Ultracentrifuge - Principle, Parts, Types, Uses

Ultracentrifuge is a high speed centrifuge which is used for separation of very small particles from a liquid medium.

It can rotate at very high speed, usually more than 100000 rpm. Due to this high speed, very strong centrifugal force is produced. This force may be more than 1000000 times of gravity.

Ultracentrifuge works on the principle of sedimentation. In this process, particles move under centrifugal force according to their size, shape and density. Thus proteins, nucleic acids, viruses and nanoparticles can be separated.

In ultracentrifuge, the rotor moves at very high speed. If the rotor is allowed to rotate in normal air, heat is produced due to friction. This heat may damage the biological samples and also disturb the separation.

To prevent this, the rotor is placed inside a sealed and strong chamber. The chamber is kept under high vacuum. This reduces air resistance. A cooling system is also present which keeps the sample at low temperature, generally 0 to 4°C.

Ultracentrifuge are mainly of two types. These are preparative ultracentrifuge and analytical ultracentrifuge.

Preparative ultracentrifuge is used to separate and purify particles from a mixture. The separated particles can be collected and used for further work.

Analytical ultracentrifuge is used to study particles during centrifugation. It has optical system which detects the movement of particles in real time. It helps to know molecular weight, shape and interaction of molecules.

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

Principle of Ultracentrifuge is based on the sedimentation of particles under very high centrifugal force.

In ultracentrifuge, the sample is rotated at very high speed around a fixed central axis. Due to this rotation, strong centrifugal force is produced. This force pushes the suspended particles away from the axis of rotation.

During this process, three forces act on the particles. These are centrifugal force, buoyant force and frictional force. Centrifugal force moves the particles outward. Buoyant force and frictional force resist this movement.

The larger and denser particles move faster towards the bottom or outer side of the centrifuge tube. These particles form a pellet. The smaller and less dense particles move slowly.

Thus, the particles are separated according to their size, shape and density. The lighter particles may remain suspended in the liquid supernatant or stay near the upper region of the tube.

Types of Ultracentrifuge

The following are the types of ultracentrifuge-

Analytical Ultracentrifuge (AUC)- Analytical ultracentrifuge is a type of ultracentrifuge which is used for analysis of particles during centrifugation. It contains optical detection system like UV-Visible absorbance, interference and fluorescence optics. The particles can be observed in real time while they are spinning. It is used to study molecular weight, size, shape and interaction of particles.
- i. Commercially Available Analytical Ultracentrifuge- It is the ready-made analytical ultracentrifuge available for laboratory use. These instruments are manufactured by companies. Beckman-Coulter Optima series is an example.
- ii. User-Made Analytical Ultracentrifuge- It is a custom made analytical ultracentrifuge. It is generally made by modifying preparative ultracentrifuge. It is used for special type of research work where normal available instrument is not enough.

Preparative Ultracentrifuge- Preparative ultracentrifuge is a type of ultracentrifuge which is used for separation and purification of particles. It is used for biological materials like cell organelles, viruses, proteins and nucleic acids. It has no real time optical detection system. The separated particles are collected for further use.
- i. Tabletop Preparative Ultracentrifuge- It is a small and compact form of preparative ultracentrifuge. Airfuge is an example of this type. It is used for very small amount of sample. It can produce very high centrifugal force for small scale separation.

Instrumentation/ Parts of Ultracentrifuge

The following are the instrumentation or parts of ultracentrifuge-

Housing and Safety Chamber- Housing is the outer protective part of ultracentrifuge. It is made up of heavy armoured steel chamber or barrier ring. It holds the rotor safely during high speed rotation. It also protects the operator if rotor breaks during running. The chamber is closed by strong structural door.

Drive Mechanism- Drive mechanism is the part which rotates the rotor. It consists of high speed electric motor, flexible drive shaft or spindle and damping units. Brushless induction motor is commonly used. The damping unit reduces vibration and helps in smooth rotation of rotor.
Rotors– Rotor is the important rotating part of ultracentrifuge. It holds the sample tubes during centrifugation. It is made up of strong materials like titanium, aluminium alloy or carbon fibre composite. The rotors are of different types i.e. fixed angle rotor, swinging bucket rotor, vertical rotor and near vertical rotor.
Vacuum System- Vacuum system is used to remove air from the rotor chamber. At very high speed, air friction can produce heat and damage the sample. So the rotor chamber is sealed and evacuated. Mechanical rotary vane pump and diffusion pump are used for creating vacuum.

