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 electroph oresis (CZE) in both bare fused-silica and acrylamide-coated capillaries is evaluated. The buffer system consists of a mixture of the aliphatic polyam ine triethylenetetramine and phosphoric acid in aqueous media. The study ha s been performed at pH 3.0, which is within the buffering capacity range in the acidic domain determined by the pK(a1) values of both the tetramine (3 .2) and the polyprotic inorganic acid (2.1). Consequently, the solution res ulting 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 compa rison of the tryptic, peptide separation performed by CZE with either triet hylentetramine phosphate or sodium phosphate buffers, both containing the s ame concentration of phosphate ions. The results show that both separation performance and electroosmotic flow are greatly influenced by the incorpora tion of triethylenetetramine in the buffer system. With the triethylenetetr amine phosphate buffer, the electroosmotic flow results in being either sup pressed or reversed from cathodic to anodic in bare fused-silica and in acr ylamide-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 phosph ate buffer is also related to the adsorption of the tetramine evidenced by the variation of the electroosmotic flow. The specific adsorption of cation ic triethylenetetramine results in masking the silanol groups and other act ive adsorption sites on the capillary wall, that are believed to be respons ible 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 in fluencing, significantly, efficiency and resolution of the tryptic peptides .