Exploring the retention characteristics of stationary phases and their application in two-dimensional high performance liquid chromatography

Abstract

Phenyl type stationary phases have the ability to undergo π-π interactions with aromatic compounds and have the potential to take advantage of this particular sample dimension in 2DHPLC. This thesis examined a series of phenyl type sta¬tionary phases that were synthesised and packed in-house, containing systematic changes in the length of the chain binding the aromatic ring to the silica sup¬port (between 0 and 4 carbon atoms). The stationary phase was characterised and tested to determine retention characteristics with both linear and non-linear chromatographic measurements. The linear measurements involved a selectivity study with homologues of lin¬ear polycyclic aromatic hydrocarbons and n-alkylbenzenes to determine the aro¬matic and methylene selectivities, respectively. It was found that there were distinctly different trends in aromatic selectivity occurring on these phases that was dependent on an even or odd number of carbon atoms in the spacer chain. For short chain lengths the even phases were more aromatic selective though for longer chain lengths the odd phases were more selective. Ab initio calculations performed on these ligands show a change in the ring orientation between even and odd chain lengths that can explain the different trends in selectivity. The changes in methylene selectivity were relatively minor. Selectivity changes were also measured for a series of propyl phenyl (3 carbon spacer chain) stationary phases with different ligand densities. It was found that aromatic selectivity increased with ligand density and methylene selectivity plateaued near a ligand density of 2.0 µmol m'2 . Adsorption isotherm measurements, by frontal analysis with independent steps, of the different chain lengths with small substituted aromatics found that there were different retention processes occurring on the even and odd phases and this affected the saturation capacity. It was found that the degree of heterogeneity on these surfaces was weak and that small degrees of lateral adsorbate-adsorbate interactions occurred that are related to π stacking. To gain quantitative information from a two-dimensional separation methods need to be employed that can identify the location of each peak. A peak picking algorithm was utilised that identifes peaks in each one-dimensional cut using derivatives, followed by comparing peak overlap and peak maximum profiles. When the peaks are located it is relatively simple to perform statistical analysis to quantitate/compare 2DHPLC separations. The propyl and butyl phenyl chain lengths (i.e. 3 and 4 spacer carbons, re¬spectively) were tested in a two-dimensional separation with caf'e espresso coffee, a complex sample matrix, to determine if there was a difference in a real separa¬tion. The performance of the two separations were quantifed using an algorithm programed in-house to identify two-dimensional peaks and then performed a ge¬ometric approach to factor analysis and calculated the normalised mean radius in the separation quadrants. Subtle differences in the separations were observed when examining and comparing the individual quadrants, however the analysis of the entire separation showed very little difference.

Date of Award	2010
Original language	English