Measuring the lenght of hydrodynamicallly injected plugs in capillary electrophoresis using the electrical current monitoring

Abstract

Although Capillary Electrophoresis (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, 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 background electrolyte leaves the capillary under the influence of the electroosmotic flow. It is demonstrated that under these conditions, the relative amplitude of this step can be used to experimentally measure the injection length. This measure can not only be used for calibration, but also to correct variations of the length injected what is demonstrated to significantly improve the quantitative accuracy and reproducibility of CE. Thus, RSD values for inter-day quantification (5 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 electroosmotic flow is used as driving force (e.g., capillary electrochromatography, micellar electrokinetic chromatography or chip based separations). Advantages and limitations of this approach in comparison to the use of internal standards are also discussed.