ARTIFACTUAL PEAK SPLITTING IN CAPILLARY ELECTROPHORESIS .2. DEFOCUSING PHENOMENA FOR AMPHOLYTES

In a previous paper (Ermakov, S. V.; et al. Anal. Chem. 1994, 66, 4034 -4042) it was reported that, when weak acids and bases in a background electrolyte containing a strong co-ion were analyzed at high sample l oads (comparable to those of the buffering ion), the analyte peak spli t into two zones, representing the same species existing in its charge d and uncharged forms, The same approach has been here applied to amph oteric species, particularly amino acids, Under similar conditions (hi gh sample load, strong titrant ion) and at operative pH values close t o the pi of the analyte, it is shown that the sample can be split into three components, representing the three different charge states of t he ampholyte: its cationic, its anionic, and its zwitterionic forms, I n the case of His, that the three forms indeed represented the same sa mple ion was demonstrated by spectral analysis of each peak, This is d ue to the fact that the strong titrant ion, present in the background electrolyte, can penetrate the sample zone and titrate it not only to the pi value but beyond it, This induces a spatial and temporal pH gra dient within the sample zone, increasing from cathode to anode, i.e., having a slope opposite to Rilbe's law of pH monotony, according to wh ich, under focusing conditions, the pH gradient should be a monotonica lly increasing function from anode to cathode, As a result this phenom enon of peak splitting can be considered, in a broad sense, a ''defocu sing'' phenomenon. Computer modeling of this phenomenon showed theoret ical profiles in good agreement with experimental ones. Additionally, for peak splitting, the ampholyte should be a a ''good carrier ampholy te'', i.e., exhibit a good buffering capacity and conductivity at the pI, a condition satisfied by a steep incline of the titration curve su rrounding the pI value. ''Poor carrier ampholytes'' with wide plateaus in titration curve close to zero charge have a lower probability for three-peak splitting, since they cannot be titrated past their pI valu e.