Fabrication and characterisation of organic monolithic columns for the separation of small molecules using HPLC-MS:The Frame Problem Revisited

摘要

Monolithic columns are continuous interconnected networks with large through-pore channels. This structure results in a decrease in the diffusion path and affords high permeability, which result in obtaining good separation efficiency. Ideally, the structure of monoliths should be bi-model consisting of meso-pores and macro-pores responsible for retention time and flow of mobile phase respectively. The structure also enhances the mechanical strength, and the large through-pore channels afford very low flow resistance. This combination therefore results in the ability of smaller diameter monolithic columns to be employed under high flow rates, increasing both sensitivity and throughput simultaneously. Additionally, the unique structure of monoliths improves permeability and mass transfer leading to a decrease in band broadening. The first stage of the project was focused on the fabrication and characterisation of an organic monolithic column namely poly (SMA-co-EDMA) followed by quantification of caffeine in Arabic coffee. Since the efficiency of the above monolith was low due to the low number of mesopores (low surface area), the second stage was centered on improving the efficiency of organic monoliths via the use of a longer crosslinker namely 1,6-HEDA. This novel monolith column poly (HMA-co-1,6-HEDA) afforded high efficiency, good porosity, high permeability and excellent reproducibility. Next, this monolith was applied to several applications namely separation of neutral non-polar analytes, weak acids, and strong bases, followed by a quantification of amitriptyline in commercial pharmaceutical tablets. Since the results obtained for this novel monolith using capillary liquid chromatography were encouraging, the third stage was investigating the possibilities of coupling narrow fused silica capillaries with mass spectrometry (MS). In this stage, the novel monolith (HMA-co-1,6- HEDA) lacked stability under high pressure due to either the low concentration of the crosslinker (1,6-HEDA) in the polymerisation mixture or the ratio between the monomer mixture (HMA and 1,6-HEDA) and porogen system (1-propanol and 1,4-butanediol). Hence, a move towards using nano-flow to couple narrow fused silica capillaries to the MS was utilised and was successful in separating two basic drugs (amitriptyline and nortriptyline). Finally, in order to widen the application of reversed- phase monoliths, a new monolithic material namely poly (GMA-co-EDMA) was synthesised followed by incorporation of high purity Congo red (CR) which contains several functional groups including SO3H, and then evaluating by separation of some reversed phase and HILIC mixtures.