毛细管筛分电泳中电动超荷电结合预填充水塞在线富集十二烷基磺酸钠-蛋白质的复合物

提出了一种应用于毛细管筛分电泳中的电动超荷电结合柱头水塞堆积样品的方法,实现了十二烷基磺酸钠-蛋白质复合物的在线富集。一般情况下,电动超荷电方法是一种将电动进样与瞬时等速电泳联用的富集技术。具体过程是,首先在毛细管中注入背景电解质,再注入适量的前导电解质,然后电动进样一段时间。最后注入后导电解质开始瞬时等速电泳及分离的过程。本文在常规的电动超荷电技术基础上,在电动进样之前先注入一段含有聚合物的水塞以进一步提高富集效果。同时,考察了电动超荷电中不同富集方法叠加联用的效果,包括聚合物的筛分效应、结合水塞和不结合水塞的场放大样品进样效果、瞬时等速电泳等。结果表明,由于十二烷基磺酸钠-蛋白质复合物的质荷比接近,电动进样中的进样歧视得到消除,电动超荷电结合含聚合物水塞堆积样品的方法可以无歧视地在线富集十二烷基磺酸钠-蛋白质复合物,检测灵敏度增强1000倍以上。该方法非常适用于低丰度蛋白质的分析,并可同时提供相对分子质量信息。     

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging preconcentration. Insight into the stacking mechanism via computer modeling

This review discusses recent progress in the application of one of the most effective in-line preconcentration techniques used in electrophoresis in capillaries and microchips, electrokinetic supercharging (EKS). Conventionally considered as a transient isotachophoresis (tITP) step put into effect after the electrokinetic sample injection (EKI), EKS presumes that the electrolyte filled into the capillary (or microchip channel) comprises a co-ion acting as a leading ion to stack the injected analytes. Subsequently, to create the tITP state, one needs an additional injection of a suitable terminating ion. As a resulting increase in sensitivity strongly depends on the performance of both EKS stages, two theoretical sections are focused on hints for proper arrangement of EKI and tITP elaborated by means of computer simulation. In particular, factors affecting the injected amount of analytes, different modes of introducing the sample, suitable combinations of leading and terminating ions, and optimization of supporting electrolyte compositions are discussed with an objective to increase the enrichment factors. A comprehensive coverage of recent EKS applications in capillary and microchip electrophoresis, including metal ions, pharmaceuticals, peptides, DNA fragments, and proteins, demonstrates attainable sensitivity enhancements up to two orders of magnitude. This should make this method exportable to other analytes and facilitate its more widespread use to applications that require low limits of detection.     

Counter-flow electrokinetic supercharging for the determination of non-steroidal anti-inflammatory drugs in water samples

Electrokinetic supercharging (EKS) has been used in the last few years as a powerful tool for separation and on-line preconcentration of different types of analytes. We have developed a valuable modification for EKS system, namely counter-flow EKS (CF-EKS) and applied it for the separation and on-line preconcentration of seven non-steroidal anti-inflammatory drugs (NSAIDs) in water samples. In CF-EKS, a hydrodynamic counter-flow is applied during electrokinetic injection of the analytes within the EKS system. This counter-flow minimises the introduction of the sample matrix into the capillary, allowing longer injections to be performed. Careful choice of the optimum counter-flow as well as the optimum injection voltage allowed the sensitivity to be enhanced by 11,800-fold, giving limits of detection (LODs) of 10.7–47.0 ng/L for the selected NSAIDs. The developed method was validated and then applied for the determination of the studied NSAIDs in drinking water as well as wastewater samples from Hobart city.     

