Page 30 - Subyek Encyclopedia - Encyclopedia of Separation Science

Sepsci*11*TSK*Venkatachala=BG
I / CENTRIFUGATION 25

centrifugal speeds. This is achieved by routing the be stable in solution;
solutions to the rotor wall through veins in the central be inert towards the fractionated materials, includ-
core. When such self-generating gradients are used, it ing biological activity;
is not necessary that the sample be layered on top of exert the minimum osmotic effect, ionic
the solution but instead it may be mixed with the strength and pH;
medium prior to loading (Figure 5). While self- be removable from the product;
generating gradients offer greater simplicity, they be readily available and either inexpensive or easily
often require a signiRcant increase in run time. For recyclable;
instance, though the advent of vertical tubes, faster be sterilizable.
centrifugal speeds, and overspeeding techniques have
reduced run times to about one-third of those re- It should not:
quired only a few years ago, runs of 3 to 12 h are still generate a prohibitively high viscosity;
typical for DNA banding experiments. interfere with the assay technique (e.g. absorb UV
Isopycnic separation is a more powerful separation or visible light);
tool than rate-zonal separation in the sense that a gen- be corrosive; or
erally greater number of particle types can be re- generate Sammable or toxic aerosols.
solved. However, rate runs may still be preferred for
separating large and/or fragile particles, since shorter From this list of properties, it is apparent that no
run times and lower centrifugal forces are used. Run single ideal gradient material exists, as each separ-
duration is crucial for a rate separation, whereas ation problem imposes its own set of requirements.
isopycnic runs simply require a minimum time for the Rather, selection can only be made after a careful
particles to reach a stationary state. It is sometimes evaluation of the gradient properties with respect to
useful to conduct a two-dimensional separation in the requirements imposed by the separation to be
which, for instance, a rate-zonal run generates frac- conducted. The list of materials that have been used
tions of particles with similar S values that are further for gradient formation is extensive with examples
fractionated according to density in an isopycnic sep- of the more commonly used materials along with
aration. The reverse process can also be performed to selected properties listed in Table 1.
yield particles of similar density but different With respect to biological inertness and low viscos-
particle size distributions. ity, the ideal aqueous gradient material is deuterium
oxide (D 2 O). However, D 2 O is expensive and has
Gradient materials The selection of an appropriate a relatively low maximum density (1.11 g cm ).
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gradient material is an important consideration as the Sucrose was used in the pioneering density-gradient
gradient properties must be compatible with the sep- work of Brakke and, due to its low cost, transpar-
aration objectives. The desired properties of an ideal ency, ready availability and nontoxic nature, is still
gradient material, as set forth by GrifRth and by the most widely used. Densities to 1.33 g cm can be
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Ridge, are summarized below. achieved, which is sufRcient for separating most
The ideal gradient material should: cells and intracellular organelles. However, sucrose
span a density range sufRcient to permit separ- solutions are not completely physiologically in-
ation of the particles of interest without overstress- active and often contain UV-absorbing components.
ing the rotor; Mannitol and sorbitol can be used as substitutes to

Table 1 Physical properties of gradient materials in aqueous solutions at 203C (from Sheeler, 1981)
Gradient material Tradename Maximum solution concentration 20% w/w solution

Concentration Density Viscocity (cP) Density Viscosity (cP)
(% w/w) (gcm ) (gcm )
3
3
Sucrose 65 1.33 182 1.08 2
Sucrose polymer Ficoll 43 1.17 600 1.07 27
Colloidal silica Ludox-SM } 1.40 } 1.13 2
Colloidal silica Percoll 23 1.13 10 1.11 8
Metrizamide 56 1.44 58 1.12 2
CsCl 65 1.91 1.3 1.17 0.9
Polytungstate salt LST 85 2.89 14 1.20 }
Polytungstate salt SPT 85 2.89 26 1.20 2