Page 29 - Subyek Encyclopedia - Encyclopedia of Separation Science

24 I / CENTRIFUGATION/ Derivatization

conducted in a density gradient. The density range of
the gradient often spans the full range of particle
densities so that particles never reach the rotor wall,
regardless of run time. Instead, particles move
through the gradient until they reach a position in
which the medium density is the same as their own
(Figure 5). As governed by the settling velocity equa-
tions (eqns [13] and [14]), particles introduced to the
top of a performed gradient sediment relatively
quickly at Rrst, with movement slowing as the dif-
ference in density between particles and gradient les-
sens and essentially stopping once the particles reach
a position in the rotor where the density of the me-
dium is equal to their own. Particles remain in this
terminal position even after the rotor is stopped; this
allows them to be recovered as density fractions.
Differences in particle size only affect their
rate of movement, though this may ultimately dictate
the required run time. When the range of particle
Figure 4 Rate-zonal separation in a swinging-bucket rotor. densities exceeds the range of the density gradient,
(Courtesy of Beckman Instruments, Inc.) then a mixture of pelleting and isopycnic separations
will occur as some particles fully traverse the rotor
separation, larger particles sediment more rapidly, and pellet while others attain their isopycnic position
just as in a pelleting run. Also similar to a pelleting and remain suspended. While most density gradients
run, the maximum medium density is lower than the are formed by the loading of solutions of successively
density of the particles being processed. However, higher density to the rotor, it is possible to form such
unlike pelleting runs, the run must be stopped before gradients in situ from a homogeneous solution at high
particles reach the bottom of the tube or rotor wall,
otherwise all sample components will simply sedi-
ment to the pellet.
Rate or setting velocity separations may be conduc-
ted with a homogeneous medium in batch or semi-
batch mode. However, the use of density-gradient
media offers several advantages. The steep gradi-
ent beneath the layer of sample suppresses premature
sedimentation as well as convection currents in the
liquid column, both of which lower the separation
efRciency. In addition, the continuous increase in
density, often accompanied by an increase in viscosity
across the rotor, serves to slow the faster-moving
particles and provide better resolution in the sample
component bands. Increasing-viscosity gradients also
lessen diffusional effects, though this ad-
vantage may be offset by an increase in the re-
quired run time. Rate-zonal separations are well
suited for mixtures of particles of similar density that
exhibit two or more well-deRned modes of size distri-
bution. However, owing to the additional steps and
equipment required for DGC as opposed to pelleting,
DGC separations are more commonly used to separ-
ate particle mixtures based on a parameter other than
size, e.g. density.

Isopycnic separations These separations, which are Figure 5 Isopycnic separation with a self-generating gradient.
based on differences in particle densities, are (Courtesy of Beckman Instruments, Inc.)