Phd in PHARMACEUTICAL SCIENCES

Thesis title: The control of the surface chemistry of porous silica and the transition towards greener enantioselective UHPLC for tackling noteworthy issues in liquid chromatography

In the field of chromatography, the need to have increasingly efficient methodologies with high selectivity and high resolution is the primary driving force. The transition from high-performance liquid chromatography to ultra-high performance would never have been possible if, alongside the continuous study of new stationary phases, there had not been the evolution of the silica support. In this sense, the introduction of sub-2µm silica particles (STM) marks a fundamental point in the evolution of the science of separations. Still, the transition lies not only in the particle size. We must also consider the evolution in the structure of the particle, from the increase in mechanical strength to enormous pressures and the high degree of purity achieved with Type-B silica. The instrumental part also had to do its part to manage the back pressure and minimise the instrumental variance. Therefore, the evolution of the chromatographic technique has always seen increasing advances both in the study of silica and in what it supports. This dissertation reports the results of the works obtained in the context of chiral and non-chiral chromatography. The improvement of the performances is the result of studies and innovations that start from the study of the surface properties of the silica particle. The thesis is divided into two parts. In the first part, various works concerning the properties and characteristics of the silica particle used as support for chiral and non-chiral stationary phases will be presented. In particular, the first topic concerns the effects of the porosity of the silica particle on the chromatographic performance of chiral columns based on zwitterionic-teicoplanin. The enantioseparation of important classes of compounds was evaluated, including chiral drugs, pesticides, and derivatised N-amino acids. The new CSP composed of superficially porous particles (SPP) with a particle diameter of 2.7 μm and large pores (160 Å) showed a significant efficiency, almost comparable to that of the column packed with the finest SPPs (particle diameter 2.0 μm) but a smaller average pore size (90 Å). The new CSP was characterised by a lower absolute loading of selector (µmol / gram of bare silica) and a higher specific density of the chiral selector (µmol / m2), which were related to lower retention and higher enantioselectivity, respectively. Always talking about the properties of the silica surface, two different strategies will be exposed to reduce non-specific interactions: dynamic electrostatic repulsive reversed phase (d-ERRP) and horizontal polymerisation. The d-ERRP is an elution modality developed and studied in our laboratory, where an electrostatic repulsion mechanism is generated during the elution thanks to two factors: i) the addition of a cationic and hydrophobic additive that adsorbs on the stationary phase; ii) and the acid pH of the mobile phase. With this modality, small basic molecules of pharmaceutical interest were analysed. The results show that d-ERRP significantly increases the efficiency and improves the asymmetry factor (150970 N/m vs 22540 N/m and As5% 1.12 vs 4.55 on amitriptyline for the same amount of k). This mode has also shown excellent results in peptides analysis, in separating glucagon from its D-His1-Gluc epimer. d-ERRP was also tested in analysing other peptides of pharmaceutical interest, such as lanreotide, octreotide, icatibant, degarelix, bivalirudin, semaglutide, and exenatide. Also, in this case, the efficiency, resolution, and peak shape results were excellent. In these works, the performances in the d-ERRP mode were systematically compared with the best commercial silica supports, specially designed to reduce the non-specific interactions between analyte and silica particle surface. The other strategy performed to control and reduce non-specific interactions is horizontal polymerisation. This work introduced two novelties in the derivatisation of silica for chromatographic use: the horizontal polymerisation on the silica surface using vinyl-triacetoxy silane and a subsequent cysteine-based stationary phase preparation by thiol-ene photo-click coupling. Due to the horizontal polymerisation, the modified silica particles offer a high load of vinyl fragments and extensive passivation of the silanols, and an end-capping procedure is not required. The derivatisation of this vinyl-silica with cysteine provided a stationary phase suitable for the hydrophilic interaction liquid chromatography (HILIC) mode. This phase was used to study the anomerization of some monosaccharides through dynamic hydrophilic interaction liquid chromatography (D-HILIC) conditions. The second part of this thesis is focused on studying the kinetic and thermodynamic performances of UHPLC chromatographic columns packed with CSP Whelk-O1 in reversed phase (RP) mode. These columns are usually used in normal phase mode; however, this poses a contradiction because, in the field of chiral drug analysis, we are dealing with molecules designed for aqueous environments. This issue, together with the effort to reduce the production of large quantities of eluent with high environmental impact (halogenated solvents and hydrocarbons, such as hexane), has prompted us to study the effects of the transition from NP to RP mode using CSP based on this selector. The results demonstrate that The NP elution mode is undoubtedly the condition for which CSP Whelk-O1 fully express its potential. The goal is to evaluate the results of the transition from NP to RP mode comprehensively and systematically and evaluate the trend of kinetic and thermodynamic performance. This work demonstrates that although the RP mode is less performing, it is still able to provide more than satisfactory results if the three organic modifiers (OMs) used are considered together. Furthermore, the results obtained in terms of chemo and enantioselectivity indicate that this modality should be considered complementary to NP.