Increasing the effect of gravity: the centrifuge. Table 1. Types of Centrifugal Separations. Differential centrifugation. Density gradient centrifugation. Rate zonal size separation Rate-zonal separation takes advantage of particle size and mass instead of particle density for sedimentation. Criteria for successful rate-zonal centrifugation: Density of the sample solution must be less than that of the lowest density portion of the gradient.
Density of the sample particle must be greater than that of the highest density portion of the gradient. The pathlength of the gradient must be sufficient for the separation to occur. Time is important. If you perform too long runs, particles may all pellet at the bottom of the tube. Isopycnic separation In this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle.
Criteria for successful isopycnic separation: Density of the sample particle must fall within the limits of the gradient densities. Any gradient length is acceptable. The run time must be sufficient for the particles to band at their isopycnic point. Excessive run times have no adverse effect. Table 2. Applications of density gradient media for isopycnic separations Table scrolls horizontally Gradient media Cells Viruses Organelles Nucleoproteins Macromolecules Sugars e.
Rotor categories Rotors can be broadly classified into three common categories namely swinging-bucket rotors, fixed-angle rotors, and vertical rotors Figure 4, Table 3. Table 3. Selection of Centrifuge Tubes.
Table 4. Selection of the appropriate centrifuge tube: Prevents sample leakage or loss Ensures chemical compatibility Allows easy sample recovery Major factor in selection of a tube plastic material: Clarity Chemical resistance Sealing mechanism if needed check product guide pages or tube packaging for notes on recommended sample volume and maximum speed. To prolong tube life and avoid breakage or collapse: Table 5. Common Centrifugation Vocabulary and Formulas. Pellet: hard-packed concentration of particles in a tube or rotor after centrifugation.
Supernatant: The clarified liquid above the pellet. Adapter: A device used to fit smaller tubes or centrifugal devices in the rotor cavities. R max : Maximum radius from the axis of rotation in centimeters.
R min : Minimum radius from the axis of rotation in centimeters. 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.
Centrifuges may be classified based on maximum speeds, measured as revolutions per minute RPM. 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.
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, x g , and ultracentrifuges often used for DNA or RNA fractionation, can achieve up to 1,, x g.
For large-capacity, low-speed applications, low-speed centrifuges reaching approximately 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. The shorter the sedimentation distance, the shorter the centrifugation time. In addition, the angle of the tubes within the rotor influences the sedimentation distance.
The radius of the rotor and the angle of the bores as well as the type of vessel respectively the adapter you use will all have an impact on the sedimentation distance and thus on the centrifugation time you will need. When processing blood for serum, manufacturers of evacuated collection tubes often recommend a period of time to allow the blood to clot prior to centrifugation. This may range from minutes depending on the manufacturer.
Some manufacturers use a clot activator, which is coated onto the interior surface of the tubes.
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