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.     

Study of injection bias in a simple hydrodynamic injection in microchip CE

The electrokinetically pinched method is the most commonly used mode for sample injection in microchip capillary electrophoresis (microCE) due to its simplicity and well-defined sample volume. However, the limited injection volume and the electrophoretic bias of the pinched injection may limit its universal usage to specific applications. Several hydrodynamic injection methods in microCE have been reported; however, almost all claimed that their methods are bias-free without considering the dispensing bias. To investigate the dispensing bias, a simple hydrodynamic injection was developed in single-T and double-T glass microchips. The sample flow was produced by hydrostatic pressure generated by the liquid level difference between the sample reservoir and the other reservoirs. The reproducibility of peak area and peak area ratio was improved to a significant extent using large-surface reservoirs for the buffer reservoir and the sample waste reservoir to reduce the Laplace pressure effect. Without a voltage applied on the sample solution, the voltage-related sample bias was eliminated. The dispensing bias was analyzed theoretically and studied experimentally. It was demonstrated that the dispensing bias existed and could be reduced significantly by appropriately setting up the voltage configuration and by controlling the appropriate liquid level difference.     

An electrical pumping approach to eliminate sample bias in capillary electrokinetic injection

A general pumping injection (PI), which involves the use of two capillaries with different diameters, was taken to evaluate systematically the effects on eliminating sample bias associated with the electrokinetic injection process in CE. One end of the separation capillary of the smaller diameter was inserted into another pumping capillary of larger diameter. When a high voltage was applied to the pumping capillary, the EOF generated inside will act as a pump to drive the solution stream in the separation capillary. The results have demonstrated that PI is suitable for both normal and reverse EOF situations. Second, the bias degree (BD) and SD of bias we presented were used to evaluate the degree of the bias under different conditions, and the factors of bias elimination have been investigated. Under optimal conditions, the bias was satisfactorily eliminated by PI. This EOF pumping system was successfully applied to the analysis of samples in CEC for a bias-free injection. Moreover, this two-capillary pumping system did not significantly affect the EOF, current, and the column efficiency of the separation process. Finally, a PI with grounded electrode was proposed and shown to be suitable for samples with low conductivity and ions with different mobility.     

A capillary electrophoresis chip with hydrodynamic sample injection for measurements from a continuous sample flow

A microchip-based capillary electrophoresis device supported by a microfluidic network made of poly(dimethylsiloxane), used for measuring target analytes from a continuous sample flow, is presented. The microsystem was fabricated by means of replica molding in combination with standard microfabrication technologies, resulting in microfluidic components and an electrochemical detector. A new hydrodynamic sample injection procedure is introduced, and the maximum number of consecutive measurements that can be made with a poly(dimethylsiloxane) capillary electrophoresis chip with amperometric detection is investigated with respect to reproducibility. The device features a high degree of functional integration, so the benefits associated with miniaturized analysis systems apply to it.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

Isoelectric focusing sample injection for capillary electrophoresis of proteins

Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.     

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.     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

Field-amplified sample injection and in-capillary derivatization for capillary electrophoretic analysis of metal ions in local wines

In-capillary derivatization and field-amplified sample injection (FASI) coupled to capillary zone electrophoresis (CZE) was evaluated for the analysis of metals (Co(II), Cu(II), Ni(II), and Fe(II)) using 2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol (Nitro-PAPS) as the derivatizing agent. For FASI, the optimum conditions were water as sample solvent, 1 s hydrodynamic injection (0.1 psi) of a water plug, 5 s of electrokinetic introduction (10 kV) of the sample. The in-capillary derivatization was successfully achieved with zone-passing strategy in order tandem injection of Nitro-PAPS reagent (0.5 psi, 7 s), a small water plug (0.1 psi, 1 s), and metal ion introduction (10 kV, 5 s). The solution of 45 mmol L^− 1 borate pH 9.7 and 1.0 × 10^− 5 mol L^− 1 Nitro-PAPS containing 20% acetonitrile was used as the running buffer. The limit of detection obtained by the proposed method was lower than those from pre-capillary derivatization about 3–28 times. The recovery of the method was comparable to pre-capillary derivatization method. In-capillary derivatization-FASI-CZE was applied to analysis of metals in wine samples. The results were compared with those obtained by CZE with pre-capillary derivatization method and atomic absorption spectrometry (AAS).     

