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.     

小型可连续进样微流控芯片分析仪的研制

报道了一种结构简单、可连续进样的小型微流控芯片分析仪的研制。顺序注射分析系统通过芯片上制作的接口将试样连续引入芯片 ,并采用自行设计的紧凑型光纤式激光诱导荧光检测器进行检测。该仪器用于芯片毛细管电泳分离实验室合成Cy5荧光染料 ,实现了连续进样和换样。峰高RSD为 1 .9% (n=1 1 ) ,试样通量 3 5 h ;相邻试样携出 <4%。     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

Analysis of non-covalent protein complexes by capillary electrophoresis--time-of-flight mass spectrometry

A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected.     

Multiplexed detection of nitrate and nitrite for capillary electrophoresis with an automated device for high injection efficiency

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 microL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.     

A microchip-based proteolytic digestion system driven by electroosmotic pumping

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.     

Capillary electrophoresis with contactless conductivity detection coupled to a sequential injection analysis manifold for extended automated monitoring applications

A capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C⁴D) based on a sequential injection analysis (SIA) manifold was refined. Hydrodynamic injection was implemented to avoid a sampling bias by using a split-injection device based on a needle valve for precise adjustment. For safety and reliability, the integrity of the high voltage compartment at the detection end was fully maintained by implementing flushing of the high voltage interface through the capillary. With this set-up, extended fully automated monitoring applications are possible. The system was successfully tested in the field for the determination of the concentration levels of major inorganic cations and anions in a creek over a period of 5 days.     

Parameters affecting reproducibility in capillary electrophoresis

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.     

毛细管电泳流动注射-负压进样装置的研究

设计了一种用于毛细管电泳系统的流动注射-负压进样装置。样品由蠕动泵输送到进样阀后再由缓冲液带到分离毛细管入口,由毛细管出口端施加的负压引入。进样时间由自制精密控时电路控制,经进样条件的优化,能获得良好的重现性。实验中两种阳离子峰面积和迁移时间的RSD(n=8)≤2.7%,优于传统重力进样,而且操作简便;与非接触电导检测器组装成流动注射-毛细管电泳系统,可实现快速、高效的在线分析。初步应用于无机阳离子的分离,取得了满意的结果。     

Headspace‐single drop microextraction with a commercial capillary electrophoresis instrument

Automated coupling of headspace‐single drop microextraction (HS‐SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS‐SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS‐SDME with an on‐line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS‐SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6‐trichlorophenol which is a precursor for 2,4,6‐trichloroanisole causing the foul odor in wine called cork taint.     

Simple interface for capillary electrophoresis-inductively coupled plasma atomic emission spectrometry

A simple interface has been developed to couple capillary electrophoresis (CE) to inductively coupled plasma atomic emission spectrometry (ICP-AES) for metal speciation. A concentric glass nebulizer with elongated tip is used as the CE-ICP interface. The CE capillary is the central tube of the nebulizer. A platinum wire is wrapped across the exit end of the CE capillary to provide electrical connection to the CE power supply. No sheath flow of buffer solution is needed. A simple cooling system has also been developed. A peristaltic pump circulates water through a plastic tube that encloses the section of the CE capillary between the CE instrument and the ICP spectrometer. Characteristics of the CE-ICP interface, e.g., elution time, nebulization and transport efficiency and peak broadening, versus carrier gas flow-rate have been studied. Comparisons to a previous design with the Pt electrode inserted into the end of the CE capillary are made where appropriate. The reproducibility (RSD) in ICP emission intensity of the system is <4%. Detection limits of Cr and Cu are approximately 5 ng/ml.     

On-line simultaneous and rapid separation of anions and cations from a single sample using dual-capillary sequential injection-capillary electrophoresis

A novel capillary electrophoresis (CE) approach has been developed for the simultaneous rapid separation and identification of common environmental inorganic anions and cations from a single sample injection. The method utilised a sequential injection-capillary electrophoresis instrument (SI-CE) with capacitively-coupled contactless conductivity detection (C⁴D) constructed in-house from commercial-off-the-shelf components. Oppositely charged analytes from a single sample plug were simultaneously injected electrokinetically onto two separate capillaries for independent separation and detection. Injection was automated and may occur from a syringe or be directly coupled to an external source in a continuous manner. Software control enabled high sample throughput (17 runs per hour for the target analyte set) and the inclusion of an isolation valve allowed the separation capillaries to be flushed, increasing throughput by removing slow migrating species as well as improving repeatability. Various environmental and industrial samples (subjected only to filtering) were analysed in the laboratory with a 3 min analysis time which allowed the separation of 23 inorganic and small organic anions and cations. Finally, the system was applied to an extended automated analysis of Hobart Southern Water tap water for a period of 48 h. The overall repeatability of the migration times of a 14 analyte standard sample was less than 0.74% under laboratory conditions. LODs ranged from 5 to 61 μg L⁻¹. The combination of automation, high confidence of peak identification, and low limits of detection make this a useful system for the simultaneous identification of a range of common inorganic anions and cations for discrete or continuous monitoring applications.     

