<p>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.</p> Graphical abstract <p></p>