Abstract

Despite the wide range of HPLC stationary phases available for reversed-phase
high-performance liquid chromatography (RP-HPLC) and the in-depth studies
using probes to highlight differences between them, there is very little in the way of
stationary phases which offer selectivity that is substantially different from that
offered by the very commonly used alkyl-silicas. Therefore, the primary aim of the
research programme was to explore and try to exploit LC stationary phases which
offered genuinely different selectivity to alkyl-silicas for typical drug applications.
Chiral stationary phases (CSP) potentially had different selectivity and in this
context a secondary aim was to explore aspects of the enantioselectivity of CSP
as well as their chemical selectivity.
Claims of orthogonal selectivity had been made for pentafluorophenyl (PFP)
phases and phases exhibiting the hydrophilic interaction liquid chromatography
(HILIC) mode. However, the Ultra PFP phase was found to be very similar in
selectivity to ACE 5 C18 for both amitriptyline and acemetacin related compounds.
The ZIC-HILIC phase was shown to behave as a reversed-phase material at high
aqueous content in the mobile phase. There was some indication of selectivity
orthogonal to that of ACE 5 C18 with low aqueous content in the mobile phase but
this occurred at low retention and with mobile phases unsuitable for use with C18
phases in coupled (column or phase) systems. Nonetheless the work carried out
shed more light on the mechanisms taking place in the HILIC mode which is
currently attracting so much interest. Also it was possible to put ZIC-HILIC to good
use for polar plant metabolites and other applications.
Chiral stationary phases (CSP) also offered the prospect of selectivity orthogonal
to that of C18 phases. Given the proliferation of such phases though and the fact
that it would be useful to use CSP that gave chiral separations for a broad
spectrum of compound classes as well as giving orthogonal separations between
different compounds, it was decided to carry out comparative studies of CSP
classes in order to identify any redundancies and to seek out CSP that were
complementary to one another. The Regis Whelk-O1 CSP was shown to be much
superior to other higher-generation Pirkle-concept CSP such as DACH-DNB and
ULMO. Also it was shown to be complementary to the Chiralcel OD derivatised
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polysaccharide CSP and that both had something to offer alongside the widely
used Chiralpak AD derivatised polysaccharide CSP. It was also found that a series
of Chiralcel OD clones were virtually identical to Chiralcel OD and similarly for
Chiralpak AD clones. Chiralpak IA, an immobilised version of Chiralpak AD, was
not markedly less enantioselective than Chiralpak AD. Chiralcel OJ was less
enantioselective than Chiralpak AD but the gap in performance was not as wide as
between Whelk-O1 and the other Pirkle-concept CSP. The information gathered
during these studies should prove to be of enormous value for further work in
chiral LC method development screening.
Before embarking on applications work utilising the stationary phase selectivity
that had been found, a study was carried out on the effectiveness of the high
efficiencies obtainable with short run times through ultra-performance liquid
chromatography (UPLC). It was found that, for a range of pharmaceutical
applications, that it was still necessary in each case to adjust selectivity before
increasing speed through working at higher temperatures with faster flow rates. In
the course of this work some exceptionally high speed separations for example for
paroxetine and related substances, benzodiazepines and flurbiprofen and related
substances, were developed.
With respect to the evaluation of CSP as orthogonal phases to alkyl silicas under
reversed-phase conditions, the Whelk-O1 CSP showed promise. However on
closer inspection it was found that the Whelk-O1 CSP had very similar selectivity
to the alkyl silica phase, ACE 5 C18, and deviation from this only occurred in
instances when there was interaction with the chiral recognition site to give a
separation of enantiomers. This prompted the notion that, rather than using Whelk-
O1 in a coupled column system with ACE 5 C18, it could be used on its own for
the separation of both trace enantiomer and all other related substances. This was
shown to be possible using (S)-naproxen, laevokalim and (S)-flurbiprofen as
illustrative examples.
The evaluation of the enantioselectivity of CSP led to an optimised resolution
(suitable for scaling up for preparative work) of the enantiomers of the former
‘legal-high’ drug, mephedrone, on Whelk-O1 under normal phase conditions. It
was also shown that the infrequently used Chiralcel OJ derivatised polysaccharide
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CSP was ideal for developing an assay to determine trace amounts of (R)-nicotine
in (S)-nicotine.
Overall, the information obtained on stationary phase selectivity and retentivity
through evaluation and application will be of great value in HPLC and UHPLC
column selection and also selection of orthogonal phases for coupled column
systems but, ultimately, moving forward, most value may be in aiding the design of
two-dimensional LC systems for complex mixture analysis. This would particularly
apply to the use of CSP with reversed-phase eluents in achiral-chiral systems.