Sequential injection sample introduction microfluidic-chip based capillary electrophoresis system

A sequential injection micro-sample introduction system was coupled to a microfluidic-chip based capillary electrophoresis system through a split–flow sampling interface integrated on the micro-chip. The microfluidic system measured 20×70×3 mm in dimension, and was produced using a non-lithographic approach with components readily available in the analytical laboratory. In the H-configuration channel design the horizontal separation channel was a 75 μm I.D.×60 mm quartz capillary, with two vertical side arms produced from plastic tubing. The conduits were embedded in silicon elastomer with a planar glass base. Sequential introduction of a series of samples with about 2.5% carryover was achieved at 48 h⁻¹ throughput with samples containing a mixture of fluorescein isothiocyanate (FITC)-labeled amino acids using SI sample volumes of 3.3 μl and carrier flow-rate of 2.0 ml min⁻¹. Baseline separation was achieved for FITC-labeled arginine, phenylalanine, glycine and FITC (laser induced fluorescence detection) in sodium tetraborate buffer (pH 9.2) within 8–80 s, at separation lengths of 25–35 mm and electrical field strengths of 250–1500 V cm⁻¹, with plate heights in the 0.7–3 μm range.     

Capillary electrophoresis of methylmercury with injection by sample stacking

A procedure for separation and quantitation of methylmercury by capillary electrophoresis using sample stacking as the injection technique is presented. The CE conditions have been optimized in order to separate the methylmercury from the excess cysteine peak and to concentrate large volumes of sample obtaining a low detection limit. Under the proposed operational conditions, the detection limit (S/N = 3) was 12 ng g⁻ and the limit of quantitation (S/N = 10) was 20 ng g⁻¹ with a linear range of 20–100 ng g⁻¹ (as methylmercury in samples). The method was tested using different reference materials with a certified methylmercury content.     

Enhancing resolution of free‐flow zone electrophoresis via a simple sheath‐flow sample injection

In this work, a simple and novel sheath‐flow sample injection method (SFSIM) is introduced to reduce the band broadening of free‐flow zone electrophoresis separation in newly developed self‐balance free‐flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C‐phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free‐flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath‐flow sample injection method.     

Head-column field-amplified sample stacking in a capillary electrophoresis-flow injection system

A simple, effective, and continuous online concentration method for the sensitive detection of alkaloids applying CE-flow injection analysis with head-column field-amplified sample stacking was developed. A series of samples was continuously introduced into the capillary by electrokinetic means without interrupting the high voltage. A short water plug was introduced by the EOF at the capillary inlet end prior to sample introduction. Under optimum conditions, 15-fold improvement in concentration sensitivity was achieved, giving an LOD of about 0.67 and 0.73 microg/mL for ephedrine (E) and pseudoephedrine (PE), respectively. The separation could be achieved within 4 min and sample throughput rate could reach up to 7/h. The repeatability (defined as RSD) was 3.62, 1.51% with peak area evaluation and 1.30, 2.58% with peak height evaluation for E and PE, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing E and PE, and the recoveries were 92.3-102.4%.     

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection

We present sequential CE analysis of amino acids and l ‐asparaginase‐catalyzed enzyme reaction, by combing the on‐line derivatization, optically gated (OG) injection and commercial‐available UV‐Vis detection. Various experimental conditions for sequential OG‐UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG‐UV/vis CE analysis, sequential online CE enzyme assay of l ‐asparaginase‐catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off‐line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.     

On‐chip pressure generation using a gel membrane fabricated outside of the microfluidic network

On‐chip generation of pressure gradients via electrokinetic means can offer several advantages to microfluidic assay design and operation in a variety of applications. In this article, we describe a simple approach to realizing this capability by employing a polyacrylamide‐based gel structure fabricated within a fluid reservoir located at the terminating end of a microchannel. Application of an electric field across this membrane has been shown to block a majority of the electroosmotic flow generated within the open duct yielding a high pressure at the channel–membrane junction. Experiments show the realization of higher pressure‐driven velocities in an electric field‐free separation channel integrated to the micropump with this design compared to other similar micropumps described in the literature. In addition, the noted velocity was found to be less sensitive to the extent of Debye layer overlap in the channel network, and therefore more impressive when working with background electrolytes having higher ionic strengths. With the current system, pressure‐driven velocities up to 3.6 mm/s were realized in a 300‐nm‐deep separation channel applying a maximum voltage of 3 kV at a channel terminal. To demonstrate the separative performance of our device, a nanofluidic pressure‐driven ion‐chromatographic analysis was subsequently implemented that relied on the slower migration of cationic analytes relative to the neutral and anionic ones in the separation channel likely due to their strong electrostatic interaction with the channel surface charges. A mixture of amino acids was thus separated with resolutions greater than those reported by our group for a similar analysis previously.     

