Reversed-phase high-performance liquid chromatography of mouse epidermal growth factor and its congeners: mobile...

Optimization of the mobile phase in reversed-phase high-performance liquid chromatography has been examined with respect to the separation of multiple components from mouse epidermal growth factor preparations. Neither trifluoroacetic acid, nor heptafluorobutyric acid afforded optimal separations when added in various concentrations as potential ion-pairing agents, requiring low ionic strengths and yielding separations that were susceptible to changes in packings due to differences in stationary phase coverage and the presence of accessible silanol groups on the C-18 phases used. Pentadecafluorooctanoic acid, on the other hand, gave a reproducible, effective separation when combined with 0.155 M sodium chloride in the primary solvent, and eluted with a gradient of acetonitrile-1-propanol as the secondary solvent. Additional selectivities were conferred by the addition of triethylamine (0.015 M) to the mobile phase containing pentadecafluorooctanoic acid. These studies illustrate the value of such mixed mobile phases in achieving complex separations of closely eluted polypeptides.