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Parameter Interaction Investigated Test Molecules
HR
Hydrophobic retention is the retention factor of a hydrophobic hydrocarbon,
pentylbenzene, which gives a broad measure of retention for a particular molecule.
This is based on the available surface area, the ligand, and its density.
HS
Hydrophobic selectivity is the selectivity factor between pentylbenzene and
butylbenzene and provides a measure for the selectivity of two molecules based on
their hydrophobicity. It is affected by the surface coverage of the phase and the ligand
density.
SS
Steric selectivity is the ability of the stationary phase to distinguish between
molecules with similar structures and hydrophobicity but different shapes. The
selectivity factor between o-terphenyl and triphenylene is indicative of steric selectivity,
as the former has the ability to twist and bend, while the latter has a fairly rigid
structure and will be retained quite differently. More rigid ligands will in general give
greater selectivity.
HBC
Hydrogen bonding capacity is the selectivity factor between caffeine and phenol,
which provides a measure of the degree of hydrogen bonding a molecule will
experience. Caffeine is a good hydrogen bonder and increasing the number of available
silanol groups will increase the retention of the caffeine molecule but not the phenol
molecule. This term is affected by the total number of silanols, which in turn is affected
by the total surface area and also the degree of endcapping.
Table 1: Hydrophobic tests
Parameter Interaction Investigated Test Molecules
IEX2.7
Ion-exchange capacity at pH 2.7 is estimated by the selectivity factor between
benzylamine and phenol, at pH 2.7. Tanaka [1] showed that the retention of protonated
amines at pH < 3 could be used to get a measure of the ion exchange sites on the
silica surface. Silanol groups (Si-OH) are undissociated at pH < 3 and therefore cannot
contribute to the retention of protonated amines, but the acidic silanols in the
dissociated form (SiO-) can. Acidic silanols can be formed with the addition of impurities
and also the different forms (in order of acidity: geminyl, bridged, vicinyl, metallic forms)
of the silanols moiety. The more acidic silanols contribute to the retention of the
protonated amines.
AI
The capacity factor and tailing factor of chlorocinnamic acid are also measured to
test the applicability of the stationary phase towards acidic interactions. These
interactions are due to impurities in the substrate material and also to certain ligands
that are used.
Table 2: Secondary interactions and ion exchange tests at low pH
Parameter Interaction Investigated Test Molecules
IEX7.6
Ion-exchange capacity at pH 7.6 is estimated by the selectivity factor between
benzylamine and phenol and is a measure of the total silanol activity on the surface of
the silica. At pH > 7 the silanol groups are fully dissociated and combine with the ion
exchange sites to influence the retention of benzylamine.
C
Silica surface metal interactions can cause changes in selectivity and peak shape for
analytes which are able to chelate. Changes in the capacity factor and tailing factor
of quinizarin, which is a chelator, are indicative of secondary metal interactions.
BA
The presence of dissociated silanols at pH > 7 can cause poor peak shapes of
protonated basic compounds such as amitriptyline. Secondary ion exchange and
silanolic interactions can cause shifts in retention and asymmetrical peaks. The
capacity factor and tailing factor of amitriptyline are indicative of the overall
performance of the column.
Table 3: Secondary interactions and ion exchange tests at neutral pH
Pentylbenzene
Butylbenzene Pentylbenzene
o-Terphenyl Triphenylene
Caffeine Phenol
Benzylamine Phenol
4-Chlorocinnamic acid
Quinizarin
Amitriptyline
Benzylamine Phenol