Pressure-assisted electrokinetic supercharging for the enhancement of non-steroidal anti-inflammatory drugs

Electrokinetic supercharging (EKS) combines field-amplified sample injection with transient isotachophoresis (tITP) to create a powerful on-line preconcentration technique for capillary electrophoresis. In this work, EKS is enhanced with a positive pressure (pressure-assisted EKS, or PA-EKS) during injection to improve stacking of non-steroidal anti-inflammatory drugs (NSAIDs). Several parameters, including buffer composition and concentration, terminating electrolyte, organic modifier, and injection voltage and injection time of both terminating electrolyte and sample were optimized. Detection limits for seven NSAIDs were determined and an enhancement in sensitivity of almost 50,000-fold was obtained. The PA-EKS method has the potential to be a simple MS compatible preconcentration method to improve the sensitivity of CE.     

Analysis of phenolic acids by non-aqueous capillary electrophoresis after electrokinetic supercharging

Electrokinetic supercharging (EKS), a new and powerful on-line preconcentration method for capillary electrophoresis, was utilized in non-aqueous capillary electrophoresis (NACE) to enhance the sensitivity of phenolic acids. The buffer acidity and concentration, leader and terminator length and electrokinetic injection time were optimised, with the optimum conditions being: a background electrolyte of 40 mM Tris-acetic acid (pH 7.9), hydrodynamic injection of 50 mM ammonium chloride (22 s, 0.5 psi) as leader, electrokinetic injection of the sample (180 s, -10 kV), hydrodynamic injection of 20 mM CHES (32 s, 0.5 psi) as terminator, before application of the separation voltage (-25 kV). Under these conditions the sensitivity was enhanced between 1333 and 3440 times when compared to a normal hydrodynamic injection with the sample volume <3% of the capillary volume. Detection limits for the seven phenolic acids were in the range of 0.22-0.51 ng/mL and EKS was found to be 3.6-7.9 times more sensitive than large-volume sample stacking and anion selective exhaustive injection for the same seven phenolic acids.     

Online preconcentration by electrokinetic supercharging for separation of endocrine disrupting chemical and phenolic pollutants in water samples

Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2‐nitrophenol (NP) and 4‐chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS‐CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at –3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100‐ to 737‐fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid‐microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07–2.39% (migration time) and 8.28–14.0% (peak area).     

Advances of a capillary electrophoretic on-line concentration technique: Electrokinetic supercharging

After comparing with electrokinetic injection (EKI) and transient isotachophoresis (t-ITP), the principles of electrokinetic supercharging (EKS) were introduced. Thereafter, the advances and applications of EKS in capillary electrophoresis were intorduced in the following aspects: EKI setups, t-ITP setups, capillary electrophoresis (CE) separation, and real sample analysis. The factors that limit its application are discussed, and the future development of EKS is also prospected.     

Performance of electrokinetic supercharging for high-sensitivity detection of DNA fragments in chip gel electrophoresis

Chip gel electrophoresis was explored for high-sensitivity detection of DNA by combining electrokinetic injection with transient isotachophoresis preconcentration (here named electrokinetic supercharging (EKS)). Low concentrations (0.2 mg/L) of DNA sample could be detected without fluorescence labeling using a conventional UV detector (at 260 nm). On a single-channel microchip, identification of PCR product was performed by exploiting both external and internal calibration methods. The deviation between the two calibration methods was about 3.6%, and the identified DNA fragment size matched with the predicted size of the template DNA. On the cross microchip the EKS preconcentration has also been achieved when changing the injection reservoir differing from the one used previously. The procedure was computer-simulated and the influence of the voltage applied to two-side reservoirs on sample preconcentration and dilution was also discussed.     

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging

Electrokinetic supercharging (EKS) is considered as one of the most powerful online preconcentration techniques in electrophoresis. It combines the efficient preconcentration power of field-amplified sample injection and the exceptional selective nature of transient isotachophoresis. It has a wide range of applications to different types of analytes ranging from small ions to large proteins and DNA fragments. This comprehensive review--up to date--provides listing for all the works, developments, and advances in EKS. The review will pay particular attention to innovations, new methodologies for manipulation, challenges for improving the detection sensitivity, and various applications of EKS in capillaries and microchips.     

