# Imaging and simulation study of electrokinetic supercharging in flow-gated capillary electrophoresis

## TL;DR

This study explores how electrokinetic supercharging works in capillary electrophoresis using imaging and simulations to improve sample concentration techniques.

## Contribution

The paper introduces a detailed analysis of electrokinetic supercharging mechanisms using fluorescence imaging and simulations in flow-gated capillary electrophoresis.

## Key findings

- Two operational modes of electrokinetic supercharging were evaluated: simultaneous and sequential.
- Sequential electrokinetic supercharging provided better control and reproducibility despite lower signal enhancement.
- The study offers practical guidelines for optimizing buffer conditions in flow-gated capillary electrophoresis systems.

## Abstract

Electrokinetic supercharging (EKS), an efficient sample preconcentration technique, has demonstrated millionfold concentration enhancement. Despite extensive studies, the detailed behavior of associated ions during EKS remains insufficiently understood. This research aims to elucidate the underlying EKS mechanisms on a flow-gated capillary electrophoresis (CE) system by using the combination of fluorescence imaging, computer simulations, and comparative analysis of electropherograms under various pairs of leading/terminating electrolytes (LE/TE). Two distinct operational modes were evaluated to highlight procedural differences: simultaneous EKS and sequential EKS. In the simultaneous mode, transient isotachophoresis (tITP) was conducted concurrently with field-amplified sample injection (FASI). For effective operation, LE anions were included in background electrolyte (BGE), while the TE anionic plug was generated by FASI from TE anions included in the sample. In the sequential mode, FASI was performed first to inject and preconcentrate analytes along with LE anions present in the sample, and the preconcentrated LE plug then functioned as LE in the subsequent tITP stage. While sequential EKS generally yielded lower signal enhancement compared to the simultaneous approach, it offered improved operational control and reproducibility. Due to its greater robustness, sequential EKS was chosen for the determination of herbicide residues in cereal samples. This study offers detailed insights into ion dynamics under various EKS configurations and provides practical guidelines for selecting LE/TE compositions and optimizing buffer conditions in flow-gated CE systems.

The online version contains supplementary material available at 10.1007/s00216-025-06116-3.