Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis

Abstract

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.