Negative pressure pinched sample injection for microchip-based electrophoresis

A simple method for injecting well-defined non-biased sample plugs into the separation channel of a microfluidic chip-based capillary electrophoresis system was developed by a combination of flows generated by negative pressure, electrokinetic and hydrostatic forces. This was achieved by using only a single syringe pump and a single voltage supply at constant voltage. In the loading step, a partial vacuum in the headspace of a sealed sample waste reservoir was produced using a syringe pump equipped with a 3-way valve. Almost instantaneously, sample was drawn from the sample reservoir across the injection intersection to the sample waste reservoir by negative pressure. Simultaneously, buffer flow from the remaining two buffer reservoirs pinched the sample flow to form a well-defined sample plug at the channel intersection. In the subsequent separation stage, the vacuum in headspace of the sample waste reservoir was released to terminate all flows generated by negative pressure, and the sample plug at the channel intersection was electrokinetically injected into the separation channel under the potential applied along the separation channel. The liquid levels of the four reservoirs were optimized to prevent sample leakage during the separation stage. The approach considerably simplified the operations and equipment for pinched injection in chip-based CE, and improved the throughput. Migration time precisions of 3.3 and 1.5% RSD for rhodamine123 (Rh123) and fluorescein sodium (Flu) in the separation of a mixture of Flu and Rh123 were obtained for 56 consecutive determinations with peak height precisions of 6.2% and 4.4% RSD for Rh123 and Flu, respectively.     

Electrokinetic injection of samples into a short electrophoretic capillary controlled by piezoelectric micropumps

Electrokinetic sample injection using two piezoelectric micropumps has been proposed for electrophoresis in short capillaries. The sample is brought to the injection end of the capillary using one of them. Then, the high‐voltage source is turned on and the sample is injected electrokinetically for a defined time. The injection is terminated by removal of the sample zone by the flowing separation electrolyte pumped by the second piezoelectric micropump. The RSD value, expressing the repeatability of the injection, does not exceed 4%. The injection apparatus does not contain any mobile mechanical components, there is no movement of the capillary and both its ends remain constantly in the solution during both the sample injection and separation. Thus, the micropumps replace the six‐way injection valve and linear pump in similar types of injection apparatuses. The injection was tested in the separation and determination of ammonium and potassium ions in two samples of mineral fertilizers. The separation was performed in background electrolyte containing 500 mM of acetic acid + 20 mM Tris + 2 mM 18‐crown‐6 (pH 3.3) in a capillary with id 50 μm and total length/length to the contactless conductivity detector of 10.5/8 cm. The injection and separation took place at a voltage of 5 kV and the separation time equaled 20 s. The measured values of the analyte contents corresponded to the value declared by the manufacturer within the reliability interval, where RSD equaled between 3.5 and 4.7%.     

Electrokinetic-driven microfluidic system in poly(dimethylsiloxane) for mass spectrometry detection integrating sample injection, capillary electrophoresis, and electrospray emitter on-chip

A novel microsystem device in poly(dimethylsiloxane) (PDMS) for MS detection is presented. The microchip integrates sample injection, capillary electrophoretic separation, and electrospray emitter in a single substrate, and all modules are fabricated in the PDMS bulk material. The injection and separation flow is driven electrokinetically and the total amount of external equipment needed consists of a three-channel high-voltage power supply. The instant switching between sample injection and separation is performed through a series of low-cost relays, limiting the separation field strength to a maximum of 270 V/cm. We show that this set-up is sufficient to accomplish electrospray MS analysis and, to a moderate extent, microchip separation of standard peptides. A new method of instant in-channel oxidation makes it possible to overcome the problem of irreversibly bonded PDMS channels that have recovered their hydrophobic properties over time. The fast method turns the channel surfaces hydrophilic and less prone to nonspecific analyte adsorption, yielding better separation efficiencies and higher apparent peptide mobilities.     