Thermal Control and Refrigeration- This system is used to maintain low temperature inside the centrifuge. It protects heat sensitive biological samples. It generally contains CFC-free compressor and temperature measuring devices. Thermistors and radiometers are used for measuring temperature. The temperature is often maintained around 4°C.
Control Console and Safety Electronics- Control console is used to set the speed, time and temperature of ultracentrifuge. It is generally a digital microcomputer system. It is connected with safety sensors. Hall-effect sensor checks rotor position. Imbalance detector detects improper balancing. Overspeed protection system prevents the rotor from crossing its safe speed limit.
Optical Detection System– Optical detection system is present only in analytical ultracentrifuge. It is used to observe the sample during centrifugation. It monitors sedimentation of particles in real time. It is not present in preparative ultracentrifuge.
- i. Measuring Cells- Measuring cells are special sample holders used in analytical ultracentrifuge. They have quartz or sapphire windows. These windows allow light to pass through the sample solution.
- ii. Light Sources and Detectors- Light source and detector are used for optical measurement. Xenon flash lamp or laser diode may be used as light source. Photomultiplier tube or CCD camera may be used as detector. These are used in UV-Visible absorbance, Rayleigh interference and fluorescence optics.
- iii. Multiplexer Unit- Multiplexer unit is used to control the light flash at proper time. It helps the instrument to observe many sample cells in one run. Thus more than one sample can be measured during the same centrifugation.

Procedure/ Steps of Ultracentrifuge

The following are the procedure or steps of ultracentrifuge-

A. General preparation and setup

Selection of Protocol- First the protocol is selected according to the particle which is to be separated. The size and density of the particle is considered. According to this, rotor, speed, RCF and time are selected. If sample density is high, the speed is reduced.
Balancing of Tubes- The centrifuge tubes are balanced before centrifugation. Opposite tubes are balanced by mass in a weighing balance. The difference should be very low, usually within ±0.1 g. The tubes should be filled up to same level and with liquid of same density.
Loading of Rotor- The balanced tubes are placed in the rotor. The tubes are placed opposite to each other. The arrangement should be symmetrical. This prevents vibration and damage during high speed rotation.

Setting of Instrument- The speed, time and temperature are set in the instrument. The rotor chamber is closed properly. Vacuum system is started before reaching maximum speed. This prevents air friction and heat production.
Starting of Centrifugation- After proper setting, the ultracentrifuge is started. The rotor slowly increases speed and reaches the selected speed. The sample particles start moving according to their size, shape and density.
Stopping and Collection- After completion of run, the rotor is allowed to stop completely. The chamber is opened after vacuum release. The tubes are removed carefully. Pellet, supernatant or bands are collected according to the type of ultracentrifugation.

B. Differential centrifugation

Homogenization of Sample- The sample is first homogenized in suitable buffer medium. This breaks the cells and releases the particles into the solution. The medium should maintain the activity and structure of particles.
First Centrifugation- The sample is placed in centrifuge tube and centrifuged at selected speed, time and temperature. In this step, larger and heavier particles sediment first. These particles form pellet at the bottom of tube.
Collection of Pellet- The pellet formed at the bottom is collected carefully. The supernatant is separated without disturbing the pellet. The pellet contains large or dense particles.

Successive Centrifugation- The supernatant is transferred into another centrifuge tube. It is again centrifuged at higher speed. In this step, smaller particles are sedimented.
Final Separation- The process is repeated by increasing the speed step by step. Different particles are separated in different pellets. The separated particles are then tested by their specific indicators.

C. Density gradient centrifugation

Preparation of Gradient- A density gradient is first prepared in centrifuge tube. Sucrose or cesium chloride solution is commonly used. Lower concentration is layered over higher concentration carefully.

Loading of Sample- The sample mixture is placed gently on the top of the prepared gradient. It should be added as a thin layer. The gradient should not be disturbed during loading.
Centrifugation- The tube is placed in the rotor and centrifuged at selected speed, time and temperature. During centrifugation, the particles move through the gradient medium.
Formation of Bands- The particles move until they reach the region where their density is equal to the density of medium. At this position, particles stop moving. They form separate bands in the tube.

Recovery of Fractions- The separated bands are removed carefully. Each layer or fraction is collected separately. These fractions are then used for further analysis or purification.

D. Analytical ultracentrifugation

Preparation of Analytical Cell- A very small amount of sample is loaded into analytical measuring cell. The cell has optical window. Quartz or sapphire window is generally used for passing light through the sample.
Centrifugation of Sample- The analytical cell is placed in rotor and centrifuged. The biomolecules move outward from the centre of rotation. The movement depends on molecular size, shape and density.

