Page 10 - BR20828_E Accucore 0713S

transferred to a Accucore 2.6µm, 100 x
2.1mm ID column, taking into account the
difference in column volume and the optimal
flow rate for the solid-core 2.6µm, 2.1mm ID
column (from the van Deemter plot),
specifically, the flow rate change takes into
consideration the columns internal diameter
and particle size. The gradient is scaled
ensuring that the number of column volumes
is kept constant. Injection volume is also
scaled down proportionally to the reduction
in column volume [8]. For the example in
Figure 7, resolution of the critical pair is
maintained (2.64 and 2.50), whilst reducing
analysis time from 17 to 6 minutes (including
column re-equilibration) and solvent
consumption from 17mL to 2.4mL per run.
Analysis time can be further halved by
reducing the solid-core column length to 50
mm, which still provides baseline resolution
of the critical pair (1.51).
Selectivity
The primary goal of developing a
chromatographic separation is to resolve a
mixture of analytes. So far in this paper the
discussion has been focused on efficiency
and the benefits this parameter can bring to
the assay. However, from the general
resolution equation it is evident that the
selectivity parameter has the greatest impact
on resolution. Selectivity can be changed by
modification of the mobile phase
composition, column chemistry or
temperature.
Accucore columns are available in a series
of chemistries to provide a wide range of
selectivities for method development;
these are:
• Optimised alkyl chain (RP-MS)
• C18
• Polar endcapped C18 (aQ)
• Phenyl-Hexyl
• Pentafluorophenyl (PFP)
• unbonded silica for HILIC.
To fully characterise the surface chemistry of
the reversed-phase materials, a series of
diagnostic chromatographic tests were used
(based on those developed by Tanaka [9]).
These tests characterise analyte/stationary
phase interactions and combine probes to
measure hydrophobicity, shape selectivity
and secondary interactions with bases, acids
and chelators. These tests are described here
in Table 2-4.
The phase characterisation data obtained
from these tests can be summarised in radar
plots (Figure 8), which allow visual
comparison of the overall selectivity of the
different stationary phase chemistries. The
hydrophobic retention and selectivity of the
C18, RP-MS and aQ are comparable, and
significantly higher that those of the PFP and
Phenyl-Hexyl phases. The steric selectivity of
the aQ phase is slightly higher than that of
the C18 or RP-MS phase but considerable
lower than that of the PFP phase, which
shows the highest steric selectivity. The
introduction of fluorine groups into the
stationary phase causes significant changes
in analyte-stationary phase interactions,
which can produce high selectivity for
Parameter Interaction investigated Test molecules
HR
Hydrophobic retention is the retention factor of a hydrophobic
hydrocarbon, pentylbenzene, which gives a broad measure of
hydrophobicity of the ligand and its density.
Pentylbenzene
HS
Hydrophobic selectivity is the selectivity factor between
pentylbenzene and butylbenzene and provides a measure of the
surface coverage of the phase; these two alkylbenzenes differ by one
methylene group and their selectivity is dependent on ligand density.
Butylbenzene
Pentylbenzene
SS
Steric selectivity (SS) 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.
o-Terphenyl
Triphenylene
HBC
Hydrogen bonding capacity (HBC) is the selectivity factor between
caffeine and phenol, which provides a measure of the number of
available silanol groups and the degree of endcapping.
Caffeine
Phenol
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 [7] 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. The latter contribute to the retention of
the protonated amines.
Benzylamine
Phenol
AI
The capacity factor and tailing factor of chlorocinnamic acid are
also measured to test the applicability of the stationary phase
acidic interactions.
4-Chlorocinnamic
acid
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
dissociated and combine with the ion exchange sites to influence the
retention of benzylamine.
Benzylamine
Phenol
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.
Quinizarin
BA
The presence of dissociated silanols at pH>7 can cause poor peak
shapes of protonated basic compounds such as amitriptilyne.
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.
Amitriptyline
Table 2. Hydrophobic tests
Table 3. Secondary interactions and ion exchange tests at low pH
Table 4. Secondary interactions and ion exchange tests at high pH