Parameters affecting reproducibility in capillary electrophoresis.

Abstract

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.