Three-step stacking of cationic analytes by field-enhanced sample injection, sweeping, and micelle to solvent stacking in capillary electrophoresis

Abstract

The sensitivity enhancement factor (SEF) in field enhanced sample injection (FESI) in capillary electrophoresis is dictated by the conductivity ratio. The higher the conductivity ratio (using very low conductivity sample diluents such as water), the higher the SEF. Here, we improved the performance of FESI by combination with sweeping and micelle to solvent stacking (MSS) in a well-defined three-step stacking procedure using model cationic drugs. The separation was by capillary zone electrophoresis (CZE) using 100mM phosphoric acid as background solution (BGS). Under the experimental conditions studied, the SEF (vs. typical injection in CZE) range of FESI using a conductivity ratio of 10, 100, and 1000 (sample diluent with conductivity 10, 100, and 1000× lower than the BGS, respectively) was 5–6, 33–50, and 272–393, respectively. The SEF range of three-step stacking was 308–891, 2188–6463, and 3088–6499, correspondingly. The SEF enhancement factor due to three-step stacking (SEF of three-step stacking divided by SEF of FESI) was from 11 to 161. We evaluated the performance of proposed procedure using a conductivity ratio of 10 (10mM phosphoric acid as diluent) which is the minimum requirement for field-enhancement. The strategy was as follows: long FESI (e.g., 420s at 10kV) to form an overloaded stacked zone; sweeping (e.g., 315s at −10kV) with 10mM sodium dodecyl sulfate micelles; and MSS by injection (6s at 50mbar) of 30% acetonitrile. The strategy was studied in terms of sweeping and MSS conditions, FESI/sweeping time ratio, and FESI time at constant FESI/sweeping ratio. Analytical figures of merit including linearity, LOD (S/N=3), and repeatability (intraday and interday) were determined. Moreover, sample matrix effect was studied using acetone treated plasma sample.