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Centrifugation separates suspended material by its motion relative to a rotating liquid. A centrifuge rotor creates an outward centrifugal acceleration in the rotating reference frame; material denser than its surroundings tends to move outward, opposed by drag force. Collected material can form a centrifugation pellet, leaving a supernatant, or remain in separated zones within a density gradient. The useful question is which physical difference the separation exploits: a difference in sedimentation coefficient or a difference in buoyant density.

Centrifugation makes otherwise slow settling useful for studying biological cells, organelles, proteins and DNA. Separation and identification are different steps: a centrifugation pellet is a collected fraction, not proof that it contains only one kind of organelle. The lysozyme wall-removal experiment illustrates a complementary readout: testing material released into the separated liquid can be more informative than inspecting the retained solids.

A centrifugation pellet is material accumulated against the vessel wall by centrifugation. Its existence establishes collection, not purity; other suspended material can be trapped or co-sediment.

A centrifuge rotor holds sample vessels around the rotation axis. A fixed-angle centrifuge rotor holds tubes at a fixed inclination; a swinging-bucket centrifuge rotor lets them swing outward during rotation. Material moves away from the axis, so a centrifugation pellet can first collect against an outer tube wall rather than travel along the entire tube axis. Geometry sets both the rotor radius and the travel distance.^[ref]

Density-gradient centrifugation uses a liquid whose density varies along the separation direction. A band does not by itself establish which mechanism produced it: rate-zonal centrifugation separates by migration rate before equilibrium, whereas isopycnic centrifugation uses equilibrium positions set by buoyant density.

The distinction is between a moving clock and a restoring balance. In rate-zonal centrifugation, two species may occupy different positions because their sedimentation coefficients differ, even when their buoyant densities agree. In isopycnic centrifugation, two differently sized species can converge to the same band because their buoyant densities agree. Conversely, equal mass does not ensure equal migration velocity: shape affects drag force, and medium density affects buoyancy.^[ref]

The worked finite-time rate-zonal separation and linear-gradient isopycnic focusing examples isolate these mechanisms in chosen mathematical models. The actual DNA experiment described under isopycnic centrifugation illustrates the second mechanism; its initial collection of biological cells into centrifugation pellets is a different operation from the later molecular banding. Collecting liquid fractions from a tube can recover material at different positions, but the position alone does not identify its molecules or establish purity.

Differential centrifugation successively separates centrifugation pellets from supernatants by differences in settling rate. A first centrifugation pellet can enrich faster-settling material; a later centrifugation pellet from its supernatant can enrich slower-settling material. The fractions overlap because particles have different starting positions and can become trapped. Differential centrifugation is consequently an enrichment method, not automatic identification or complete purification.^[ref]

Consider two dilute populations of spheres with the same buoyant density and radii $b$ and $2b$. Adopt the fixed linear density gradient and constant local centrifugal acceleration of linear-gradient isopycnic focusing, with constant dynamic viscosity and negligible diffusion. Stokes's law makes $V/f$ proportional to $b^2$, so doubling radius multiplies the migration rate parameter by four, although the mass increases eightfold.

Use $u=z/L$, $\tau =t/T$ and $q=({\rho }_b-{\rho }_0)/(GL)=2$. The liquid occupies $0\le u\le 1$, so the matching density position lies beyond its outer boundary. Both populations begin at $u(0)=0.1$; choose ${\lambda }_A=0.15$ and ${\lambda }_B=0.6$. Their band-center trajectories areAt $\tau =0.5$, they lie at approximately $0.2373$ and $0.5924$; at $\tau =1$, at $0.3647$ and $0.9573$. Equal buoyant density has not prevented a useful finite-time separation. Solving $u_B(\tau )=1$ using the natural logarithm gives first boundary arrival at $\tau =\ln (1.9)/0.6\simeq 1.0698$. The corresponding arrival for A is $\ln (1.9)/0.15\simeq 4.2790$. Trajectories describe migration only until contact with the boundary; subsequent collection into a centrifugation pellet is not represented by extending the curve beyond $u=1$.

This is the useful contrast with isopycnic centrifugation: stopping time helps determine the recovered positions in rate-zonal centrifugation, whereas a stationary matching density position sets the limiting band center in isopycnic centrifugation. Real resolution also depends on initial zone width, diffusion and variations in particle properties; distinct calculated centers alone do not prove that fractions will be pure.

In isopycnic centrifugation, a species concentrates near the position where the liquid's density matches its buoyant density. In a density gradient increasing outward, material displaced inward encounters less-dense liquid and tends to move outward; material displaced outward encounters denser liquid and tends to move inward. Diffusion broadens the resulting band. If the density gradient never reaches the species' buoyant density, a matching equilibrium position need not exist within the vessel.^[ref]

The 1958 paper The replication of DNA in Escherichia coli by Matthew Meselson and Franklin W. Stahl (pp. 672–676, Figures 1–4) makes the distinction concrete. Its separation apparatus was a Spinco Model E ultracentrifuge with an Escherichia coli lysate in a cesium chloride density gradient. Ultraviolet wave absorption photographs of the rotating sample were scanned with a recording microdensitometer: peak position reported buoyant density, while signal above baseline reported DNA concentration.^[ref]

Cultures received labeled nitrogen; chilled biological cells were collected as centrifugation pellets and disrupted before molecular banding.^[ref]

Mixed lysates labeled fully with the nitrogen isotope ${}^{15}\mathrm{N}$ and unlabeled lysates supplied reference bands; a mixture including intermediate material checked its position relative to those references. Figure 1 followed band formation over time, helping distinguish a migrating distribution from an approximately stationary one. These controls address resolution, calibration and approach to equilibrium. They do not make every ultraviolet-absorbing component identifiable as DNA, or turn a band into a direct image of individual strands. The DNA replication article develops the copying inference; here the key result is a separation by buoyant density, not a claim that heavier-labeled DNA is physically larger.^[ref]

Rate-zonal centrifugation resolves an initially narrow sample zone by differences in sedimentation coefficient during a finite interval. Its bands remain transient: extending the interval can carry material into a centrifugation pellet. Thus a density gradient need not imply an equilibrium buoyant density measurement.^[ref]

A typical preparation layers a narrow sample above a preformed density gradient, often containing sucrose. The initial zone must be narrow enough that differences in starting position do not overwhelm the later separation. The run ends while useful bands remain inside the liquid. In the simple case where all particles are denser than every part of the column, none has an interior isopycnic centrifugation position.^[ref][ref]

The sedimentation coefficient $s=(m-{\rho }_{f}V)/f$ explains why separation is not an ordering by mass alone. At fixed local centrifugal acceleration, increasing displaced volume can increase buoyancy, while a less compact shape can increase the linear friction coefficient and reduce velocity. Even for a sphere, Stokes's law gives $s=2b^2({\rho }_p-{\rho }_f)/(9\eta )$, so both the density difference and the dynamic viscosity matter. The resulting sedimentation coefficient can change along a density gradient.

A supernatant is the liquid remaining above settled or pelleted material. It can contain dissolved molecules and particles that have not reached the centrifugation pellet; a visually clear supernatant need not be chemically empty.