Part 1: What is a centrifuge used for?
Centrifuges are used in various laboratories to separate fluids, gases, or liquids based on density. In research and clinical laboratories, centrifuges are often used for cell, organelle, virus, protein, and nucleic acid purification.
An example of centrifuge use in a clinical setting is for the separation of whole blood components. Different assays necessitate serum or plasma, which may be obtained with centrifugation.
Serum is obtained by letting a whole blood sample clot at room temperature. The sample is then centrifuged and the clot is removed, leaving a serum supernatant.
Unlike serum, plasma is obtained from whole blood that is not left to clot, and contains serum along with clotting factors. To obtain plasma, a whole blood sample is collected in tubes treated with anticoagulants. Following centrifugation, cells are removed and plasma supernatant remains.
Part 2: How does centrifugation work?
Principles of centrifugation
A centrifuge is used to separate particles suspended in a liquid according to particle size and density, viscosity of the medium, and rotor speed.
Within a solution, gravitational force will cause particles of higher density than the solvent to sink, and those less dense than the solvent to float to the top. Centrifugation takes advantage of even minute differences in density to separate particles within a solution.
As the rotor spins around a central axis, it generates a centrifugal force acting to move particles away from the axis of rotation. If the centrifugal force exceeds the buoyant forces of liquid media and the frictional force created by the particle, the particles will sediment.
Centrifuge Rotor Types
There are two very common rotor designs: fixed angle, and swinging bucket. The fixed angle rotor is designed to hold tubes in a fixed position at a fixed angle relative to the vertical axis of rotation (up to about 45°). Centrifugation will cause particles to sediment along the side and bottom of the tube. The swinging bucket design allows the tubes to swing out from a vertical resting position to become parallel to the horizontal during centrifugation. As a result, sediment will form along the bottom of the tube.
Fixed angle rotors are ideal for pelleting applications either to remove particles from a suspension and discard the debris or to recover the pellet, whereas swinging bucket rotors are best for separating large volume samples at low speeds and resolving samples in rate-zonal (density) gradients.
Part 3: How do you choose a centrifuge?
Centrifuges may be classified based on maximum speeds, measured as revolutions per minute (RPM). Speeds range from 0-7,500 RPM for low-speed centrifuges, all the way to 20,000 RPM or higher.
Centrifuge rotor speed is often expressed as RCF in units of gravity (x g) for various procedures. However, many centrifuges display speed as revolutions per minute (RPM), necessitating conversion to ensure the correct experimental conditions. The following formula is used to convert RPM to RCF, where R is the rotor radius (cm) and S is the speed (RPM):
g = (1.118 x 10-5) R S2
Centrifuges are available as various benchtop or floor-standing models.
Floor-standing models offer greater sample capacity and can achieve high speeds. Superspeed centrifuges can achieve a maximum g-force (relative centrifugal force, RCF) of over 70,000 x g, and ultracentrifuges often used for DNA or RNA fractionation, can achieve up to 1,000,000 x g. For large-capacity, low-speed applications, low-speed centrifuges reaching approximately 7000 x g are available.
Benchtop models have a smaller footprint, and general-purpose models are ideal for a wide range of applications. There are many benchtop models available, including high-speed, microcentrifuge, clinical, and cell washer models. Clinical benchtop models and cell washers typically operate at lower speeds, and are suited to diagnostic applications, and washing debris from red blood cells.
Centrifuges for different applications
It is essential to select a centrifuge that is suited to the specific application. When purchasing a centrifuge, it is important to consider the following questions:
- What sample volumes are you working with? For processes involving large or varying volumes, a floor-standing model with higher capacity and different rotor configurations may be the best solution.
- Are samples temperature sensitive? If so, a centrifuge with refrigeration and temperature control options is required.
- Will the centrifuge be used for processing clinical or blood banking samples? Cell washers or clinical models are available for these specific applications.
- How much laboratory space is available vs the centrifuge footprint?
- What is the maximum g-force the centrifuge is capable of generating? Low-speed centrifuges are ideal for separating whole cells, while ultracentrifuges are necessary for separating DNA and RNA.
Part 4: What safety precautions should be taken when working with a centrifuge?
Ensure a sturdy, level worksurface
Always ensure the centrifuge is on an appropriate surface prior to operation.
Balance the centrifuge
Running an unbalanced centrifuge may cause significant damage, and injure the operator and other laboratory personnel. The total mass of each tube should be as close as possible- this becomes increasingly important at very high rotor speeds. Balancing masses to the nearest 0.1 gram is advisable, and it is important to balance tubes by mass, not volume. For example, do not balance a sample consisting of liquid with a higher or lower density than water with an equal volume of water.
Do not open the lid while the rotor is moving
Many centrifuges have a “safety shutoff”. However, this will only stop power to the rotor, which will still spin due to its own inertia for some time until it is slowed to a stop by friction.
If the centrifuge is wobbling or shaking, pull the plug
A little vibration is normal, but excessive amounts can mean danger. First, double check that the tubes are correctly balanced. If this does not resolve the issue, do not operate the centrifuge until it has been serviced by the manufacturer or dealer.
Part 5: How do you balance a centrifuge?
Why you need to balance a centrifuge
Prior to starting the centrifuge, it is necessary to load it correctly. Balancing the centrifuge prevents potential damage to the instrument, and is crucial for safe operation.
How to balance a centrifuge
- Ensure all sample tubes are evenly filled. If additional tubes are required for balancing, fill them with water or a liquid of similar density to the sample, and ensure the mass is balanced to the nearest 0.1 grams.
- For each tube inserted in the rotor, add a tube of equal weight directly opposite it. This will ensure the center of gravity remains in the center of the rotor.
- Rotate the rotor 90° and add two additional tubes directly opposite one another.
How to balance 3 tubes, 5 tubes, or 7 tubes in a centrifuge with 12 positions
There are two ways to balance three tubes. The first option is to insert three sample tubes next to each other, and create three balance tubes to be situated directly across from the sample tubes.
Alternatively, three sample tubes may be spaced evenly around the rotor.
To balance five tubes, create one balance tube and place two sets of three tubes across from each other.
To balance seven tubes, create one balance tube and place two sets of four tubes across from each other.
Part 6: How do you maintain a centrifuge?
Centrifuge care and maintenance
A few simple steps can keep a centrifuge functioning properly and reduce the risk of damage or injury.
- Keep the centrifuge properly lubricated. O-rings are the main source of protection against sample leakage, and must be lubricated prior to installation of a new rotor or following cleaning. Any threaded components should also be cleaned regularly and lubricated with an approved grease to ensure proper operation and to prevent cross-threading and corrosion.
- Ensure all users are aware of how to properly operate the centrifuge, including ensuring buckets are properly seated in their pins, balancing tubes in the rotor, operating rotors within stated guidelines for speed and maximum compartment mass, and avoiding scratching the rotor.
- Inspect critical components, and look for signs of wear including scratches, or effects of chemical exposure on the rotor.
- Pay close attention to noise, vibration, shaking, or grinding and stop the unit immediately if this occurs.
Regularly clean the centrifuge with neutral cleaning solutions (alcohol or alcohol-based disinfectant) applied with a soft cloth to rotors and accessories. Daily cleaning should include the interior portion of the centrifuge, the rotor chamber, and surfaces with electronic components, such as touchscreens and keypads.
It is important to be aware of the different types of samples used with the centrifuge and any specific products or protocols necessary for cleaning spills.