Maojun Gong

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. Graphical abstract

A low-conductivity sample buffer plug was electroosmotically injected, and then the FASI-EKS was conducted at a reversed voltage polarity. When the conentrated analyte region arrived at the capillary inlet edge, a normal voltage was applied for separations.

On-line sample preconcentration is often required to achieve sensitive detection of specific analytes in capillary electrophoresis. However, available on-line techniques for concentrating high-salt samples such as cerebrospinal fluid (CSF) are mainly limited to sweeping and dynamic pH-junctions, etc. Electrokinetic supercharging (EKS) for sample preconcentration combines the techniques of field-amplified sample injection and transient isotachophoresis that may generate tremendous signal enhancement while maintaining high separation efficiency. This paper reports a novel EKS method for the on-line preconcentration of high-salt CSF samples in flow-gated capillary electrophoresis. The basic procedures include three major steps. First, a sample plug was hydrodynamically injected under vacuum; second, a voltage with the reversed polarity was applied to perform EKS with the assistance of a vacuum during the sample plug push-out; and finally a normal-polarity voltage was applied for separation. Specifically, amino acids were fluorogenically derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The optimal final sample medium included 20% CSF (or aCSF in volume), 50% acetonitrile, 12.0 mM borate, 5.0 mM KCN, 5.0 mM NDA, and 2.0 mM EDTA; and the separation buffer was composed of 40.0 tetraborate pH 9.2, 50.0 mM SDS, and 3.5 mM hydroxypropyl-β-cyclodextrin. The enhancement factors for a series of amines were in the range of 30–100 fold, and the detection limits were estimated to be below 0.10 nM. The developed method was coupled with alternate injections for the one-point standard addition method to determine γ-aminobutyric acid (GABA) in CSF from rat brain striatum. The results demonstrated detection sensitivity and accuracy for the rapid analysis of high-salt CSF samples.

Flow-gated capillary electrophoresis (CE) is a hybrid of conventional and microchip CE since it employs a fused silica capillary as the separation channel while taking advantage of the rapid flow-gated injection that is mainly used in microchip CE. Compared with conventional CE, flow-gated CE has excellent flexibility to be combined with online sample pre-treatment procedures such as microdialysis, analyte derivatization, sample cleanup and enrichment via solid phase extraction, as well as 2D separation coupled with HPLC or CE. Additionally, the rapid sample injection may improve the separation efficiency and analytical throughput. Compared with electrophoresis on a microchip, flow-gated CE has advantages such as high separation efficiency, low capillary cost, convenient coupling with different detectors, and using fresh separation buffer. To build flow-gated CE systems, several configurations of flow-gating interfaces have been developed, out of which the transverse flow-gating configuration predominates with advantages such as small void volumes and easy preparation. Flow-gated CE has shown cutting-edge applications in in vivo measurements of neurotransmitters and in vitro monitoring of chemicals released by cultured cells or tissues. In addition, flow-gated CE, as the second dimensional separation method, has been successfully coupled with HPLC and CE to perform 2D separation. This review covers the basic instrumental setup and characteristics of flow-gated CE, flow-gating interfaces, and major applications, as well as coupling with various analytical techniques. Finally, a brief statement about the future direction of flow-gated CE is discussed. The review aims to provide the scientific community with meaningful knowledge and progress about flow-gated CE thus promoting its further development and applications.

Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was > 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids.

Amino acid analysis is an important procedure to measure free amino acids (AAs) in various samples including proteins, blood plasma, urine, foods and beverages. Due to the complexity of these samples, chemical separations are required to determine the composition and quantities of AAs. Here, we report a fast and accurate approach to quantify AAs in beers. This method was based on a flow-gated capillary electrophoresis (CE) system coupled with alternate injections enabled by a micro-fabricated switch. AAs in beer samples and standard solutions were fluorogenically derivatized with 2,3-naphthalenedicarboxaldehyde (NDA) in the presence of cyanide. The two derivatized mixtures were then loaded into two separate micro-syringes and were alternately delivered to the flow gate for rapid injections and separations. Here, the standards functioned as side-by-side calibration and quantitation references. The separation of 21 AA derivatives was achieved within 90 seconds under an electric field of 1470 V cm⁻¹ by using optimized background electrolytes consisting of 40 mM sodium tetraborate, 60 mM sodium dodecyl sulfate, and 2 mM 2-hydroxypropyl-β-cyclodextrin, and theoretical plates of >300k for most AAs were obtained. The beer matrix effect was studied, and the quantitation of AAs was conducted by directly comparing the peak heights of analytes with those of their individual self-standards. The method was validated with respect to the linearity, precision, and accuracy, and was applied to the measurements of 21 AAs in eight different beer samples. The results demonstrated the quantitative capability of flow-gated CE coupled with alternate injections for side-by-side calibration that was able to compensate for potential drift of the CE system. The developed method is expected to be valuable for high-throughput amino acid analysis of a variety of samples.

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high‐throughput analysis with flow‐gated CE. Here, we report a fast fluorescein‐labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow‐gated CE. This scheme was based on the reaction between primary amines and o‐phthalaldehyde in the presence of a fluorescent thiol, 2‐((5‐fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE‐SH). The short reaction time (<30 s) was suited for on‐line mixing and derivatization that was directly coupled with flow‐gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE‐SH, Tris(2‐carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow‐gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on‐chip labeling of proteins retained on support in SPE.