Characterization and quantification of organic anions with capillary zone electrophoresis using direct and indirect...

A comparison of the separation and quantification capabilities of capillary zone electrophoresis (CZE) using direct and indirect detection of organic anions was conducted. A conventional CZE separation (normal polarity, electroosmotic flow toward the cathode) of phenol, benzoic acid, and 2,4-dihydroxybenzoic acid utilized direct UV absorption at 215 nM. A separation of myo-inositol 1,4,5-trisphosphate and myo-inositol hexakisphosphate utilizing a reversed polarity and an electroosmotic flow modifier (flow toward the anode) was monitored by indirect UV absorption at 250 nM. The separation buffers utilized in this study consisted of 50 mM borate buffer (pH 8.3) and IonPhor Anion PMA Electrolyte Buffer (pH 7.7) (Dionex Corp., Sunnyvale, CA, U.S.A.) for studies utilizing direct and indirect detection methods, respectively. The effect of separation voltage on the theoretical plate numbers observed for the separations was linear for both the direct and indirect systems. Sample introduction parameters investigated included electromigration injection voltage and duration, and gravity injection duration. The conventional CZE separation using direct detection gave superior precision and better agreement with theoretical predictions than the separation using indirect detection. Both systems were evaluated for quantitative accuracy using electromigration, pressure, and gravity sample introduction modes. The conventional CZE system showed superior performance with regard to sensitivity and limits of detection. Accuracy and precision in the quantitation of known standards was greatest for both systems when the gravity sample introduction mode was used.