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.     

Development and validation of a capillary electrophoresis method for the determination of phenothiazines in human urine in the low nanogram per milliliter concentration range using field-amplified sample injection

A capillary zone electrophoresis (CZE) method with ultraviolet-visible detection has been established and validated for the determination of five phenothiazines: thiazinamium methylsulfate, promazine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, and promethazine hydrochloride in human urine. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 150 mM tris(hydroxymethyl)aminomethane and 25% acetonitrile at pH 8.2, with temperature and voltage of 25 degrees C and 20 kV, respectively. Naphazoline hydrochloride was used as an internal standard. Field-amplified sample injection (FASI) has been applied to improve the sensitivity of the detection. Considering the influence of parameters affecting the on-line preconcentration (nature of preinjection plug, sample solvent composition, injection times, and injection voltage) and due to the significant interactions among them, in this paper we propose for the first time the application of a multivariate approach to carry out the study. The optimized conditions were as follows: preinjection plug of water for 7 s at 50 mbar, electrokinetic injection for 40 s at 6.2 kV, and 32 microm of H3PO4 in the sample solvent. Also, a solid-phase extraction (SPE) procedure is developed to obtain low detection limits and an adequate selectivity for urine samples. The combination of SPE and FASI-CZE-UV allows adequate linearities and recoveries, low detection limits (from 2 to 5 ng/mL), and satisfactory precisions (3.0-7.2% for an intermediate RSD %).     

Split injection: A simple introduction of subnanoliter sample volumes for chip electrophoresis

The ability to accurately inject small volumes of sample into microfluidic channels is of great importance in electrophoretic separations. While electrokinetic injection of nanoliter scale volumes is commonly utilized in microchip capillary electrophoresis (MCE), mobility and matrix bias makes quantitation difficult. Herein, we describe a new injection method based on the simple patterning of the crossing of channels that does not require sophisticated instrumentation. The sample volume injected into the separation channel is dependent on the ratio of the widths of the crossing channels. This injection method is capable of introducing, into a separation channel, multiple plugs of sample on a large scale. This injection technique is tested for zone electrophoresis in native and surface modified poly(dimethylsiloxane) (PDMS) chips.     

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.     

Study on characteristics of bias caused by FI-CE split flow electrokinetic injection

The characteristics of bias caused by split-flow electrokinetic injection (SEKI), a new type of sample injection method used in coupled flow injection-capillary electrophoresis system (FI-CE), was investigated using pseudoephedrine hydrochloride, a basic drug, and ibuprofen, an acidic drug, as model analytes. It was found that bias imposed by SEKI under the condition of continuous sample matrix/running buffer was similar to that done by electrokinetic injection (EKI). The linearity of calibration curve provided by SEKI was similar to that offered by non-bias hydrodynamic injection (HDI) but significantly better than that obtained by EKI. These features were exploited to improve analytical performances in simultaneous determination of the minor ingredient of pseudoephedrine hydrochloride and the major ingredient of ibuprofen in a pharmaceutical preparation. Detectability of 0.7 mg/l for pseudoephedrine hydrochloride was achieved at a sample throughput rate of 24 times per hour, which is 30% lower than that obtained by HDI-based conventional CE. Relative standard deviations (RSDs) of 2.8% for the minor ingredient and 1.2% for the major ingredient were produced in 11 runs of a test solution containing 13.1 mg/l pseudoephedrine hydrochloride and 81.4 mg/l ibuprofen. This is an improvement compared to that obtained by HDI-based conventional CE. Analytical results for two batches of compound ibuprofen tablets by the SEKI-based FI-CE approach were in good agreement with that obtained by a conventional high performance liquid chromatographic method. __________ Translated from Chinese Journal of Chromatography, 2005, 23(2) (in Chinese)