Reversed-phase systems for the separation of pentapeptides by high-performance liquid chromatography

Summary Reversed-phase systems using octyl modified silica as such and as a support for dynamically coated ion-exchangers, were investigated for their ability to separate pentapeptides. Normal reversed-phase adsorption with C-8 bonded silica in combination with citrate bufferpropanol-1 mixtures were found useful for the separation of a number of pentapeptides. The separation of pentapeptides differing widely in retention can be speeded up by applying an organic modifier and/or sodium citrate gradient. A solvent generated cation-exchange system with sodium dodecylsulfate as surfactant showed a high selectivity for the pentapeptides under investigation and is better for analytical purposes than the normal reversed-phase adsorption systems investigated. With respect to the detection of pentapeptides with fluorescamine, the use of dry pyridine as a basic buffer and as diluent for the fluorescamine was also investigated. Compared to the commonly used diluent acetone, pyridine is better when using acidic eluents of moderate buffer strength. At pH>6 no significant differences in sensitivity between acetone and pyridine could be noticed.