High Performance Liquid Chromatographic Analyses of Sulphonamides and Dihydrofolate Reductase Inhibitors. II. Separations in Acetonitrile Modified Solutions, Ternary Gradient Studies & Flow Programming

Acetonitrile has been investigated as the organic modifier for the reverse phase separation of twenty-two sulphonamides and three commonly used dihydrofolate reductase inhibitors. Isocratic analyses indicate that 10% acetonitrile is approximately isoeluotropic with 16% methanol. This allows a comparison of selectivities. Relative to methanol, in gradient elution involving acetonitrile only, unfavourable changes in the relative retentions of some pairs of analyses outweighed the advantages of favourable selectivities. Insertion of acetonitrile into methanol gradients did not produce the improved separations expected. However, the imposition of a program of reduced flow upon an established methanol gradient demonstrated significantly increased efficiencies in the front half of the chromatogram. This permitted the resolution of the first thirteen compounds, including the difficult-to-separate pair of sulphathiazole and sulphapyridine.