Measuring the length of hydrodynamically injected plugs in capillary electrophoresis using the electrical current monitoring

Although CE is nowadays a worldwide separation technique, it is generally recognized that one of its main limitations is its poor robustness for quantitative analysis. Although this limitation can partially be surpassed using internal standards (ISs), it is well known that to find adequate standards is a very difficult task when too complex mixtures have to be analyzed. In this work, an alternative method to improve quantitation by CE is presented using the electrical current profile monitored during any CE run. Thus, an abrupt step in the current monitoring is observed when a hydrodynamically injected plug of conductivity different from the BGE leaves the capillary under the influence of the EOF. It is demonstrated that under these conditions, the relative amplitude of this step can be used to measure experimentally the injection length. This measure can not only be used for calibration, but also to correct variations of the length injected which is demonstrated to improve significantly the quantitative accuracy and reproducibility of CE. Thus, RSD values for interday quantification (five experiments a day for 5 days) were improved from 10.5 to 4.2%. Moreover, it is also demonstrated that accuracy of quantitative determinations by CE can greatly be improved by using this procedure. The method can also be implemented in other separation techniques where the EOF is used as driving force (e.g., CEC, MEKC or chip-based separations). Advantages and limitations of this approach in comparison to the use of ISs are also discussed.