Simultaneous enantioseparation and sensitivity enhancement of basic drugs using large-volume sample stacking

Simultaneous enantioseparation with sensitive detection of four basic drugs, namely methoxamine, metaproterenol, terbutaline and carvedilol, using a 20-mum ID capillary with native beta-CD as the chiral selector was demonstrated by the large-volume sample stacking method. The procedure included conventional sample loading either hydrodynamically or electrokinetically at longer injection times without polarity switching and EOF manipulation. In comparison to conventional injections, depending on the analyte, about several hundred- and a thousand-fold sensitivity enhancement was achieved with the hydrodynamic and the electrokinetic injections, respectively. The simple method developed was applied to the analysis of racemic analytes in serum samples and better recovery was achieved using hydrodynamic injection than electrokinetic injection.     

An automated electrokinetic continuous sample introduction system for microfluidic chip-based capillary electrophoresis

An automated and continuous sample introduction system for microfluidic chip-based capillary electrophoresis (CE) was developed in this work. An efficient world-to-chip interface for chip-based CE separation was produced by horizontally connecting a Z-shaped fused silica capillary sampling probe to the sample loading channel of a crossed-channel chip. The sample presentation system was composed of an array of bottom-slotted sample vials filled alternately with samples and working electrolyte, horizontally positioned on a programmable linearly moving platform. On moving the array from one vial to the next, and scanning the probe, which was fixed with a platinum electrode on its tip, through the slots of the vials, a series of samples, each followed by a flow of working electrolyte was continuously introduced electrokinetically from the off-chip vials into the sample loading channel of the chip. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine and phenylalanine with LIF detection, by continuously introducing a train of different samples. Employing 4.5 kV sampling voltage (1000 V cm(-1) field strength) for 30 s and 1.8 kV separation voltage (400 V cm(-1) field strength) for 70 s, throughputs of 36 h(-1) were achieved with <1.0% carryover and 4.6, 3.2 and 4.0% RSD for arginine, FITC and phenylalanine, respectively (n = 11). Net sample consumption was only 240 nL for each sample.     

Detection of chlorogenic acid in honeysuckle using infrared-assisted extraction followed by capillary electrophoresis with UV detector

In this study, a novel infrared-assisted extraction method coupled capillary electrophoresis (CE) is employed to determine chlorogenic acid from a traditional Chinese medicine (TCM), honeysuckle. The effects of pH and the concentration of the running buffer, separation voltage, injection time, IR irradiation time, and anhydrous ethanol in the extraction concentration were investigated. The optimal conditions were as follows: extraction time, 30 min; extraction solvent, 80% (v/v) ethanol in water solution; and 50 mmol/L borate buffer (pH 8.7) was used as the running buffer at a separation voltage of 16 kV. The samples were injected electrokinetically at 16 kV for 8 s. Good linearity (r(2) > 0.9996) was observed over the concentration ranges investigated, and the stability of the solutions was high. Recoveries of the chlorogenic acid were from 95.53% to 106.62%, and the relative standard deviation was below 4.1%. By using this novel IR-assisted extraction method, a higher extraction efficiency than those extracted with conventional heat-reflux extraction was found. The developed IR-assisted extraction method is simple, low-cost, and efficient, offering a great promise for the quick determination of active compounds in TCM. The results indicated that IR-assisted extraction followed by CE is a reliable method for quantitative analysis of active ingredient in TCM.     

Single potential electrophoresis microchip with reduced bias using pressure pulse injection

We propose two variants of a new injection technique for use in electrophoresis microchips, called "front gate pressure injection" and "back gate pressure injection", that both enable a controlled and reproducible sample introduction with reduced bias compared to electrokinetic gated injection. A continuous flow of a test solution of fluorescein/rhodamine B in 20 mM Tris/boric acid buffer (pH 8.6) sample test solution is electrokinetically driven near to the entrance of the separation channel, using a single voltage (3 kV) that is constant in time. A sample plug is injected in the separation channel by a pressure pulse of the order of 0.1 s. The latter is generated using the mechanical deflection of a PDMS membrane that is loosely placed on a dedicated chip reservoir. The analysis of the peak area ratio of the separated compounds demonstrates a nearly constant sample composition when using pressure-based injection. A small remaining injection bias for the shortest membrane deflection times can be attributed to a dilution effect of the charged compound due to the presence of an electrical field transverse to the sample flow boundary in the channel junction.     

