Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C18, have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH2 (TAPP), H-Tyr-(D)Ala-Phe-NH2 (TAP), H-Phe-Phe-NH2 (PP) and H-Phe-NH2 (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H2O as BGE, operating at 20 °C and 20 kV high voltage. For the C18 stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 °C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.