Low flow high-performance liquid chromatography solvent delivery system designed for tandem capillary liquid chromatography-mass spectrometry

A solvent delivery system is described that is designed to increase the efficiency of liquid chromatography-mass spectrometry (LC/MS) analyses. Gradients formed by using two low pressure syringe pumps are stored in a length of narrow bore tubing (gradient loop) mounted on a standard high pressure switching valve. The preformed gradient is pushed through the column by using a high pressure syringe pump. The system is fully automated and can be controlled with either a personal computer or the mass spectrometer data system. Advantages include gradient operation without the use of split flows, pressure programed flow control for rapid sample loading and recycling to initial conditions, and a flow rate range of 0.1–20 μL/min, which is suitable for packed capillary columns 50–500 μm in diameter. The system has been used extensively for rapid molecular weight determinations of intact protein samples, as well as LC/MS and liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures.     

Pressure-assisted capillary electrophoresis for cation separations using a sequential injection analysis manifold and contactless conductivity detection

Pressure assisted capillary electrophoresis in capillaries with internal diameters of 10 μm was found possible without significant penalty in terms of separation efficiency and sensitivity when using contactless conductivity detection. A sequential injection analysis manifold consisting of a syringe pump and valves was used to impose a hydrodynamic flow in the separation of some inorganic as well as organic cations. It is demonstrated that the approach may be used to optimize analysis time by superimposing a hydrodynamic flow parallel to the electrokinetic motion. It is also possible to improve the separation by using the forced flow to maintain the analytes in the capillary, and thus the separation field, for longer times. The use of the syringe pump allows flexible and precise control of the pressure, so that it is possible to impose pressure steps during the separation. The use of this was demonstrated for the speeding up of late peaks, or forcing repeated passage of the sample plug through the capillary in order to increase separation.     

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire that is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N = 3) was achieved for detection of myoglobin utilizing a 75-µm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550–1300 Da. The advantages of this new design include (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Application of poly(methacrylic acid-ethylene glycol dimethacrylate) monolith microextraction coupled with capillary zone electrophoresis to the determination of opiates in human urine

A novel poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction method coupled with CZE was proposed for rapidly determining a mixture of opiates comprising heroin, 6-monoacetylmorphine, morphine, codeine, papaverine, and narcotine in human urine. The extraction device contained a regular plastic syringe, the poly(MAA-EGDMA) monolithic capillary tube (530 microm id x 3 cm) and a plastic pinhead, which connected the monolithic capillary tube and the syringe without leakage. In the polymer monolith microextraction, the sample solution was ejected via the monolithic capillary tube by a programmable syringe pump, followed by desorption with an aliquot of appropriate solution, which was collected into a vial for the subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1 M disodium hydrogen phosphate (adjusted to pH 4.5 with 1 M hydrochloric acid) and 20% methanol v/v with temperature and voltage of 25 degrees C and 25 kV, respectively. By applying electrokinetic injection with field-enhanced sample stacking, detection limits of 6.6-19.5 ng/mL were achieved. Excellent method of reproducibility was found over a linear range of 80-2000 ng/mL.     

Field amplified sample stacking coupled with chip-based capillary electrophoresis using negative pressure sample injection technique

A multi-T microchip for integrated field amplified sample stacking (FASS) with CE separation to increase the chip-based capillary electrophoresis (chip-based CE) sensitivity was developed. Volumetrically defined large sample plug was formed in one step within 5s by the negative pressure in headspace of the two sealed sample waste reservoirs produced using a syringe pump equipped with a 3-way valve. Stacking and separation can proceed only by switching the 3-way valve to release the vacuum in headspace of the two sample waste reservoirs. This approach considerably simplified the operations and the equipments for FASS in chip-based CE systems. Migration time precisions of 3.3% and 1.3% RSD for rhodamine123 (Rh123) and fluorescien sodium salt (Flu) in the separation of a mixture of Flu and Rh123 were obtained for nine consecutive determinations with peak height precisions of 4.8% and 3.4% RSD, respectively. Compared with the chip-based CE on the cross microchip, the sensitivity for analysis of FlTC, FITC-labeled valine (Val) and Alanine (Ala) increased 55-, 41- and 43-fold, respectively.     

Quality evaluation of six bioactive constituents in goji berry based on capillary electrophoresis field amplified sample stacking

Goji berry, fruits of the plant Lycium barbarum L., has long been used as traditional medicine and functional food in China. In this work, a simple and easy‐operation on‐line concentration capillary electrophoresis (CE) for detection flavonoids in goji berry was developed by coupling of field amplified sample stacking (FASS) with an electroosmotic (EOF) pump driving water removal process. Due to the EOF pump and electrokinetic injection showing different influence on the concentration, the analytes injection condition should be systemically studied. Thereafter, the verification of the analytes injection conditions was achieved using response surface experimental design. Under the optimum conditions, 86–271 folds sensitivity enhancement upon normal capillary zone electrophoresis (CZE, 50 mbar × 5 s) were achieved for six flavonoids, and the detection limits ranged from 0.35 to 1.82 ng/mL; the LOQ ranged from 1.20 to 6.01 ng/mL. Eventually, the proposed method was applied to detect flavonoids in 30 goji berry samples from different habitats of China; and the results indicated that the flavonoids were rich in the eluent of 30–60% methanol, which provided a reference for extraction of goji berry flavonoids.