Counterflow centrifugation, also known as centrifugal elutriation (CCE), works by separating particles based on their size and density by balancing centrifugal force with a counteracting fluid flow.
Here's a breakdown of the process:
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Setup: A cell or particle suspension is introduced into a specialized centrifuge rotor chamber (the elutriation chamber). This chamber is designed to allow fluid flow in a direction opposite to the centrifugal force.
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Centrifugal Force: As the rotor spins, particles in the chamber experience a centrifugal force pushing them towards the outer edge of the chamber (away from the center of rotation). The magnitude of this force is dependent on the particle's size, density, and the rotor's speed (RPMs). Larger, denser particles experience a greater centrifugal force.
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Counteracting Fluid Flow: A buffer or liquid medium is pumped through the elutriation chamber in a direction opposite to the centrifugal force, i.e., towards the center of the rotor. This fluid flow exerts a drag force on the particles, attempting to wash them out of the chamber.
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Separation Principle: The key to separation lies in balancing these two opposing forces.
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Smaller particles experience a relatively smaller centrifugal force and are easily washed out of the chamber by the fluid flow at a lower flow rate.
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Larger particles experience a larger centrifugal force, which counteracts the fluid flow, allowing them to remain in the chamber.
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Fractionation: By gradually increasing the fluid flow rate, particles of progressively larger sizes can be selectively washed out of the chamber and collected as separate fractions. The rate of flow is carefully controlled. As the flow increases, the equilibrium position for differently sized particles shifts inward toward the axis of rotation until a point where the counterflow exceeds the centrifugal force on a population and they are washed out.
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Collection: The effluent containing the separated particles is collected in fractions for further analysis or use.
In essence, counterflow centrifugation dynamically balances centrifugal force with a controlled fluid flow, allowing for high-resolution separation of particles based on their physical properties. This technique is particularly useful for separating cells in a heterogeneous population based on size. The cell suspension passes through a chamber containing a dense liquid medium flowing at a controlled rate. Smaller particles are pushed out, and larger particles remain.
