Centrifuge & Sedimentation

Spinning replaces waiting

Left alone under ordinary gravity, particles of different density and size in a suspension eventually settle out — but for small particles like blood cells or protein complexes, that natural settling can take hours or days. A centrifuge replaces Earth's 1g with an artificial field that can reach hundreds of thousands of g, turning a settling process that would take days into one that takes minutes.

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Relative centrifugal force

Spinning a sample at angular velocity ω at radius r from the axis subjects it to a centripetal acceleration of ω²r, conventionally reported as a multiple of Earth's gravity — relative centrifugal force, RCF, colloquially just '×g':

RCF = omega^2 . r / g          omega = 2.pi.N / 60   (N = rotor speed in rpm)

example: N = 10,000 rpm, r = 0.08 m  ->  omega ~ 1047 rad/s  ->  RCF ~ 9,300 x g

Because RCF scales with the square of rotor speed, doubling the rpm quadruples the force — which is why a centrifuge's speed dial has an outsized effect on separation time, and why the same rotor at 5,000 versus 15,000 rpm can be the difference between a gentle plasma separation and one violent enough to lyse cells.

Stokes' law: how fast a particle actually moves

A particle does not separate instantly just because a force acts on it — it moves at a terminal velocity set by the balance between that force and the viscous drag of the surrounding fluid. For a small sphere in laminar (low-Reynolds-number) flow, Stokes' law gives that terminal sedimentation velocity, and the centrifugal version simply replaces gravitational acceleration g with the local centrifugal acceleration ω²r:

v = d^2 . (rho_p - rho_f) . omega^2 . r / (18 . eta)

d       = particle diameter
rho_p, rho_f = density of particle, of fluid
eta     = fluid viscosity

Two consequences fall straight out of that equation. Velocity scales with the square of diameter, so a particle twice the size sediments four times as fast — which is why differential centrifugation separates by size and density in stages, spinning briefly at low speed to pellet the largest structures first, then progressively faster and longer to pull down smaller and smaller ones. And a particle whose density exactly matches the surrounding fluid (ρ_p = ρ_f) has zero net velocity no matter how hard it is spun — the basis of isopycnic ('equal density') separation.

Differential centrifugation: separating blood

A single blood sample spun at moderate speed for several minutes separates cleanly into three visible layers purely by density difference. Red blood cells, the densest component, pellet at the bottom. Above them sits a thin, pale buffy coat of white blood cells and platelets, intermediate in density. On top floats the straw-coloured plasma, the least dense, essentially water with dissolved proteins and clotting factors. This same staged principle — spin, decant the supernatant, resuspend the pellet, spin again at a different speed — is the standard workflow for isolating everything from blood components to subcellular organelles.

Density-gradient centrifugation: sorting by density alone

For finer separation, samples are layered on top of a pre-formed density gradient (commonly sucrose or caesium chloride, dense at the bottom and dilute at the top) before spinning. In rate-zonal centrifugation, particles are separated primarily by size and shape, migrating at different speeds through the gradient and the run is stopped before anyone reaches equilibrium. In isopycnic (equilibrium) centrifugation, the run continues until every particle stops migrating altogether — settling at the exact point in the gradient where the surrounding fluid density matches its own — which separates purely by density, independent of size or shape, and is precise enough to separate DNA of slightly different densities, as in the classic Meselson-Stahl experiment that confirmed semiconservative DNA replication.

Frequently asked questions

Why does doubling a centrifuge's rpm do so much more than doubling its speed?

Relative centrifugal force scales with the square of angular velocity, RCF = omega^2 r / g, so doubling the rotor speed quadruples the applied force. That squared relationship is why small adjustments to the speed dial have an outsized effect on both separation time and the risk of damaging fragile samples.

Why do larger particles separate out faster than smaller ones at the same speed?

Stokes' law makes sedimentation velocity proportional to the square of particle diameter, so a particle twice as large moves through the fluid four times as fast under the same centrifugal force. This is exactly why differential centrifugation works in stages: a short, gentle spin pellets only the largest structures, and each subsequent longer or faster spin pulls down progressively smaller ones.

What is the difference between rate-zonal and isopycnic density-gradient centrifugation?

Rate-zonal centrifugation is stopped before particles reach equilibrium, so it separates mainly by size and shape as particles migrate through the gradient at different speeds. Isopycnic centrifugation runs until every particle settles at the exact gradient position matching its own density and stops moving there, which separates purely by density and is precise enough to resolve molecules that differ only slightly in density, such as DNA labelled with different isotopes.

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Centrifuges: Using Rotation to Force Sedimentation