of 150,000 RPM.

    Relative Centrifugal Force (RCF)

    Relative Centrifugal Force (RCF) or g-force (both are the same; RCF is expressed in units of gravity) is a measurement of the gravitational force that a sample experiences.

    The force is generated by the rotor spinning, which then exerts this force outward on the centrifuge tube. RCF not only considers the speed of rotation, but it also measures the distance from the centre of rotation to calculate g-force.

    The preferred method of measurement is RCF because it will remain constant even if you use a different centrifuge with a different rotor size.

    The formula below allows you to convert RPM to RCF, but you must first determine the radius. In general, the rotor manufacturer will provide three radius values: maximum, minimum, and average, which are the distances from the rotor’s centre to the top, bottom, and middle of the centrifuge tube.

    RCF (g Force) = 1.118 × 10-5 × r × (RPM)2

    Principle

    In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it floats to the top. The greater the difference in density, the faster they move. If there is no difference in density (isopycnic conditions), the particles stay steady.

    To take advantage of even tiny differences in density to separate various particles in a solution, gravity can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.

    A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward).

    The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and move to the center.

    In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low- density substances rise to the top.

    Centrifuge Rotors

    The centrifuge rotor is a critical component of the device because it determines not only sample size but also how particles migrate and distribute in solution after centrifugation techniques.

    Centrifuge rotors are designed to generate rotational speed that allows components in a sample to separate. In a centrifuge, there are three main types of rotors, which are as follows:

    Swinging-bucket rotors

    The rotor buckets rotate in the same direction as the centrifugal force, elevating the sample up to 90º relative to the rotation axis. This rotor is primarily used for rate zonal centrifugation techniques, i.e., particle separation based on size and density, where maximum particle separation resolution is required.

    Swinging-bucket rotors can also be used for isopycnic centrifugation, or separation based solely on density. As particles with small sizes migrate to the complete extension of the sample tube, the time to reach the tube bottom is higher than fixed-angle or vertical-tube rotors.

    Different Types of Centrifugation Techniques: (A) example of a swinging-bucket rotor (image credit: Beckman Coulter Life Sciences); (B) Schematic representation of the centrifugal field and particle movement in swinging-bucket rotors (image credit: microbionotes).

    Fixed-angle rotors

    Tubes are held at a fixed angle, usually 45º to the rotational axis. As a result, particles migrate in a downward spiral and settle at the bottom of the tube. More diffuse sediment is produced by smaller rotor angles (pellet).

    Fixed-angle rotors are appropriate for fractionating samples where the sedimentation rates of the various components differ significantly, such as the separation of cellular components such as mitochondria, cell nuclei, and cytoplasmic content.

    Fixed-angle rotors typically accommodate a greater number of samples than swing-bucket rotors, making them more suitable for high throughput applications. Fixed rotors can resist much higher gravitational forces, with minimal metal stress, and are used for the separation of biological macromolecules such as RNA, DNA, and protein.

    Different Types of Centrifugation Techniques: (A) example of a fixed-angle rotor (image credit: Beckman Coulter Life Sciences); (B) Schematic representation of the centrifugal field and particle movement in swinging-bucket rotors (image credit: microbionotes).

    Vertical rotors

    Tubes are held at 0º – 9º from the axis of rotation, representing the shortest radial distance and thus the shortest path length for particles during centrifugation techniques. Particles settle throughout the tube wall of vertical and near-vertical rotors.

    Centrifugation techniques time is reduced due to the shorter radial distance, which may be important for certain biological samples. However, the particle separation resolution is significantly reduced: particles sediment through the tube wall during centrifugation; however, when the rotor deaccelerates and stops, the sedimented particles fall off the tube wall and contaminate the separated sample zones.

    Different Types of Centrifugation Techniques: (A) example of a vertical rotor; (B) example of a near-vertical rotor (image credit: Beckman Coulter Life Sciences); C. Schematic representation of the centrifugal field and particle movement in swinging-bucket rotors (image credit: microbionotes).

