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.     

Electrophoretic injection bias in a microchip valving scheme

The pinched injection strategy, implemented on microfabricated fluidic devices (microchips), was investigated for an electrophoretic injection bias. Both the sample loading and dispensing steps were found to contribute to the injection bias whereby neutral species were injected preferentially to anionic species. In the sample loading step, neutral species filled a larger volume in the cross intersection than anionic species. Similarly, in the dispensing step, a larger volume of neutral analyte was injected than anionic analyte. Up to a 27% difference in injected volumes was observed. Fluorescently labeled amino acids were used as model analytes.     

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.     

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.     

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.     

Pressure-driven sample injection with quantitative liquid dispensing for on-chip electrophoresis.

A novel pressure-driven sample injection method was developed as an alternative to electrokinetic injection, and electrophoretic separation was carried out on a microfabricated device employing this method. This method enables a defined volume of liquid dispensing, followed by instantaneous injection driven by pneumatic pressure, greatly simplifying the injection procedure. A particular microstructure, called a "metering chamber", has been designed for the quantitative dispensing of an ultra-low volume of sample liquid; a "hydrophobic passive valve" equipped with an air vent channel is employed for injecting a dispensed sample into the separation channel. The reproducibility of dispensing was 3.3% (n = 15), expressed by the variation of dispensed volumes. The electrophoretic separation of DNA fragments was performed using this injection method, varying the injection volumes from 0.45 to 4.0 nL, and the separation efficiencies were compared. This precise injection method, easily variable in injection volumes, is highly suitable for quantitative as well as qualitative electrophoretic analyses.     

Effects of the electric field distribution on microchip valving performance

Valving characteristics on microfluidic devices were controlled through manipulation of the electric field strengths during both the sample loading and dispensing steps. Three sample loading profiles for the constant volume valve (pinched injection) in conjunction with four dispensing schemes were investigated to study valving performance. The sample confinement profiles for the sample loading step consisted of a weakly pinched sample, a medium pinched sample, and a strongly pinched sample. Four dispensing schemes varied the electric field strengths in the sample and sample waste channels relative to the analysis channel to control the volume of the sample dispensed from the valve. The axial extent of the sample plug decreased as the electric field strengths in the sample and sample waste channels were raised relative to the analysis channel. In addition, a trade-off existed between sample plug length and sensitivity.     

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3?psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100?pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1?Hz to > 2?Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 103 theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.     

Improving precision of manual hydrodynamic injection in capillary electrophoresis with contactless conductivity detection

Reproducible injection in capillary electrophoresis has been difficult to achieve with manual injection techniques using simple injection devices, such as gravity injection (siphoning) or hydrodynamic sample splitting. We demonstrate that the injection reproducibility can be improved using very simple means. With hydrodynamic sample splitter, a passive micro-metering valve can be inserted in-line to regulate the sample flow rate through the splitter interface. A significant improvement of both reproducibility and repeatability was achieved. The reproducibility of RSD of the peak areas improved from 25.4% to 4.4%, while the repeatability was below 4.1% when micro-metering valve was used. Additional simple correction that can be used to further improve the variability of injected sample volumes in any hydrodynamic injection mode in CE with conductivity detection was proposed and verified. The measured EOF peak can serve as a simple indicator of the injected volume and can be effectively used for additional correction. By a linear function between the injection volume and the peak area of the EOF, the RSD values of peak areas for both manual gravity injection and hydrodynamic sample splitter were further improved below 2% RSD. The linearity of the calibration curve was also significantly improved. The proposed correction works even with slight differences in matrix composition, as demonstrated on the analysis aqueous soil extract of model mixture of five nerve agent degradation products.     

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.     

Pressure-assisted field-amplified sample injection with reverse migrating micelles for analyzing trace steroids in MEKC

This paper describes a novel method that applies pressure-assisted field-amplified sample injection with reverse migrating micelles (PA-FASI-RMM) for the online concentration of neutral analytes in MEKC with a low-pH BGE. After injection of a plug of water into the separation capillary, negative voltage and positive pressure were simultaneously applied to initialize PA-FASI-RMM injection. The hydrodynamic flow generated by the positive pressure compensated the reverse EOF in the water plug and allowed the water plug to remain in the capillary during FASI with reverse migrating micelles (FASI-RMM) to obtain a much longer injection time than usual, which improved stacking efficiency greatly. Equations describing this injection mode were introduced and were supported by experimental results. For a 450-s online PA-FASI-RMM injection, three orders of magnitude sample enhancement in terms of peak area could be observed for the steroids and an achievement of detection limits was between 1 and 10 ng/mL.     

