Abstract
Semi-preparative high performance liquid chromatography facilities have been developed, capable of separating up to 100mg of material per injection. Stirred slurry column packing techniques were studied in detail. The effects of packing pressure, slurrying solvent, slurry concentration and the duration of the packing process were examined. The optimised packing technique was successfully used to pack columns of 12.5 to 50cm length and 4.9 to 10.9mm internal diameter. Analytical columns (4.9mm I.D.) were packed to evaluate twenty-two commercially available packing materials on a cost-performance basis. Semi-preparative columns (8.1 or 10.9mm I.D.) were packed with materials which gave either high efficiency or showed high performance to cost benefit. The performance of these columns compares favourably with the best results reported in the literature. The sample injection technique was studied using dye mixtures. Experimental results showed that the valve injection technique implemented the full loading capacity of the semi-preparative columns. Mass and volume overload studies were carried out to optimise conditions for semi-preparative chromatography. Column performance was measured in terms of peak resolution (RS) and the time yield factor (TYF). The effects of variation in capacity factor, column geometry, and particle size were examined. The relative loading capacities of silica and chemically bonded silica materials were also studied. A separation strategy for semi-preparative chromatography is proposed and various practical considerations are discussed. A wide range of applications are described illustrating the use of semi-preparative high performance liquid chromatography to separate novel compounds of pharmaceutical interest. The majority of the separations were effected using a low cost irregular silica material (~20mum). However, the most difficult separations were performed using 5mum spherical silica or chemically bonded silica materials.