Anion-selective exhaustive injection-sweeping microemulsion electrokinetic chromatography

In this study, anion-selective exhaustive injection-sweeping (ASEI-sweeping) technique, which is a selective on-line sample concentration technique, was first proposed in microemulsion electrokinetic chromatography (MEEKC) for analyses of eight acidic phenolic compounds. In contrast to a capillary that is typically filled with nonmicellar background solution in conventional ASEI-sweeping MEKC method, in the proposed ASEI-sweeping MEEKC method, a capillary is filled with a low pH microemulsion solution (pH 2.0), and then with a short acid plug (pH 2.0, 1.9 cm) before field-amplified sample injection. This proposed design has two functions. First, the microemulsion solution that is present at the front of capillary column is able to avoid phase separation of microemulsion solution during MEEKC separation. Second, the presence of the short acid plug would effectively limit the partition behavior of acid analytes with the oil droplets in the microemulsion during field-amplified sample injection; otherwise, the stacking effect of acid analytes would be markedly reduced. This optimal ASEI-sweeping MEEKC method afforded about 96,000-fold to 238,000-fold increases in detection sensitivity in terms of peak areas without any separation efficiency loss when compared to normal MEEKC separation. Furthermore, trace levels (about 3 ng/g) of gallic acid and catechin in foods were also detected successfully by the proposed ASEI-sweeping MEEKC technique.     

Analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) by electrokinetic supercharging preconcentration, CZE separation, and in-capillary derivatization

The analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) in heat exchanger fluids of nuclear power plants is needed to monitor corrosion. A method involving preconcentration with electrokinetic supercharging (electrokinetic injection with transient ITP), CZE separation, and in-capillary derivatization with ortho-phenanthroline (o-Phe) for direct UV detection was thus developed. First, a multizone BGE was loaded into the capillary by successive hydrodynamic introduction of zones of (i) o-Phe-containing BGE, (ii) BGE for the zonal separation, and (iii) ammonium-based leading electrolyte. Metal cations were electrokinetically injected and stacked at the capillary inlet behind this last leading zone. Finally, a terminating electrolyte zone was hydrodynamically introduced. When a constant voltage was applied, metal ions kept on concentrating isotachophoretically, then separated in CZE mode, were complexed by migrating through an o-Phe zone, and finally detected by direct absorbance. To detect extremely thin peaks, it was attempted for the first time to focus the derivatization reagent by inducing a second transient ITP, before labeling analytes, already separated in CZE mode. With this arrangement, LODs were about 30 ppt in pure water. In heat exchanger fluid matrices containing 1000 ppm bore and 2 ppm lithium, only Fe(II) cation was detected among the three cations of interest at the 1 ppb level using the present method, and its LOD was about ten times higher, due to the lower loading rate during electrokinetic injection.     

基于微芯片电泳的脱氧核糖核酸片段的浓缩和分离

采用超负荷电动供给(electrokinetic supercharging, EKS)预浓缩技术,在微芯片电泳(MCE)上对脱氧核糖核酸（DNA）片段进行浓缩和分离。EKS是集合样品电动进样(EKI)和过渡等速电泳(tITP)的一种在线浓缩方法。研究表明：采用该方法后,在40.5 mm长的单通道芯片上能够实现对低浓度样品的大量进样、浓缩和基线分离。在普通的紫外检测条件(检测波长为260 nm)下,对DNA片段的平均检出限(S/N=3)约为0.07 mg/L,仅为十字芯片上的微芯片电泳检出限的1/40。本文还对浓缩过程中的一些关键因素和定性分析进行了探讨。     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

Electrokinetic supercharging with a system-induced terminator and an optimized capillary versus electrode configuration for parts-per-trillion detection of rare-earth elements in CZE

