Page 32 - Subyek Encyclopedia - Encyclopedia of Separation Science

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I / CENTRIFUGATION 27

In a linear gradient, density increases linearly with mated fractionators that select cut points and auto-
distance from the axis of rotation (Figure 6A), and matically switch collection vessels rely on such in-line
for cylindrical swing-out rotors, with increasing detectors.
gradient volume as well. In an exponential gradient,
the density increases or decreases exponentially Analytical Centrifugation
across the rotor, producing convex or concave This is the only type of centrifugal separation in
shapes, respectively, when plotted as a function of which the primary objective is not to purify or de-
radial distance (Figure 6B). Isokinetic gradients are water one or more of the feed components. Rather,
designed to produce a uniform sedimentation velocity this method is used to monitor particle sedimentation
throughout the gradient by counterbalancing the in- behaviour. Analytical centrifugation is used to char-
crease in centrifugal force particles experience as they acterize particle properties such as molecular weight,
traverse the gradient with an increase in the density diffusion and sedimentation coefRcients, buoyancy
and viscosity of the medium. Such gradients are often density, etc. The critical component in this technique
used in analytical rotors to study sedimentation be- is the addition of a transparent window, e.g. quartz or
haviour. Simple linear sucrose gradients loaded in sapphire, to the centrifuge rotor to permit in situ
a swinging rotor provide a near isokinetic gradient. optical measurements. Sample movement is typically
Various methods are used to form gradients. The monitored by UV absorption or refractive index dur-
simplest approach is to form the gradient in situ, i.e. ing high speed separations in ultracentrifuges. Experi-
self-generating, by mixing the sample with a single- ments are conducted in batch mode using very small
density medium prior to loading, then forming the sample volumes, as low as 5 L for some rotors. Two
gradient at high centrifugal speeds. While this is the classes of experiments are conducted in an analytical
simplest approach, higher speeds and longer run ultracentrifugation } sedimentation velocity and sedi-
times are often required. Step gradients are also easily mentation equilibrium } anlogous to rate and isopyc-
formed by simply pumping targeted volumes of suc- nic experiments in preparative ultracentrifugation.
cessively denser solutions to the rotor wall. Inexpen- Of these, sedimentation velocity is the more common.
sive peristaltic pumps provide the simplest means of Analytical centrifugation is less common today than
loading step gradients. The simplest liner-gradient in the 1950s when this was the principal method for
generators consist of two equivalent cross-section cy- molecular weight determinations (1}10 kDa). How-
linders that contain an initial and a limiting solution, ever, the method is still used, primarily in biological
respectively. The chambers are interconnected at the applications, for studying phenomena such as interac-
base with liquid from the limiting solution being tions between macromolecules and ligand-induced
drawn into and mixed with the initial solution as binding events. More recently, this technique has
material from the initial-solution chamber is loaded. experienced somewhat of a renaissance in drug dis-
Exponential gradient generators are similar except covery applications.
that the cross-sectional area of one of the chambers
changes in a predetermined manner as the chambers
are depleted, thereby changing the relative volume Continuous Centrifugation
contributed from the two chambers with time. More These separations are similar to those previously dis-
sophisticated gradient pumps are available including cussed in the sense that separations are based on size
mechanical pumps that use cams to mix variable or density differences. However, unlike batch-
amounts of low and a high density solution prior to mode separation, in continuous centrifugation the
loading or programmable pumps, e.g. a liquid sample mixture is introduced continuously to a spin-
chromatograph pump, to generate the targeted gradi- ning rotor as the supernatant stream continuously
ent curve shape. exits. For pelleting separations, the denser product
Several approaches are used to analyse and/or frac- may either accumulate on the rotor wall from where
tionate the rotor efSuent. The simplest is to split it is recovered after the rotor capacity is reached
the gradient into fractions according to volume, then (semi-batch) or continuously discharged as the rotor
subsequently analyse each fraction by chemical (den- spins (continuous mode). Continuous-feed centri-
sity, absorbance, refractive index, Suorescence) or fuges may be used for rate, pelleting, Rltration, or
scintillation methods. However, this approach may isopycnic banding separations. They are best suited
be somewhat limited in resolution if the collected for applications in which large volumes of sample
fractions are large, and thus represent a wider range must be processed, the stream to be recovered is at
in density. An alternative approach is to route the low concentration, the particle sedimentation coef-
efSuent through one or more in-line, low volume Rcient is high (less than about 50 S), or long acceler-
Sow cells to monitor the gradient properties. Auto- ation/deceleration times are required.