and non-compressible are particularly useful in separations where the mobile phase is pressurized as in
HPLC (p. 118 et seq).
The Separation Process
Columns of gel beads are more often used than thin layers. The process can be considered analogous to
a partition system wherein molecules which are completely excluded from the gel have a distribution
ratio of 0 whilst those small enough to penetrate all parts of the structure have a value of 1. Adsorption
or ion-exchange effects can cause the distribution ratios of polar molecules to exceed 1. Components of
a mixture are characterized by their retention volume, VR, which is determined by the distribution ratio.
A solute always has the same retention volume for a given gel. It is virtually independent of
temperature and flow rate because separation occurs by selective diffusion within a single liquid phase.
True partition is an equilibrium process involving the transfer of a solute between two immiscible
phases. For solutes whose molecular size or mass falls within the fractionation range of the gel, i.e. for
which distribution ratios lie between 0 and 1, retention volume is approximately a linear function of the
logarithm of the relative molecular mass, RMM (molecular weight) (Figure 4.50). Molecules that are
completely excluded from the gel are eluted with a retention volume equal to the void or dead volume
of the column (p. 85); molecules which freely permeate all parts of the gel network are eluted by a
volume equal to the dead volume + the volume of liquid within the gel particles. A column is calibrated
by eluting substances of known RMM or molecular mass range. Elution profiles are Gaussian because
of diffusion effects; peak broadening can be minimized by eluting with a solvent of low viscosity, e.g.
tetrahydrofuran. Efficiency is determined only by peak width, as retention volumes are constant (cf.
other column techniques, p. 86). Eluted components are detected by monitoring a physical property of
the column effluent such as refractive index or UV absorption or by collecting and analysing separate
fractions.
Figure 4.50
Relation between molecular weight and retention volume
for proteins at pH = 7.5 (Sephadex G-200 column).