Supercritical fluid chromatography - mass spectrometry

Abstract

High performance liquid chromatography (HPLC) is the most widely used
separation technique within the pharmaceutical industry. Due to the growing need for
high speed and high quality separations other techniques, such as supercritical fluid
chromatography (SFC), are now being considered. The key advantage of SFC is minimal
solvent waste, which is particularly important in preparative SFC, leading to fast sample
recovery. Hence it is important to explore whether SFC, which promises to be cheaper
and more environmentally friendly than conventional HPLC, can be applied more widely
as a complementary method.
SFC coupled to mass spectrometry, with an electrospray ion source, was used to
analyse diverse series of test compounds. The ionisation of samples in the absence of
high voltage, i.e. ionisation voltages, was observed when a SFC was coupled to
electrospray ionization (ESI) source. This novel ionisation process was further
investigated and an attempt was made to explain this ionisation phenomenon and the
improved sensitivity quantified. To probe this ionization mechanism, specific test
compounds were analysed and data acquired with high voltages on (electrospray) or off
(Novospray). Ammonium acetate and formic acid were introduced as buffer to see if
Novospray ionization is thermo spray type or driven by charged residue model. The
Novospray data was comparable or better than the classical ESI and atmospheric pressure
chemical ionization (APCI) methods. The ionisation is neither thermospray type nor
driven by charged residue model. Yet another possibility was sonic spray, where the ion
intensity strongly depends on the gas flow velocity consentient with high pressure flow
from SFC.
One of the objectives of this project has been to determine whether a generic set
of rules can be applied to choosing the best technique for the separation and analysis of a
given sample based on the chemistries of compounds involved. In an attempt to develop
generic analytical and preparative methods, a diverse series of test compounds were
analyzed on different stationary phase columns with use of a modifier, primarily
methanol. To improve the chromatography on certain stationary phases additives have
been used. For some compound types two peaks were observed upon injection, this
appears to be linked to compound type and the injection procedure. Data attempting to
explain this phenomenon is presented and in particular how the choice of injection
solvent affects possible compound interactions with the stationary phase and peak shape.
Thus, a direct comparison of HPLC and SFC was undertaken with a diverse
series of test compounds using the same conditions, to highlight the effectiveness of the
two techniques in terms of speed and more importantly compound coverage. HPLC and
SFC data are presented, comparing a generic analysis protocol with compound specific
analyses. Preliminary findings showing the overlap between the two separation
techniques is discussed and specific differences observed with the different column types
used is outlined

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Identifiers

ORCID for G. John Langley: orcid.org/0000-0002-8323-7235

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