Suppressed ion chromatography of organic acids with universal detection
Creators: name: Karu, N
Publisher: name: University Of Tasmania
Year: 2023

description: This work presents an investigation into the challenges involving the utilisation of ion chromatography (IC) for the identification of impurities in pharmaceutical compounds, which is an essential task in the pharmaceutical industry. In IC, the use of a suppressor results in insensitive conductivity responses when applied to weak acids. In an attempt to circumvent this problem, signal enhancement through a post-suppressor reaction was performed by introducing a low concentration of a basic reagent, via a tee-connector, into the suppressor effluent. This approach exhibited enhancements of up to 500-fold for weak acids with `pK_a &gt;4.7`. However, signal enhancement was limited to high concentrations and sample volumes (at least 10 nmol on column), and did not greatly improve the limits of detection due to 50-100 times increase in baseline noise after reagent mixing. pH detection was also assessed, either after suppression or after base introduction, yet it hardly exhibited any signal enhancement of weak acids and at best resulted in limits of detection 4-times lower than suppressed conductivity. Universal detection methods suitable for coupling to IC were then investigated. However, the non-volatile ionic eluents commonly used in IC pose an obstacle in coupling to mass spectrometry (MS) and aerosol-based detectors, as the high ionic content can cause severe interferences in these detectors. A detailed study of the use of commercially-available chemical or electrolytic suppressors for desalting eluents comprising isocratic or gradient steps and containing organic solvents was undertaken. First, chemical and electrolytic suppressors were evaluated for baseline drift, noise and efficiency of suppression using aqueous/organic eluents containing up to 40% (v/v) methanol or acetonitrile. Chemical suppression of aqueous/organic eluents showed minimal noise levels, uniform low baseline and low gradient drift. Electrolytic suppression gave good performance, but with higher baseline conductivity levels and baseline drift than chemical suppression. The elevated baseline was found not to be caused by incomplete suppression of the eluent, but was attributed to chemical reactions involving the organic solvents and facilitated by high electric currents and heat generation. It was demonstrated that suppressed ion-exchange separation using a complex KOH elution profile could be coupled with an evaporative light-scattering detector (ELSD), with the suppressor effectively de-salting the eluent, producing a stable baseline. Second, the interactions between the suppressor and weak organic acid analytes, including pharmaceutically-related compounds, were investigated for eluents containing organic solvents. Correlations were observed between analyte recovery rates after electrolytic suppression and the eluent composition, the suppression conditions, and the physico-chemical properties of the analytes. These results suggest that hydrophobic adsorption interactions occur in the electrolytic suppressor and that these interactions are ameliorated by the addition to the eluent of high levels of organic solvents, especially acetonitrile, leading to 5-15% analyte losses. Use of eluents containing 80% acetonitrile resulted in very low losses of analyte during suppression (1-8%). Recovery experiments conducted in various compartments of the electrolytic suppressor showed that some analytes permeated through the suppressor membrane into the regenerant chambers, but this could be prevented by adding organic solvent to the regenerant solution. It was also noted that analyte losses increased with aging of the electrolytic suppressors, to levels of 15-35% loss. Chemical suppression avoids some of the analyte losses observed with an electrolytic suppressor, but when used under the correct conditions, electrolytic suppressors gave close to equivalent performance to chemical suppressors. Following the above studies, three new prototype designs for the electrolytic suppressors comprising high ion-exchange capacity screens and membranes were developed. These designs aim to minimise hydrophobic interactions of the suppressor with organic analytes and to provide higher compatibility with eluents containing acetonitrile. In comparison with a commercially-available electrolytic suppressor and also a commercially-available chemical suppressor, the new high-capacity suppressor showed superior performance, exhibiting minimal interactions with a test set of analytes under the examined conditions. This led to the attainment of high recoveries of the analytes after suppression (93-99% recovery) and significantly reduced band broadening during suppression. The new suppressor has been shown to perform well under both isocratic or gradient elution conditions. For proof of concept, IC was coupled to an electro-spray-ionisation mass spectrometer (ESI-MS), a corona charged aerosol detector (CAD), an evaporative light-scattering detector (ELSD), and a UV detector, which served as a reference detection technique. Suppression of the ionic gradient containing moderate concentrations of organic solvent was conducted by employing the new electrolytic suppressor design, and compared to a chemical suppressor. Complex elution profiles could be applied for separation, without the complications of organic solvent gradients typical to reversed-phase (RP) HPLC. The limits of detection were not greatly compromised by the suppressed system, yielding values of low ng/mL with MS detection, low to sub-˜í¬¿g/mL levels with the CAD and 2-20 ˜í¬¿g/mL with the ELSD. When coupled to MS, CAD and UV detectors, the modified electrolytic suppressor showed precision in peak areas of 0.4%-2.5%, outperforming to the chemical suppressor which yielded averages of 1.5-3 fold higher %RSDs. The modified electrolytic suppressor also generally exhibited wider linear response ranges than the chemical suppressor. Most importantly, complementary selectivity to reversed-phase separation was demonstrated for the test analytes as well as sample impurities, showcasing the use of IC as an orthogonal separation technique in the pharmaceutical industry.