Column reaction:n(Resin-N+R 3 HCO 3 - ) +Xn-Æ(Resin-N+R 3 )nXn-+ nHCO 3 -where Xn-= F-, Cl-, NO 3 - , SO 42 - , PO 43 - etc.Suppressor reactions:Na+HCO 3 - +ÆH 2 O +CO 2 ++ Æ +
Similar reactions form the basis of the separation of cations. An example of
the separation of inorganic anions at the ppm level is shown in Figure 3(b).
● Size exclusion chromatography (SEC). This is suitable for mixtures of
solutes with relative molecular masses (RMM) in the range 10^2 –10^8 Da.
Stationary phases are either microparticulate cross-linked co-polymers of
styrene and divinyl benzene with a narrow distribution of pore sizes, or
controlled-porosity silica gels, usually end-capped with a short alkyl chain
reagent to prevent adsorptive interactions with solutes.Exclusionis not a
true sorption mechanism because solutes do not interact with the stationary
phase (Topic D2). They can be divided into three groups:
(i) Those larger than the largest pores are excluded completely, and are
eluted in the same volume as the interstitial space in the column, Vo.
(ii) Those smaller than the smallest pores, can diffuse throughout the entire
network and are eluted in a total volume, Vtot.
(iii) Those of an intermediate size separate according to the extent to which
they diffuse through the network of pores, of volume Vpand are eluted
in volumes between Voand Vtot.
Only those solutes in the third group will be separated from one another,
and their retention volumes are directly proportional to the logarithm of
their relative molecular mass(RMM; molecular weight). Columns can be
calibrated with standards of known RMM before analyzing unknowns.
Figure 4 shows a typical plot of elution volumeagainst log (RMM)and a
chromatogram of a mixture with a range of solutes of differing molecular
mass. SEC is of particular value in characterizing polymer mixtures and in
separating biological macromolecules such as peptides and proteins. It is also
used for preliminary separations prior to further analysis by other more
efficient modes of HPLC.
● Chiral chromatography. Chiral stationary phases (CSP) enable enantiomers
(mirror image forms) of a solute to be separated. Several types of these
stereoselective materials have been investigated and marketed commercially,
some of the most useful being cyclodextrinsbonded to silica. The cyclodex-
trins are cyclic chiral carbohydrates with barrel-shaped cavities into which
solutes can fit and be bound by H-bonding, p-pand dipolar interactions.
Where the total adsorptive binding energies of two enantiomers differ, they
will have different retention factors and can be resolved. Steric repulsion and
the pH, ionic strength and temperature of the mobile phase all affect the
resolution. Although of great interest to the pharmaceutical industry for thenNa+
removed via
a membraneHn+Xn-
detected by
conductancenH+
introduced via
a membraneNan+Xn-
separated by
the columnNa+
removed via
a membraneH+
introduced via
a membrane170 Section D – Separation techniques