High-throughput characterization for organic pollutants in environmental waters using a capillary electrophoresis chip.

To evaluate organic pollution in water, we did preliminarily studies on high-throughput characterization of organic pollution in water using microchip-based capillary electrophoresis (CE) with laseer-induced fluorescence (LIF) detection. The applied voltage was investigated to control the gated valve injection and CE separation for conventional cross type microchips using a self-made personal computer (PC)-based controller as the voltage supply. We obtained high-throughput data for the reproducible separation of fluorescein isothiocyanate (FITC)-labeled river-water samples using a zwitter-ion based buffer solution to avoid adsorption of the labeled sample onto the channel of a microchip made from quartz glass. We used real samples from the Hino River that flows into Lake Biwa, from ten sampling points and obtained several reproducible peaks in different separation patterns for each sample within 2 min. We successfully demonstrated high-throughput characterization of dissolved organic carbon (DOC) in environmental water using the microchip.     

Simultaneous determination of aripiprazole and its active metabolite,dehydroaripiprazole, in plasma by capillary electrophoresis combining on‐column field‐amplified sample injection and application in schizophrenia

A sensitive high‐performance CZE combining on‐column field‐amplified sample injection (FASI) has been developed for simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in human plasma. A sample pretreatment by means of liquid–liquid extraction (LLE) (diethyl ether) with subsequent quantitation by FASI‐CZE was used. The separation of aripiprazole and dehydroaripiprazole was performed using a BGE containing 150 mM phosphate buffer (pH 3.5) with 40% methanol and 0.02% PVA as a dynamic coating to reduce interaction of analytes with the capillary wall. Before sample loading, a methanol plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (10 kV, 30 s) to introduce sample cations and the applied voltage was 20 kV with on‐column detection at 214 nm. Several parameters affecting the separation and sensitivity of the drug and its active metabolite were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer. The linear ranges of the method for test drug and its active metabolite, in plasma using amlodipine as an internal standard, were over the range 5.0–100.0 ng/mL. One female volunteer (25 years old) was orally administered a single dose of 10 mg aripiprazole (Abilify^®, Otsuka) and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied to monitor the concentration of aripiprazole and dehydroaripiprazole in plasma collected after oral administration of 20 or 30 mg aripiprazole (Abilify^®, Otsuka) daily at steady state in one schizophrenic patient.     

提高芯片毛细管电泳系统检测信噪比的哈达玛变换法

采用门控进样,在简单的十字通道微流控玻璃芯片上实现了假随机多次进样,研究了利用哈达玛变换提高微流控毛细管电泳分析系统信噪比的方法.在实验中,以7阶127步假随机二进制序列作为进样模板,将缓冲液和Cy₅衍生后的氨基酸试样交替注入到分离通道中,检测到的电泳信号经过哈达玛反变换还原使信噪比提高5倍(理论上5.6倍)的电泳谱,各组分的出峰时间、峰高和峰形均完全还原,毛细管电泳分离的采样频率不受影响.     

Rapid and variable-volume sample loading in sieving electrophoresis microchips using negative pressure combined with electrokinetic force