    Different Types of Centrifugation Techniques

    Benchtop centrifuges

    Benchtop centrifuges are a type of centrifuge that is distinguished by its modest footprint on the bench. A range of various aspects can be addressed depending on the study need. RCFs can have maximum speeds ranging from a few hundred to over 50,000 x g. Tube volumes can range from as little as 1 mL (as in PCR tubes) to several litres. Rotors of various sorts, such as fixed angle, swinging bucket, and continuous flow, and frequently interchangeable.

    Refrigerated Centrifuges

    Refrigerated Benchtop Centrifuges are small, portable centrifuges that are perfect for centrifuging temperature-sensitive materials such live cells, animals, and proteins. Many have replaceable rotors and adaptors to accommodate sample quantities ranging from a few millilitres to a few litres. Speeds can also differ, with some models reaching 60,000 x g.

    Clinical Centrifuges

    Clinical benchtop centrifuges are low-speed centrifuges that are perfect for separating whole blood components such serum, plasma, buffy coat, red blood cells, and other biological fluids. Their rotational speeds can range from 200 to 6,000 revolutions per minute. Most clinical centrifuges will accept standard blood draw tubes, but check with each vendor for specific tube sizes or adaptors.

    Microcentrifuges

    Microcentrifuges are commonplace in many research labs since they can handle small tube contents including 2 mL, 1.5 mL, 0.5 mL, and PCR tubes. Microcentrifuges used in common laboratory processes typically spin at 16,000 x g, whereas more specialised devices can spin at 30,000 x g. Interchangeable rotors and tube adaptors may also be available from manufacturers.

    Vacuum Centrifuges

    Vacuum Centrifuges / Concentrators can extract liquid solvent using vacuum, centrifugal force, temperature, and/or gas for sample concentration or desiccation. For a variety of scientific applications, this device is appropriate for purification or processing of samples such as nucleic acids, proteins, peptides, and other substances. Vacuum centrifuges usually include built-in heating systems for evaporating liquids.

    Ultracentrifuge

    Ultracentrifuges are centrifuges that spin at extremely high speeds, allowing much smaller molecules such as ribosomes, proteins, and viruses to be separated. In such centrifuges, refrigeration systems are installed to help balance the heat generated by the intense spinning.

    These centrifuges can spin at up to 150,000 revolutions per minute. It can be utilised for both analytical and preparatory tasks. In addition to separation, ultracentrifuges can be used to determine macromolecule parameters such as size, shape, and density.

    Preparative Ultracentrifuges

    These are frequently used to separate particles based on their densities, isolate denser particles for pellet collection, and clarify particle-containing solutions. They aid in the isolation of macromolecules and lipoprotein components from plasma, as well as the de-protonation of physiological fluids for amino acid research. Varied types of rotors can be used in a preparative ultracentrifuge to spin multiple samples at different angles and speeds.

    Analytical Ultracentrifuges

    These are equipped with a light-based optical detection technology that allows for real-time sample monitoring while they spin. The sedimentation process is visible to the users. They may observe the material as it concentrates as the centrifugal force increases. The light absorption system, the alternate Schlieren system, and the Rayleigh interferometric system are some of the optical systems utilised for analysis.

    Analytical centrifugation

    Analytical centrifugation techniques for determining the shape, molecular weight, and purity of pure chemicals and macromolecules. Rather than fractions, as in preparative centrifugation, it is principally concerned with the research of sedimentation features of biological macromolecules.

    Principle

    Optical detection systems are installed in analytical ultracentrifuges, allowing the researcher to monitor the centrifugation techniques process in real time. Ultraviolet (UV) light absorption or refracting index interference (RII) optical detection systems are used in these systems. RII measures changes in the refraction index of a substance compared to the solvent it is dissolved in, whereas UV detection directly measures the absorbance (abs) of a substance at a certain wavelength.

    Applications

    Analytical centrifugation techniques serves a different purpose than other types of centrifugation. Although analytical centrifugation can isolate components, the purpose of this technique is to acquire data to characterise the sample that is spun (sedimentation velocity, viscosity, concentration, etc.).

    It is feasible to track changes in sample concentration as a function of the applied centrifugal force using analytical centrifugation techniques. This method is employed in two different types of experiments: sedimentation velocity and sedimentation equilibrium studies, both of which are important in macromolecular characterisation.