Real Time Observation- During centrifugation, the sample is observed by optical system. Schlieren optics, UV-Visible absorbance or Rayleigh interference optics may be used. The position and movement of particles are recorded continuously.
Data Analysis- The sedimentation data is analysed after or during the run. The solute concentration is compared with radial distance. From this, sedimentation coefficient, molecular mass and shape of molecule are calculated.

Uses of Ultracentrifuge

The following are the uses of ultracentrifuge-

Isolation of Cellular Components- Ultracentrifuge is used for separation of cell organelles from cell extract. It separates nucleus, mitochondria, ribosomes and cell membrane. These parts are separated according to their size and density.

Purification of Nucleic Acids- It is used for isolation and purification of DNA and RNA. Different forms of nucleic acids can be separated by density gradient method. It is useful for molecular biology work.
Protein Analysis and Purification- Ultracentrifuge is used for study and purification of proteins. It helps to know molecular weight, shape and purity of proteins. Protein complexes and their interaction can also be studied without destroying their native state.
Isolation of Viruses- It is used for separation and purification of viruses. Viral particles can be separated from cell debris and other proteins. It is important in virology, vaccine preparation and viral research.

Manufacturing of Viral Vectors and Vaccines- Ultracentrifuge is used in preparation of viral vectors and vaccines. It helps to separate active full viral capsids from empty capsids. These purified particles are used in gene therapy and vaccine production.
Characterization of Lipid Nanoparticles (LNPs)- Ultracentrifuge is used to study lipid nanoparticles. It helps to know size, stability and encapsulation efficiency of nanoparticles. It is also used to study mRNA containing lipid nanoparticles.
Sorting of Synthetic Nanomaterials- It is used for separation of synthetic nanomaterials. Carbon nanotubes, colloidal nanocrystals and other nanoparticles can be purified. The separation is done according to their physical properties.

Study of Biomolecular Interaction- Analytical ultracentrifuge is used to study interaction between biomolecules. It shows how macromolecules associate and dissociate. It also helps to study changes caused by pH, temperature and other conditions.
Separation of Macromolecules- Ultracentrifuge is used to separate large molecules from mixture. Proteins, polysaccharides, nucleic acids and lipoproteins can be separated. It is useful in biochemical and biophysical studies.
Research Use- Ultracentrifuge is widely used in research laboratory. It is used in cell biology, molecular biology, biochemistry, virology and nanotechnology. It helps in purification, analysis and characterization of small particles.

Advantages of Ultracentrifugation

The following are the advantages of ultracentrifugation-

Analysis in Native State- Ultracentrifugation is used to analyse macromolecules in their native state. The molecules remain in solution. No supporting matrix or column is required. So the particles are not damaged or sheared during the process.
Non-Destructive Method- It is a non-destructive method. The sample is not chemically changed during centrifugation. So the sample can be recovered after analysis and used for other work.
No Standard Required- Ultracentrifugation does not always need calibration standard. It is based on physical and thermodynamic properties of molecules. So molecular properties can be studied directly from the sedimentation behaviour.

Gives Many Molecular Information- It gives many information from one experiment. Molecular weight, size, shape and purity can be known. Association constant and stoichiometry of molecule can also be studied.
High Resolution- Ultracentrifugation gives high resolution separation. It can detect difference between closely related particles. It is useful for heterogeneous samples and complex biological mixtures.
High Precision- The result obtained from ultracentrifugation is precise. Small difference in sedimentation behaviour can be measured. It is useful for protein aggregation study and macromolecular analysis.

Real Time Monitoring- Analytical ultracentrifuge gives real time observation. It has optical detection system. The movement of particles can be observed during centrifugation. So sedimentation process can be studied directly.
Flexible Sample Requirement- It can be used with different buffer systems. The sample can be analysed in wide concentration range. Very small sample volume is also sufficient, about 0.1 ml in some cases.
High Capacity Processing- Preparative ultracentrifuge can process large amount of sample. It is used for bulk separation of cells, organelles, viruses and other particles. It is useful for purification work.

Useful for Biological Samples- Ultracentrifugation is suitable for biological macromolecules. Proteins, nucleic acids, viruses and lipid particles can be studied. It helps to keep their structure and activity in better condition.

Limitations of Ultracentrifugation

The following are the limitations of ultracentrifugation-

High Cost- Ultracentrifuge is a costly instrument. Analytical ultracentrifuge is more expensive. The instrument has special rotor, vacuum system, cooling system and safety chamber. So the purchase cost is high.
Difficult Maintenance- Maintenance of ultracentrifuge is difficult. The parts are delicate and highly specialized. Rotor, motor, vacuum pump and refrigeration system need regular checking. Repairing also needs expert service.

