A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.

DOI: https://doi.org/10.1002/elps.201900399 The cover picture shows the concept of the multiple stacking preparative capillary electrophoresis (MSP‐CE) for the purification of anionic fluorescent probes in aqueous solution. This new method was developed by combining an on‐capillary concentration‐separation technique based on capillary transient isotachophoresis of a large volume sample with multiple injections, and an accurate and precise fraction collection technique using a two‐point detection CE system. Fluorescent probe solutions of more than 99% purity were successfully obtained by MSP‐CE. This purification method is useful for the preparation of high‐purity anionic fluorescent reagent solutions available for ultratrace analysis in CE‐LIF.