principles of centrifigation
Answers
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The centrifuge works using the sedimentation principle, where the centrifugal 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.
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Answer:
Principles of centrifigation
Explanation:
Basics in Centrifugation
Centrifugation is a technique that helps to separate mixtures by applying centrifugal force. A centrifuge is a device, generally driven by an electric motor, that puts an object, e.g., a rotor, in a rotational movement around a fixed axis.
A centrifuge works by using the principle of sedimentation: Under the influence of gravitational force (g-force), substances separate according to their density. Different types of separation are known, including isopycnic, ultrafiltration, density gradient, phase separation, and pelleting.
Pelleting is the most common application for centrifuges. Here, particles are concentrated as a pellet at the bottom of the centrifuge tube and separated from the remaining solution, called supernatant. During phase separation, chemicals are converted from a matrix or an aqueous medium to a solvent (for additional chemical or molecular biological analysis). In ultrafiltration, macromolecules are purified, separated, and concentrated by using a membrane. Isopycnic centrifugation is carried out using a "self-generating" density gradient established through equilibrium sedimentation. This method concentrates the analysis matches with those of the surrounding solution. Protocols for centrifugation typically specify the relative centrifugal force (rcf) and the degree of acceleration in multiples of g (g-force). Working with the rotational speed, such as revolutions per minute (rpm), is rather imprecise.
Important definitions
In general, applications for centrifugation specify the degree of acceleration to be applied to the sample rather than specifying a specific rotational speed such as revolutions per minute. The acceleration is typically given in gravity [× g] (or multiples of x g or g-force), the standard acceleration value due to gravity at the Earth’s surface (9.81 m/s2). The distinction between rpm and rcf is important, as two rotors with different diameters running at the same rotational speed (rpm) will result in different accelerations (rcf).
Why?
As the motion of the rotor is circular, the acceleration force is calculated as the product of the radius and the square of the angular velocity. Historically known as “relative centrifugal force” (rcf), this is the measurement of the acceleration applied to a sample within a circular movement. This process is measured in units of gravity
(× g).
Example*
Rotor A Rotor B
Speed 14,000 rpm 14,000 rpm
Radius 5.98 cm 9.50 cm
Gravity 13,100 × g 20,817 × g
*using the formula above
As mentioned, when using
rotors with different radii for centrifugation, the same rcf
(g-force) should be used.
A small k-factor represents a more rapid separation. The value of the k-factor is primarily determined by the rotor diameter. Compared to rpm/rcf, the usage of the k-factor has become less important for general centrifugation processes. Especially for ultracentrifugation, the k-factor is still relevant.
How to select the right centrifuge for your application
If you follow a given protocol, make sure to use the same type of rotor and apply the given relative centrifugal force (rcf) as well as the same temperature and running time. In general, the following major parameters have to be determined for a successful centrifugation run:
A: Type of sample
B: Vessel selection
C: Type of centrifuge
D: Type of rotor
E: Determination of desired relative centrifugal force
F: Defined temperature during centrifugation
Fixed-angle or swing-bucket rotors
Swing-bucket rotor
This kind of rotor is highly flexible for using different tube formats, including SBS-format plates, based on a broad range of adapter systems and a high sample capacity. The moving swing-bucket parts result in increased metal stress for the rotor and the buckets as the bucket weight places a load on the two pivots and grooves. Compared with a fixed-angle rotor, therefore, a swing-bucket rotor is limited to a lower maximum g-force, which leads to longer centrifugation times. Based on the swing-bucket principle, the pellet is located in the bottom of the tube (horizontal position of tube during the run). The recovery by the user is facilitated compared to pellets located at the side of the tube.
The centrifuge
In general, centrifuges are classified either as floor-standing or bench-top models.
Eppendorf solutions
Centrifuges/eShop
Floor-standing centrifuges
Bench-top centrifuges
Bench-top centrifuges are available in different sizes:
Microcentrifuges
Microcentrifuges are optimized for low-volume tubes, have a small footprint, and provide 14,000 to 30,000 × g for up to 48 microtubes. Some devices can even be used for a few 15 mL or 50 mL conical tubes or 2 SBS-format plates. Many suppliers offer non-refrigerated and refrigerated versions and different sizes of devices based on their tube capacity.