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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.

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.