    Different Types of Centrifugation Techniques

    Preparative Ultracentrifuges

    Biological materials are processed in preparative ultracentrifuges for subsequent examination. Preparative ultracentrifugation is most commonly used in tissue and subcellular fractionation to extract increasingly smaller components of biological samples.

    1. Differential Centrifugation

    Differential centrifugation techniques separates distinct components primarily based on particle size. Simple pelleting and partially pure production of subcellular organelles and macromolecules are popular applications.

    Principle

    Differential centrifugation techniques is a technique for separating the components of a solution based on differences in the sedimentation rates of the various components. The size and density of a substance, as well as the density of the solvent, affect its sedimentation properties.

    Applications

    The progressive increase of the applied centrifugal field divides crude tissue homogenates comprising organelles, membrane vesicles, and other structural pieces into various fractions in medical and biology labs. In addition, differential centrifugation techniques is commonly used to isolate non-living material such as nanoparticles, colloids, and viruses.

    2. Density gradient centrifugation

    Differential centrifugation techniques does not separate particles as well as density gradient centrifugation. It’s ideal for separating particles with comparable diameters but differing densities.

    Principle

    A method for purifying subcellular organelles and macromolecules with varying densities and sizes is density gradient centrifugation techniques. Density gradients are made by centrifuging layers of gradient media, such as sucrose, in a tube with the heaviest layer at the bottom and the lightest layer at the top, in either a discontinuous or continuous mode.

    Applications

    For the purification of large volumes of biomolecules, density gradient centrifugation techniques can be used. It can also be used to purify certain viruses, which benefits in their further research. This method can be used as a separation method as well as a method for determining the densities of distinct particles.

    3. Rote-zonal centrifugation

    Particle separation depends mostly on particle mass. Zones, or bands, are generated, each containing a particle fraction of a specific mass.

    Principle

    For sedimentation, rate-zonal separation uses particle size and mass (sedimentation rates) rather than particle density. As a result, particles migrate over the gradient in a predictable pattern, separating themselves into different zones or bands (if they were layered as a thin zone onto the top of the gradient). It is depending on the passage of time.

    Applications

    Proteins, macromolecules, antibodies, viruses, DNA-RNA hybrids, hormones, enzymes, ribosomal subunits, and organelles can all be separated using this method.

    The density of the sample solution must be less than that of the lowest density portion of the gradient; the density of the sample particle must be greater than that of the highest density portion of the gradient. The gradient path length must be sufficient for the separation to occur, and the time factor is critical for successful rate zonal centrifugation.

    4. Isopycnic Centrifugation

    Isopycnic centrifugation techniques (equilibrium) – particle separation is exclusively based on density. Particles are mixed with the gradient solution in isopycnic separation, and during centrifugation, they travel until they reach the gradient phase, which equals their density (isopycnic or equilibrium point).

    Principle

    This method is used to distinguish molecules based on their density (isopycnic means equal density). For this separation, fixed angle rotors or swinging bucket rotors can be employed. Due to equilibrium sedimentation, a self-generating density gradient is generated, and analytes molecules are concentrated as bands with molecular densities that match the surrounding solution.

    Applications

    To separate plasma lipoproteins, isopycnic gradient ultracentrifugation is commonly performed. The density of the sample particle must lie within the gradient densities for successful isopycnic centrifugation techniques, the run duration must be sufficient for the particles to band at their isopycnic point, and excessive run times and gradient lengths have no negative effects.

    Application

    1. Centrifugation techniques is used in current research and clinical applications (in the fields of biochemistry, biology, chemistry, molecular biology, biotechnology, and pharmaceutical sciences) to isolate cells, subcellular organelles, and macromolecules for various analyses.

    2. Centrifugation techniques are used in the culinary, chemical, and mineral sectors to separate water from various materials, separate cream from milk, and purify water.

    3. In clinical laboratories and small medical institutions, centrifugation techniques are a must-have item. They are used to separate compounds with varying densities and to remove chylomicrons.

    4. The development of manned space travel missions has relied heavily on centrifugation techniques studies. Human volunteers are placed in very large centrifuges and spun at high speeds to simulate the centrifugal field experienced by spacecraft during launch.

    Further Readings

    Reference

    Different Types of Centrifugation Techniques