Page 31 - Subyek Encyclopedia - Encyclopedia of Separation Science

26 I / CENTRIFUGATION/ Derivatization

compensate for these deRciencies, but use of these for these materials include the separation of graphitic
sugars has disadvantages including higher viscosity carbon and mineral components from Sy ash. When
and lower maximum densities. Polysaccharides also using such high density salt solutions, the user should
have a low osmotic pressure, but again are more be aware that at high concentration, salts may pre-
viscous than sucrose solutions of equal density and cipitate on the rotor wall, thereby generating high
may induce aggregation of the suspended sample via point densities and the potential for catastrophic ro-
charge interactions. tor failure.
Silica sols (e.g. Ludox and Percoll ), also called For nonaqueous gradients, organic liquids such as
colloidal silica, are prepared from small silica par- toluene, methanol or kerosene may be blended to
ticles in mildly alkaline solution. They provide low attain gradient densities lower than that of water
3
viscosities and osmotic pressures, even at high densit- (1.0 g cm ). Of these, methanol presents an addi-
ies, and are transparent and inexpensive. Silica sols tional advantage of being water-soluble, thereby
3
provide densities to 1.40 g cm . Their disadvantages allowing gradients to be formed from a combination
include a tendency to gel at pH (7 and problems in of the two. On the other end of the density scale,
complete removal from the sample. Percoll , pre- halogenated liquids such as diodomethane, bromo-
pared by coating the silica particles with a polymer, form and tetrabromoethane can be used to prepare
3
eliminates the gelling problem and provides low vis- very dense solutions over 2.8 g cm . Problems asso-
cosity, low osmotic pressure solutions, greater stabil- ciated with Sammability, toxicity and attack of trans-
3
ity at low pH, and densities to 1.21 g cm . However, fer lines and seals must be considered when using
this material is relatively expensive and removal from these materials.
the sample can be a problem.
Salts are used to generate very high density aqueous Gradient formation and shape Gradient shape re-
solutions. Cesium chloride is by far the most widely fers to the density proRle across the tube or rotor as
used of this class. CsCl solutions can reach densities a function of gradient volume (Figure 6). Its choice is
3
of &1.9 g cm at saturation while providing a very important as it governs the sedimentation rate in both
low viscosity at lower concentrations. Although ex- rate and isopycnic experiments as well as the terminal
pensive, CsCl can be readily recovered and puriRed. position in isopycnic runs.
CsCl solutions also have a high osmotic pressure and Gradients may be classiRed as step or continuous,
are corrosive, though the titanium rotors generally as deRned by the method of preparation. Step (discon-
used with this solute are relatively resistant. CsCl tinuous) gradients are prepared by the stepwise addi-
gradients are commonly used in applications ranging tion of solutions of successively higher density to the
from the separation of viruses and dense cellular outer wall or bottom of the rotor. Steps gradients
macromolecules such as DNA, to geological poly- have the advantages that they may be formed without
mers found in coal or oil shale. Other salts that have the need for a gradient generator. These gradients
been used to produce high density gradients include may also be readily tailored to provide larger volumes
sodium bromide, sodium iodide, cesium bromide, of separation media in the ranges that correspond to
cesium sulfate, cesium formate, cesium triSuoro- the density proRle of the particles to be separated,
acetate, rubidium bromide and rubidium chloride. thereby, permitting higher sample loadings. For con-
Though expensive, tungstate polymers such as so- tinuous gradients, including the self-generating
dium polytungstate (SPT) and lithium heteropolytun- variety, the medium density varies in a continuous
gstate (LST) have recently been used to generate manner across the rotor or tube. Continuous gradi-
3
aqueous gradients well over 2.5 g cm . Applications ents are classiRed as linear, exponential or isokinetic.

Figure 6 Gradient shapes: (A) linear; (B) exponential; and (C) isokinetic.