Supercritical fluid chromatography - mass spectrometry

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

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2011

Author dc:creator

• Thite, Mohini A.

Advisor dc:contributor.advisor

• Langley, G. John