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