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A brominated thymidine analogue incorporated during DNA synthesis. Substitution changes buoyant density, allowing copied strands to be distinguished from unsubstituted strands.

Buoyant density is the surrounding liquid density at which a species has no net centrifugal drift under the stated conditions. It depends on interactions with the medium, including associated solvent, rather than necessarily equaling the dry substance's density. Equal buoyant densities can give overlapping bands even for differently sized proteins or DNA fragments.^[ref]

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.

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.

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]

Why does an isopycnic centrifugation band return after displacement? Choose a dilute mathematical model with outward coordinate $z$, constant local centrifugal acceleration $a>0$, fixed displaced volume $V>0$, linear friction coefficient $f>0$, and an imposed liquid density ${\rho }_f(z)={\rho }_0+Gz$ with $G>0$. Treat the species' buoyant density ${\rho }_b$ as constant in this medium. The sedimentation coefficient force balance gives the ordinary differential equationIts exponential function solution is $z(t)=z_{\ast }+(z(0)-z_{\ast })e^{-kt}$. The sign of the velocity reverses across $z_{\ast }$, proving stable equilibrium when $z_{\ast }$ lies inside the liquid. Changing $V/f$ changes the approach time $1/k$, while $z_{\ast }$ depends on buoyant density and the imposed density gradient. A real centrifuge rotor has radially varying centrifugal acceleration; the constant-$a$ approximation describes a short radial interval. Here the density gradient is held fixed, rather than modeling its formation.

For an original dimensionless example, let $u=z/L$ and $\tau =t/T$, where $L$ is column length and $T$ a chosen time scale. Write $q=z_{\ast }/L$ and $\lambda =kT$, so $u(\tau )=q+(u(0)-q)e^{-\lambda \tau }$. Species A and B have the same $q=0.45$ but different friction-dependent rates: $({\lambda }_A,u_A(0))=(0.4,0.1)$ and $({\lambda }_B,u_B(0))=(1.6,0.95)$. A moves outward and B inward, both toward $u=0.45$. Species C has $(q_C,{\lambda }_C,u_C(0))=(0.8,0.8,0.1)$ and approaches a separate band at $u=0.8$. At $\tau =4$, their positions are approximately $0.3793$, $0.4508$ and $0.7715$: a finite-time photograph would still show A short of its limiting position.

An equilibrium band has finite width because diffusion opposes focusing. In the same local model, let $c(z)$ be concentration and let the constant diffusion coefficient be $D>0$. Combining the restoring drift with Fick's laws, zero net flux requiresDividing by $c$ and integrating proves the Gaussian function profile, whose standard deviation is ${\sigma }_z=\sqrt{D/k}$. This local result assumes that the band is narrow relative to its distance from the boundaries and that $D$, $k$ and the density gradient vary negligibly across it. Equal buoyant densities therefore imply coincident centers in this model, not identical widths. A band outside the available density range has no such interior restoring point.

Base composition changes both the initial buoyant density and the size of the BUdR-induced density increment. The fraction-number axis runs toward lower density. The unlabelled viral peak lies at a lower fraction number than the corresponding host peak, consistent with viral DNA having a higher GC content. BUdR replaces thymidine in newly synthesized strands, so relatively AT-rich DNA incorporates more brominated nucleoside per unit length and undergoes a larger density increase.

Consequently the more AT-rich host DNA can overtake the viral DNA in the fully substituted class: the host fully heavy peak is slightly farther toward the dense end, although host unlabelled DNA was less dense. The hybrid peaks are intermediate. A useful qualitative model is ${\rho }_{LL}={\rho }_0(g)$, ${\rho }_{LH}={\rho }_0(g)+\Delta \rho (g)/2$ and ${\rho }_{HH}={\rho }_0(g)+\Delta \rho (g)$, where $g$ is GC fraction, ${\rho }_0$ increases with $g$, and $\Delta \rho$ decreases as the thymidine fraction decreases. Unequal substitution efficiencies could contribute, but different replication mechanisms are unnecessary. These are equilibrium density positions, not molecular-length measurements.

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.