A further improvement of electrokinetic supercharging (EKS) methodology has been proposed, with the objective to enhance the sensitivity of the conventional CZE-UV method down to a single-digit part per trillion (ppt) level. The advanced EKS procedure is based on a novel phenomenon displaying the formation of a zone with an increased concentration of the hydrogen ion, capable to perform the function of a terminator, behind the sample zone upon electrokinetic injection. In combination with a visualizing co-ion of BGE, protonated 4-methylbenzylamine, acting as the leading ion, such system-induced terminator a effected the transient ITP state to efficiently concentrate cationic analytes prior to CZE. Furthermore, to amass more analyte ions within the effective electric field at the injection stage, a standard sample vial was replaced with an elongated vial that allowed the sample volume to be increased from 500 to 900 μL. Alongside, this replacement made the upright distance between the electrode and the capillary tips prolonged to 40.0 mm to achieve high-efficiency electrokinetic injection. The computer simulation was used for profiling analyte concentration, pH, and field strength in order to delineate formation of the terminator during sample injection. The proposed preconcentration strategy afforded an enrichment factor of 80,000 and thereby the LODs of rare-earth metal ions at the ppt level, e.g. 0.04 nM (6.7 ng/L) for erbium(III).     

On-line preconcentration of weak electrolytes by electrokinetic accumulation in CE: experiment and simulation

A new on-line preconcentration technique was developed that makes possible to determine nanomolar concentrations of weak acidic analytes in CE. The method consists of long-running electrokinetic sample injection and stacking (electrokinetic immobilization) of the analytes at a boundary of two electrolytes with different pH values (pH 9.5 and 2.5) and consequent mobilization of the stacked uncharged analytes in a micellar system (containing SDS micelles). Several factors including buffer concentration, pH, applied voltage, time of preconcentration, and SDS concentration were tested to optimize the analysis method. An about 4600-fold increase of the sample concentration (in comparison with the standard CZE) can be achieved during the preconcentration step. Two preservatives applied in food industry -- benzoic acid and sorbic acid were used as model samples. The applicability of the proposed method in food analysis was demonstrated by determination of nanomolar concentrations of benzoic acid in sunflower oil. An extended version of the computer program Simul was used for modeling both the preconcentration and mobilization processes taking place in the capillary.     

Electrokinetic supercharging preconcentration prior to CGE analysis of DNA: Sensitivity depends on buffer viscosity and electrode configuration

Aiming to high sensitivity DNA analysis by CGE, electrokinetic supercharging (EKS) approach was adopted in this article. EKS is known as an online preconcentration technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). Herein, two factors of buffer viscosity and electrode configuration were studied to further improve EKS performance. An ultralow‐viscosity Tris‐Boric acid‐EDTA (TBE) buffer solution, consisted of 2% low‐molecular‐weight hydroxypropyl methyl cellulose (HPMC) and 6% mannitol and with pH 8.0 adjusted by boric acid, was applied. The boric acid would make a complex with mannitol and generates borate polyanion, which acts as the leading ion for tITP process. The new electrode configuration, a Pt ring around capillary, was modified on Agilent CE system to lead large amount sample introduction during EKS. The standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The 170 000‐fold highly diluted sample at concentration of 3.0 ng/mL was enriched by EKS and detected by normal UV detection method. The obtained LOD of the weakest peak of 72 bp fragment was around 7.7 pg/mL, apparently improved more than 10 000‐fold in comparison with conventional CGE with UV detection.     

Separation and sweeping of flavonoids by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants

In this report, a novel means for the separation and sweeping of flavonoids (quercetin, rutin, calycosin, ononin and calycosin-7-O-β-d-glucoside) by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants as modified pseudostationary phase was presented. The optimized background electrolyte consisted of 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 10 mM sodium borate or 25 mM phosphoric acid. We systematically investigated the separation and preconcentration conditions, including the concentrations of surfactant, types of sweeping, sample matrix, the effect of high salt or acetonitrile, and sample injection volume. It was found that the use of mixed surfactants significantly enhanced the separation efficiency through the change of the efficient electrophoretic mobility of analytes. Compared with normal sample injection, 185-508-fold sensitivity enhancement in terms of limit of detection was achieved through effective sweeping of large sample volume at 50 mbar pressure (up to 45% capillary length). At last, the proposed method was suitable for the determination of Radix Astragali sample.