System peaks in capillary zone electrophoresis. 3. Practical rules for predicting the existence of system peaks in capillary zone electrophoresis of anions using indirect spectrophotometric detection.

A theoretical and experimental study of the existence and evolution of system peaks in capillary zone electrophoresis (CZE) with indirect spectrophotometric detection is presented with respect to the effect of the number of coions present in the background electrolyte (BGE). It is shown that in BGEs having only one coion (i.e., the UV-absorbing probe anion), the sample produces only negative peaks due to each analyte anion and no system peaks, with the number of sample peaks corresponding to the number of analytes present in the sample injected. In BGEs containing two coions, a sample with one analyte anion produces one negative indirect detection peak and one system peak. The transition between BGEs having one coion and those with two coions has also been studied and it has been shown that an addition of ca. 5% of the second coion to a single coion BGE causes the resulting BGE to behave macroscopically as a regular two-coion BGE. A descriptive model is proposed, based on transient isotachophoresis (transient stacking) of the sample species and of the coion from the BGE which has the closest mobility to the sample ion. This model explains qualitatively the formation and evolution of the sample peak (containing the sample species and being detected by indirect detection due to displacement of the UV-absorbing probe in its zone) and the system peak (containing no sample species and being a vacancy in the continuum of coions of the BGE). It is shown that the system peak may be positive or negative as it corresponds to the situation where the vacancy of one component of the BGE results in an enhanced concentration of the other component. It has been demonstrated that the system peak is created by a vacancy of that component of the BGE which has the greatest difference in mobility relative to that of the sample species. On indirect detection in BGEs containing two coions the sample displaces predominantly the BGE coion which has a mobility closest to that of the analyte anion. In systems with BGEs containing two coions, a sample having n analytes produces n sample peaks and one system peak, the sign and magnitude of which are dependent on the sum of the UV absorbances of the analytes involved. The effect of bicarbonate from atmospheric CO2 has also been studied and it has been shown that weakly alkaline BGEs with a single anionic UV-absorbing coion, such as those currently used for anionic analyses with indirect detection, may suffer from the presence of system peaks due to bicarbonate.