Centrifugation

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.

Apparatus that rotates samples to separate components according to their sedimentation behaviour.

Analytical ultracentrifugation observes concentration profiles during rotation, rather than only recovering a separated pellet. Sector-shaped sample cells rotate past optical detectors. Absorbance scans measure attenuation at selected radii. A Rayleigh interferometer compares a sample sector with a solvent reference, converting concentration-dependent refractive-index changes into fringe shifts. Repeating scans follows a sedimentation boundary or the approach to a stationary distribution.^[ref]

For a dilute solute of buoyant mass $m_b$ and translational friction $f_h$, radial force balance gives $s=m_b/f_h$, where $s$ is the sedimentation coefficient. Diffusion supplies $D=k_{B}T/f_h$. A velocity experiment resolves boundary motion and broadening; a sedimentation equilibrium experiment instead measures $d\ln c/d(r^2)=m_b{\omega }^2/(2k_{B}T)$. This follows from the Boltzmann distribution in centrifugal potential $-m_b{\omega }^2{r}^2/2$. The two approaches separate frictional size information from buoyant mass information; shape, hydration and solvent properties must be specified to infer a unique geometric dimension.

Let $M$ be molar mass in $\mathrm{kg}\mathrm{mo}{\mathrm{l}}^{-1}$, $\bar{v}$ partial specific volume, ${\rho }_s$ solvent density, and $N_A$ the Avogadro constant. The buoyant molecular mass is $m_b=M(1-\bar{v}{\rho }_s)/N_A$. Combining sedimentation coefficient $s=m_b/f_h$ and the thermal diffusion-friction relation $D=k_{B}T/f_h$ eliminates the unknown friction and proves the displayed formula, with $R_{\mathrm{gas}}=N_A{k}_B$. Once friction is known, $R_h=f_h/(6\pi \eta )$ defines a hydrodynamic radius using Stokes's law. Mass and radius are different inferences: an extended molecule need not occupy the volume of that friction-equivalent sphere.

A microcentrifuge performs centrifugation of small sample tubes, as used to concentrate biological cells and separate debris in the PV92 teaching experiment.

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]

The rotor radius $r$ is the perpendicular distance from the rotation axis to a specified sample position, not the tube's length. A filled tube spans a range $r_{\min }\le r\le r_{\max }$. Consequently, one rotational speed produces different local centrifugal accelerations along that range. A reported relative centrifugal force needs a stated reference position, such as the outermost sample radius.

Relative centrifugal force is the dimensionless ratio of the local centrifugal acceleration magnitude $a$ to the conventional gravitational reference $g_0=9.80665\mathrm{m}{\mathrm{s}}^{-2}$. Despite its name, it is an acceleration ratio: the actual force on a specified mass $m$ is $ma$. With angular speed $\omega$ and rotor radius $r$, $a={\omega }^{2}r$. If $n$ is the numerical rotational speed in revolutions per minute and $r_{\mathrm{cm}}$ the numerical rotor radius in centimetres, unit conversion givesThus rpm alone does not specify the centrifugal acceleration.^[ref]

For a stipulated $3000\mathrm{rpm}$, rotor radii of $5$ and $10\mathrm{cm}$ give approximately $503$ and $1006$ times $g_0$. Doubling the rotor radius doubles relative centrifugal force; doubling rpm quadruples it. Even equal relative centrifugal forces do not guarantee equal separation: travel distance, elapsed time, viscosity, sample density and particle shape still matter. These are comparisons of a mathematical model, not instructions for running an instrument.

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 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.

The sedimentation coefficient $s$ is radial drift velocity divided by local centrifugal acceleration. It has units of time and combines particle properties with those of the surrounding liquid. In a dilute, deterministic mathematical model with negligible particle inertia, an effective displaced volume $V$, particle mass $m$, local liquid density ${\rho }_f$ and linear friction coefficient $f>0$ giveThe subtraction represents buoyancy. Equal mass does not imply equal $s$: changing shape changes drag force, and changing the medium changes both buoyancy and viscosity.^[ref]

For an isolated sphere of radius $b$ and uniform density ${\rho }_p$, Stokes's law supplies $f=6\pi \eta b$, where $\eta$ is dynamic viscosity, and $m=(4\pi /3)b^3{\rho }_p$. ThereforeThis original force-balance check predicts fourfold $s$ on doubling $b$ with everything else fixed, and zero drift when ${\rho }_p={\rho }_f$. It applies to dilute isolated spheres in Stokes flow, not arbitrary folded proteins or crowded suspensions. If $s$ and $\omega$ remain constant in a uniform medium, the ordinary differential equation $dr/dt=s{\omega }^{2}r$ has the exponential function solution $r(t)=r(0)e^{s{\omega }^{2}t}$. Arrival at an outer boundary therefore depends on starting position as well as $s$; a separation has no universal size cutoff.

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]

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.

Separation in which a particle's buoyant properties cause it to move toward a lower-density part of a gradient. A liposome fraction can thereby be separated from unassociated soluble proteins; copurification alone does not demonstrate correct membrane topology.

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.

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]

A cesium chloride density gradient forms when the salt $\mathrm{CsCl}$ redistributes in a rotating chemical solution. It can supply matching-density positions for DNA during isopycnic centrifugation.^[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.

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.