Need of Skilled Person- Ultracentrifuge cannot be operated by untrained person. Proper knowledge of rotor, balancing, speed and vacuum system is needed. Analytical ultracentrifuge needs more skill because optical system and data analysis are involved.
High Energy Requirement- Ultracentrifuge needs high amount of energy. Very high speed rotation requires more power. Vacuum system and refrigeration system also use energy. So running cost becomes high.
Strict Operating Condition- Ultracentrifuge works under strict condition. At high speed, air friction can produce heat and damage the biological sample. So high vacuum and cooling system must be maintained during the run.

Limited Sample Capacity- Analytical ultracentrifuge uses very small amount of sample. Usually sample volume is less than 1 ml. So it is not suitable for processing large amount of sample.
No Real Time Data in Preparative Type- Preparative ultracentrifuge does not have optical detection system. So it cannot show real time movement of particles during centrifugation. It is mainly used for separation and collection of particles.
Difficulty in Very Light Particles- Very light particles are difficult to separate by ultracentrifugation. Particles having very low mass may not sediment properly. So separation becomes slow or incomplete.

Difficulty in Similar Molecular Weight- Some analytical methods cannot separate components if their molecular weights are very close. Sedimentation-equilibrium method needs clear difference in molecular weight. Otherwise, the result becomes difficult to interpret.
Noise and Vibration- Ultracentrifuge rotates at very high speed. Due to this, noise and vibration may occur. Improper balancing increases vibration. So it needs proper installation place and stable surface.
Risk of Rotor Damage- Rotor is under high stress during rotation. If rotor is old, cracked or improperly used, it may break. Rotor failure is dangerous. So rotor inspection and correct handling is required.

Precautions of Ultracentrifugation

The following are the precautions of ultracentrifugation-

Proper Balancing of Rotor- The sample tubes should be balanced properly before centrifugation. The balancing should be done by weight and not only by seeing the volume. The opposite tubes should have almost same weight, usually within 0.1 g. The tubes should be placed symmetrically in the rotor.
Proper Arrangement of Buckets- In swinging bucket rotor, all buckets should be attached during the run. Empty buckets should also be placed if all positions are not used. This keeps the rotor balanced and prevents vibration.
Speed Limit- The rotor should not be run above its maximum rated speed. Each rotor has a fixed safe speed limit. More speed may damage the rotor and may cause accident.

Speed Reduction for Dense Solution- The speed should be reduced when high density solution is used. Cesium chloride and other dense solutions need derated speed. Heavy tubes like stainless steel tubes also need reduced speed.
Vacuum Safety- Vacuum should be made before the rotor reaches high speed. Vacuum decreases air friction and heat formation. The refrigeration system should not be operated at atmospheric pressure. Otherwise moisture may condense inside and causes corrosion.
Refrigeration Safety- The cooling system should be used properly to protect biological samples. The temperature should be set before starting the run. Sudden temperature change should be avoided because it may affect the sample and instrument.

Aerosol and Biohazard Safety- Infectious, toxic or radioactive samples should be handled with care. Sealed tubes and aerosol-tight rotor lids should be used. The tubes should be filled, sealed and opened inside a biological safety cabinet.
Safe Opening of Chamber- The chamber door should not be opened while rotor is moving. The rotor should be allowed to stop completely. It should never be stopped by hand.
Cleaning of Rotor- Rotor should be cleaned after use. Mild neutral detergent of pH 6-8 should be used. Hard brush should not be used because it may scratch the protective surface. Soft plastic coated brush may be used.

Drying and Storage of Rotor- Rotor should be dried completely before storage. Water or chemical residue should not remain on rotor. The rotor should be stored in proper position and not in upside down condition.
Checking of Seals and O-rings- O-rings and gaskets should be checked regularly. If there is crack, cut or wear, they should be replaced. Silicone vacuum grease should be applied lightly to keep air tight sealing.
Care of Rotor Threads- Metal threads of rotor should be clean and properly lubricated. Dirty or dry threads may become tight and difficult to open. It may also damage the rotor.

Inspection of Tubes and Adapters- Tubes, bottles and adapters should be checked before use. Cracked, brittle, discoloured or damaged tubes should not be used. Fine cracks may break during high centrifugal force.
Correct Handling of Samples- Samples should not be overfilled in tubes. Proper caps should be used. The outside of the tube should be clean and dry before placing in rotor. This prevents leakage and contamination.

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Ultracentrifuge – Principle, Parts, Types, Uses