New materials and techniques for miniaturised chromatography

This thesis describes two bodies of work in which new methods were developed to aid the miniaturisation and integration of chromatography. The first body of work deals with the development of new stationary phases for boronate affinity chromatography. Porous polymer monoliths were developed for use as microscale boronate affinity extraction materials. The monoliths were prepared in situ from poly(butyl methacrylate-co-ethylene dimethacrylate) confined inside 100 ˜í¬¿m ID fused silica capillaries. A 2-step sequential photoinitiated grafting procedure was then used to create a layer of poly(glycidyl methacrylate) on the pore surface of the monoliths. Finally, the pendant glycidyl groups on this grafted layer were functionalised by ring-opening reactions with either p-hydroxyphenylboronic acid or p-(aminomethyl)phenylboronic acid, yielding boronate extraction columns with capacities of 2.3 ˜í¬¿mol/mL or 0.03 ˜í¬¿mol/mL respectively. The p-hydroxyphenylboronic acid functionalised column was stable at up to 250 bar pressure. It was interfaced to an electrospray ionization mass spectrometer where its selectivity was demonstrated by separation of glycated and non-glycated peptides. The broad diol selectivity of the material was further demonstrated by extraction of 11 nucleosides and by extraction of guanosine from a spiked urine sample. The second body of work deals with the conception and development of a new approach to controlling eluent composition gradients in chromatography. Gradient liquid chromatography typically relies on systems with multiple pumps that mix stock solutions at varied ratios or systems with electrolytic eluent generation. This thesis introduces an entirely new method in which a photosensitive chemical is dissolved in the eluent and irradiated at variable intensities as it is pumped through a photoreaction tube to create isocratic or gradient eluent profiles. Six different acid-generating photochemical reagents were tested and it was found that 2-chloro-1-(2,5-dimethyphenyl)ethanone was the most suitable chemical for generating acid concentration gradients. The system was demonstrated for capillary scale inorganic cation exchange chromatography with suppressed conductivity detection and pH gradient reversed phase chromatography with on-line mass spectrometry detection. The advantages of this photochemical approach to eluent generation, including greater solvent compatibility than electrochemical methods and greater design simplicity for simpler miniaturised chromatography systems, are discussed.