Retention Mechanism for Reversed Phase and Hydrophilic Interaction Liquid Chromatography: Development and Characterisation of Modified Silica Particles

Bailes, Sophie (2014). Retention Mechanism for Reversed Phase and Hydrophilic Interaction Liquid Chromatography: Development and Characterisation of Modified Silica Particles. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f064

Abstract

Silanophilic interaction chromatography columns can give rise to complementary selectivity compared to the more commonly used hydrophobic interaction based HPLC columns. However, the former types of phase have historically caused issues of tailing and irreversible retention with polar or basic analytes. An investigation was therefore undertaken into the fundamental nature of their electrostatic, dipole-dipole and hydrogen bonding interactions. This was particularly focussed on interactions that are induced by the presence of residual silanols on the surface of HPLC phases, in order to fully understand the retention mechanism and the practical limitations of commercial stationary phases. Manufacturer's literature for these types of phase often contains specific applications and claims, however full independent testing of a range of acid, basic, phenolic, and polar compounds had not previously been demonstrated. Research in this area will aid in the development of new industrial applications by highlighting the stationary phase type with the most suitable mechanism to separate any combination of polar and non-polar compounds.

Initial investigations evaluated silanophilic interactions present in a set of Extended Polar Selectivity (EPS) phases utilising a set of known characterisation probes. Structural elucidation of these phases confirmed the intensity of these interactions is due to the accessibility of the silanols and the purity of the base silica. Shorter chains and sparsely bonded hydrocarbon ligands were shown to reduce hindrance and increase the degree of interaction. Endcapping was shown to decrease the accessibility of residual silanols and mask silanophilic interactions. High purity base silica was shown to reduce silanophilic interactions compared to similar phases bonded to a TYPE-A silica.

The control and restriction of silanophilic interactions is of great importance for the analysis of basic and polar molecules. Commercial TYPE-C™ phases are claimed to minimise silanophilic interactions through the "silanization" process. This converts the surface silanols to silica hydride prior to derivatisation with a functionalised ligand. Characterisation of the TYPE-C™ Bidentate C18 phase showed higher silanophilic interactions than a typical TYPE-B octadecyl bonded phase with a trimethyl silane endcap. 29Si NMR on the unpacked TYPE-C™ Bidentate C18 silica material confirmed the presence of silanols. These silanophilic interactions were shown to be coming from unreacted hydroxyl "wings" on polymeric silica hydride surface due to incomplete cross-linking of the triethoxy silane (TES) monomer during the "silanization" process.

Reduced efficiency observed in the characterisation of the TYPE-C™ Bidentate C18 phase was shown to be due to a high A Term in the van Deemter equation, due to a high level of Eddy dispersion in the silica bed. SEM imaging of the unpacked TYPE-C™ Bidentate C18 silica material showed a number of irregular particles present, these were identified as amorphous polyhydrosiloxane (PHS) formed through a cross-linking reaction of the TES monomer.

Investigation into the "silanization" reaction using TES indicated the process requires strict moisture control and monitored addition of the TES to reduce PHS formation Columns packed from the optimised "silanization" process showed lower silanophilic interactions in HILIC characterisation than the commercial TYPE-C™ phases. HILIC mode van Deemter analysis indicated the A Term contribution is comparable with unmodified bare silica; this suggests no added Eddy dispersion due to PHS particles in the packed silica bed.

HILIC analysis on the TYPE-C™ Diamond Bond and Silica-C phases coupled with 29Si NMR spectroscopy confirmed the presence of accessible silanols on the surface of the silica. Principal component analysis found these phases to be similar to experimental and literature characterisation values for TYPE-B style bare silica. The efficiency and van Deemter contributions of these phases were comparable to unmodified TYPE-B silica indicating these phase are not prepared by "silanization" by TES but by a two step chlorination and reduction process.

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