Improved peptide mapping by capillary zone electrophoresis using triethylenetetramine phosphate buffer as the electrolyte solution

An acidic separation buffer for peptide mapping by capillary zone electrophoresis (CZE) in both bare fused-silica and acrylamide-coated capillaries is evaluated. The buffer system consists of a mixture of the aliphatic polyamine triethylenetetramine and phosphoric acid in aqueous media. The study has been performed at pH 3.0, which is within the buffering capacity range in the acidic domain determined by the pKa values of both the tetramine (3.2) and the polyprotic inorganic acid (2.1). Consequently, the solution resulting by combining phosphoric acid and triethylenetetramine at pH 3.0 is a buffering system exhibiting two conjugate acid-base pairs, both effective at controlling the protonic equilibra at this pH value. The suitability of the triethylenetetramine phosphate buffer at performing efficient peptide mapping by CZE has been evaluated on a tryptic digest of the protein cytochrome c from horse heart. The study is based on the comparison of the tryptic peptide separation performed by CZE with either triethylentetramine phosphate or sodium phosphate buffers, both containing the same concentration of phosphate ions. The results show that both separation performance and electroosmotic flow are greatly influenced by the incorporation of triethylenetetramine in the buffer system. With the triethylenetetramine phosphate buffer, the electroosmotic flow results in being either suppressed or reversed from cathodic to anodic in bare fused-silica and in acrylamide-coated capillaries, respectively. This effect is indicative of the specific adsorption of positively charged molecules of triethylenetetramine into the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. The improvement of peak shape observed with the triethylenetetramine phosphate buffer is also related to the adsorption of the tetramine evidenced by the variation of the electroosmotic flow. The specific adsorption of cationic triethylenetetramine results in masking the silanol groups and other active adsorption sites on the capillary wall, that are believed to be responsible for the poorer performance obtained with sodium phosphate buffer. Thus, the results demonstrate that besides being an effective component of the buffering system, triethylenetetramine acts as a dynamic coating reagent influencing, significantly, efficiency and resolution of the tryptic peptides.