A rapid and variable-volume sample loading scheme for chip-based sieving electrophoresis was developed by negative pressure combined with electrokinetic force. This was achieved by using a low-cost microvacuum pump and a single potential supply at a constant voltage. Both 12% linear polyacrylamide (LPA) with a high viscosity of 15000 cP and 2% hydroxyethylcellulose (HEC) with a low viscosity of 102 cP were chosen as the sieving materials to study the behavior and the versatility of the proposed method. To reduce the hydrodynamic resistance in the sampling channel, sieving material was only filled in the separation channel between the buffer waste reservoir (BW) to the edge of the crossed intersection. By applying a subambient pressure to the headspace of sample waste reservoir (SW), sample and buffer solution were drawn immediately from sample reservoir (S) and buffer reservoir (B) across the intersection to SW. At the same time, the charged sample in the sample flow was driven across the interface between the sample flow and the sieving matrix into the sieving material filled separation channel by the applied electric field. The injected sample plug length is in proportion with the loading time. Once the vacuum in SW reservoir was released to activate electrophoretic separation, flows from S and B to SW were immediately terminated by the back flow induced by the difference of the liquid levels in the reservoirs to prevent sample leakage during the separation stage. The sample consumption was about 1.7 x 10(2) nL at a loading time of 1 s for each cycle. Only 0.024 s was required to transport bias-free analyte to the injection point. It is easy to freely choose the sample plug volume in this method by simply changing the loading time and to inject high quality sample plug with non-distorted shape into the separation channel. The system has been proved to possess an exciting potential for improving throughput, repeatability, sensitivity and separation performance of chip-based sieving electrophoresis.     

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.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

毛细管区带电泳峰形变化规律的研究

采用指数修正的高斯模型（ＥＭＧ）,研究了进样时间、分离电压及柱长等一系列操作条件对毛细管区带电泳峰形参数的影响。实验结果表明,进样时间对半峰宽（Ｗ１／２）和峰不对称度（Ｂ／Ａ）有显著影响;电压对以长度表示的半峰宽及峰拖尾参数无显著影响;ｔｍ与σ、τ、Ｗ１／２（ｓ）之间都有很好的线性关系;随着柱长的增大,σ、τ、Ｗ１／２都略有增加。     

Sample pre-concentration by isotachophoresis in microfluidic devices

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.     

Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.     

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.     

Improved hydrostatic pressure sample injection by tilting the microchip towards the disposable miniaturized CE device

A simple method of hydrostatic pressure sample injection towards a disposable microchip CE device was developed. The liquid level in the sample reservoir was higher than that in the sample waste reservoir (SWR) by tilting microchip and hydrostatic pressure was generated, the sample was driven to pass through injection channel into SWR. After sample loading, the microchip was levelled for separation under applied high separation voltage. Effects of tilted angle, initial liquid height and injection duration on electrophoresis were investigated. With enough injection duration, the injection result was little affected by tilted angle and initial liquid heights in the reservoirs. Injection duration for obtaining a stable sample plug was mainly dependent on the tilted angle rather than the initial height of liquid. Experimental results were consistent with theoretical prediction. Fluorescence observation and electrochemical detection of dopamine and catechol were employed to verify the feasibility of tilted microchip hydrostatic pressure injection. Good reproducibility of this injection method was obtained. Because the instrumentation was simplified and no additional hardware was needed in this technology, the proposed method would be potentially useful in disposable devices.     

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.     

阳离子选择性耗尽进样-胶束电动色谱法对可非糖浆中盐酸异丙嗪与磷酸可待因的测定

在胶束电动色谱法的基础上,联用阳离子选择性耗尽进样技术,对盐酸异丙嗪和磷酸可待因同时测定的方法进行了研究。考察了pH值、有机溶剂、SDS浓度、进样时间、进样电压等实验条件对分离效果的影响。最佳实验条件为:缓冲体系16%乙腈+80 mmol/L SDS+20 mmol/L NaH2PO4(pH2.4),分离电压为-18 kV,测量波长214 nm,萃取液pH2.4,进样电压10 kV,进样时间100 s。在优化实验条件下,两种物质在8 min内出峰,峰面积RSD不大于4.6%。盐酸异丙嗪、磷酸可待因的线性范围分别为0.50~81.3、0.78~62.5μg/L,检出限分别为0.16、0.12μg/L,相关系数分别为0.998 9、0.998 8。将方法用于可非糖浆中盐酸异丙嗪与磷酸可待因的测定,回收率为96%~106%。