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

Comparison of field‐enhanced and pressure‐assisted field‐enhanced sample injection techniques for the analysis of water‐soluble vitamins using CZE

A new online concentration method, namely pressure‐assisted field‐enhanced sample injection (PA‐FESI), was developed and compared with FESI for the analysis of water‐soluble vitamins by CZE with UV detection. In PA‐FESI, negative voltage and positive pressure were simultaneously applied to initialize PA‐FESI. PA‐FESI uses the hydrodynamic flow generated by the positive pressure to counterbalance the reverse EOF in the capillary column during electrokinetic sample injection, which allowed a longer injection time than usual FESI mode without compromising the separation efficiency. Using the PA‐FESI method, the LODs of the vitamins were at ng/mL level based on the S/N of 3 and the RSDs of migration time and peak area for each vitamin (1 μg/mL) were less than 5.1%. The developed method was applied to the analysis of water‐soluble vitamins in corns.     

Evaluation of sample injection precision in respect to sensitivity in capillary electrophoresis using various injection modes

A comparative study was conducted to assess the injection precision in capillary electrophoresis for cationic analytes (arecoline, codeine, papaverine). The precision was measured in respect to methods sensitivity in various injection modes in capillary electrophoresis: standard hydrodynamic injection (3.45 kPa for 6 s), large volume sample stacking (3.45 kPa for 40 s), and field‐amplified sample injection (10 kV for 65 s). All measurements were conducted for aqueous solutions of standards to minimize the errors linked to the sample preparation step. The methods were submitted to precision assessment at three concentration levels: at the limit of quantification, three‐fold and ten‐fold of limit of quantification. The results were compared to those from high‐performance liquid chromatography as a reference technique. The field‐amplified sample injection method was shown to provide greatest sensitivity (quantification limits down to 4 ng/mL for all three tested compounds) but the lowest precision. High‐performance liquid chromatography was established as the most reliable technique (coefficient of variation in all intraday experiments was below 1%). It was also shown that with a use of large volume sample injection technique, similar sensitivity as in high‐performance liquid chromatography can be easily reached.     

A new sequential injection analysis‐capillary electrophoresis system with amperometric detection

A novel and fully automated sequential injection analysis manifold coupled to a capillary electrophoresis apparatus with amperometric detection, is described. The sequential injection manifold was isolated from the high voltage by inserting an air plug into the circuit. Small buffer reservoirs were used to avoid the need to pump fresh buffer to the interface during the electrophoretic separation. No decoupling device was used to mitigate the interference from the high voltage electric field, instead the potential shift induced by the separation voltage, was accounted for. The new hydrodynamic injection method presented is based on the overpressure created in the circuit when a pinch valve is closed for a predetermined time. The injection method yields RSD values of peak height and area below 2.55 and 1.82%, respectively, at different durations of valve closure (n = 5). The capillary and working electrode alignment was achieved by adapting a commercial available capillary union. When the electrode was replaced, the alignment method proved to be very reliable, yielding RSD values of peak height and area lower than 2.64 and 2.08%, respectively (n = 8). Using this system with a gold microelectrode, dopamine, and epinephrine could be quantified within the concentration range of 1–500 μM and detected at a concentration of 0.3 μM. The methods here presented could be applied for the development of new capillary electrophoresis systems with amperometric detection and/or to the design of fully automated systems for online process monitoring purposes.     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

肌肽类生物活性肽的毛细管电泳在线富集技术

建立了毛细管区带电泳(CZE)在线富集3种肌肽类活性肽(肌肽、鹅肌肽和高肌肽)的两种简便有效的方法。一种是大体积进样反向压力排除基体富集（LVSRP）技术,即通过流体动力学进样,在不改变电源极性的条件下,利用反向压力排除样品基体,电堆积富集后进行CZE分离;另一种是大体积进样电渗流排除基体富集（LVSEP）技术,即通过流体动力学进样,于运行缓冲液中加入溴化十六烷基三甲基铵(CTAB)动态修饰毛细管表面,通过电渗流排除样品基体,改变电源极性后进行CZE分离。与常规CZE相比,LVSRP技术和LVSEP技术使检测灵敏度提高了40~60倍。对影响两种富集过程的一些因素进行了研究,在最优富集条件下考察本方法的线性范围为0.080~5.0 μmol/L。对3种生物活性肽的检测限(S/N=3)分别为LVSRP 41~58 nmol/L,LVSEP 35~43 nmol/L。     

Reduction in sample injection bias using pressure gradients generated on chip

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 μm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4–6 for the analyte peaks in a 3 cm long analysis channel.     

The solvent venting injection technique in capillary supercritical fluid chromatography

A solvent venting technique for injection of volumes up to 1 μl on 50 μm i.d. SFC columns has been compared to direct injection methods. The peak broadening and peak splitting observed with direct injection have been examined and found to be related to the starting pressure, the column temperature, and the sample solvent, in addition to the sample volume. The solvent venting technique removed peak splitting and improved the column efficiency. With a proper selection of experimental conditions, the sample recovery was 100%. The major part of the solvent was eluted in 15–20 s. Several applications have been demonstrated.