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Abstract

A non-discriminating, robust and economical detection system, which can be
easily coupled with different modes of separation, is highly desirable in the field of
liquid chromatography. While the evaporative light scattering detector (ELSD) and the
corona-charged aerosol detector (C-CAD) hold great promise to meet these
requirements, the widespread applicability of these techniques has been hindered due to
lower sensitivity and solvent dependency of the detection response. This work presents
an investigation into different experimental approaches to overcome these limitations,
so as to extend the field of applicability of ELSD and C-CAD.
Hyphenation of high temperature liquid chromatography (HTLC) using waterrich
mobile phases with ELSD / C-CAD is an attractive solution to the solvent
dependency limitations of these detectors and also offers a better detection alternative
for HTLC. Therefore, experiments were conducted to investigate the effect of HTLC
conditions using water-rich mobile phases on the detection response of ELSD and CCAD.
Flow-injection studies showed that eluent temperature marginally influenced the
detection response. However, in chromatographic separations the response of the
ELSD for the same analyte eluted at different retention times was increased up to 5-fold
by increasing the separation temperature from 30°C to 180°C. Compared to the ELSD,
the response of the C-CAD was found to remain relatively unaltered with variation in
retention time. This increase in ELSD response was found not to result from the eluent
temperature, but rather from compression of the elution band-width at elevated
temperatures and hence shorter retention times. The relationship between elution bandwidth
and the response mechanism of the ELSD was then explained using logarithmic
response curves obtained by flow-injection experiments. Furthermore, it was
demonstrated that a temperature gradient could be used to counteract the effects of
varying bandwidths associated with isocratic-isothermal separations.
Considering the advantages of temperature gradients in attaining elutropic
strength comparable to the solvent gradient, the possibility of employing isocratic
separations with a combination of temperature and flow-rate variation to achieve
uniform detection response of the C-CAD was investigated. Using a flow-injection study, it was demonstrated that the response of the C-CAD remain relatively unaltered with flow-rate variation when used with water-rich eluents. Based on these findings
two separation approaches were developed and their utility for C-CAD response
normalisation was demonstrated using a mixture of eight analytes. In the first
approach, a temperature gradient was applied under isocratic conditions, followed by
response enhancement through the post-column addition of organic solvent. In the
second approach, flow-rate programming was used to improve the speed of separations
performed using isocratic elution coupled with a temperature gradient. The response
homogeneity and applicability of these approaches were compared to the inverse
solvent gradient technique for quantitative analysis. Good peak area reproducibility
(RSD < 15%) and linearity (R2 > 0.994, on a log-scale) over the sample mass range of
0.1 – 10 μg were achieved. The response deviation across an equi-mass mixture of
eight analytes at seven concentration levels was 6-13% compared to 21-39% when a
conventional solvent gradient was applied and this response deviation was comparable
to that obtained in the inverse gradient solvent compensation approach. The
applicability of these approaches for typical pharmaceutical impurity profiling was
demonstrated at a concentration of 5 μg/mL (0.1% of the principal compound).
Following the above studies, the applicability of nebuliser gas flow-rate
programming and inverse gradient techniques was investigated for improving the
performance of the ELSD. The investigations showed that nebuliser gas flow-rate
programming could be used to compensate solvent effects; however it caused
significant loss in sensitivity and hence has limited applicability for the water rich
eluents. Moreover, in inverse gradient experiments, elution bandwidth variability
across the separation was found to contribute to response irregularity. This led to the
investigation of two-dimensional liquid chromatographic (2-D LC) peak modulation
approaches to improve the performance of ELSD. Experiments were conducted to
assess the feasibility of elution band-width normalisation by means of post-separation
flow-rate modulation using a switching valve. Furthermore, for proof of concept, utility
of the switching valve as a peak sampling device to overcome solvent gradient
limitations of the ELSD was demonstrated. However, some limitations of this approach
were identified, especially in terms of the ability to detect peak segments of low analyte
concentration.

Item Type:	Thesis - PhD
Authors/Creators:	Khandagale, M
Keywords:	ELSD, C-CAD, HTLC, 2-D LC modulation, Response normalisation, Dual gradient
Copyright Holders:	The Author
Copyright Information:	Copyright 2015 the author
Additional Information:	Chapter 3 appears to be the equivalent of the post-print version of an article finally published as: M. M. Khandagale, J. P. Hutchinson, G. W. Dicinoski, P. R. Haddad, 2013, Effects of eluent temperature and elution bandwidth on detection response for aerosol-based detectors, J. Chromatogr. A, 1308, 96-103. Chapter 4 appears to be the equivalent of the post-print version of an article finally published as: M. M. Khandagale, E. F. Hilder, R. A. Shellie, P. R. Haddad, 2014, Assessment of the complementarity of temperature and flow-rate for response normalisation of aerosolbased detectors, J. Chromatogr. A, 1356, 180-187.
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