Novel coupling mechanism-based imaging approach to scanning electrochemical microscopy for probing the electric field distribution at the microchannel end

A novel coupling mechanism-based imaging approach to scanning electrochemical microscopy (SECM) was used to image the distribution of electric field at the end channel of a poly(dimethylsiloxane) (PDMS) capillary electrophoresis (CE) microchip in the absence of redox species. The coupling imaging mechanism was systematically investigated and qualitatively illustrated. It was proved that the distribution of solution potentials within the scanning plane caused a different reduction rate of water at the tip electrode, which led to the variation in tip current. Within the scanning plane, the solution potentials measured in the central area of the microchannel were usually higher than those measured outside. The SECM images showed a strong dependence on tip potential, tip-to-channel distance, and separation potential. According to the Tafel equation, SECM images were converted to parameters that directly showed the distribution of solution potential. Change in the solution potential along the central axial line of the microchannel was also continuously sensed by allowing the tip to approach the microchannel in the presence of high voltage. Using dopamine as a model compound, the effect of solution potential on electrochemical detection was estimated by detecting separation parameters.     

Very fast electrophoretic separation on commercial instruments using a combination of two capillaries with different internal diameters

A capillary formed by connecting a 9.7 cm‐long separation capillary with id 25 μm with an auxiliary 22.9 cm‐long capillary with id 100 μm (coupled capillary) was tested for electrophoretic separation at high electric field intensities. The coupled capillary was placed in the cassette of a standard electrophoresis apparatus. It was used in the short‐end injection mode for separation of a mixture of dopamine, noradrenaline, and adrenaline in a BGE of 20 mM citric acid/NaOH, pH 3.2. An intensity of 2.7 kV/cm was attained in the separation part of the capillary at a separation voltage of 30 kV, which is 2.9 times more than maximum intensity value attainable in a capillary with the same length with uniform id. At these high electric field intensities, the migration times of the tested neurotransmitters had values of 12.3–13.3 s and the attained separation efficiency was between 2350 and 2760 plates/s. It is thus demonstrated that an effective separation instrument ‐ a coupled capillary ‐ can be used for very rapid separation in combination with standard, commercially available instrumentation.     

Microfluidic preparative free‐flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free‐flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular‐shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion‐permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50×70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free‐flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule‐heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370±20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.     

Optimization of Electrode Alignment for Electrochemical Detection in Capillary Electrophoresis Using a Scanning Electrochemical Microscope

Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End‐capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal‐to‐noise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using scanning electrochemical microscopy (SECM), the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal‐to‐noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal‐to‐noise does not dramatically decrease until the electrode is more than 30 μm above the 10 μm inner‐diameter capillary.     

Application of a Square‐Wave Potential Program for Time‐Dependent Amperometric Detection in Capillary Electrophoresis

Use of a square‐wave potential program for time‐dependent amperometric detection of analyte zones in capillary electrophoresis (CE) is described. Electrochemical detection for CE requires that the separation field be isolated from that of the electrochemical detection. This is generally done by physically separating the CE separation field from that of the detection. By applying a time variant potential program to the detection electrode, the detector current has a time dependence that can be used to help isolate the electrochemical detection current from that of the separation. When using a 20 μm inner‐diameter capillary, we find that a square‐wave potential program decreases the RMS baseline current from 4.5×10^?10 A, found with a constant potential amperometric detection, to 1.1×10^?10 A when using a square‐wave potential program. With a 75 μm inner‐diameter capillary, the improvement is even more dramatic, from 2.3×10^?9 A with amperometric detection to 2.06×10^?10 A when using a 1 Hz square‐wave potential program. When not using the time‐dependent detection with the 75 μm capillary, the analyte zones were beneath the S/N for the system and not detected. With the square‐wave potential program and time‐dependent detection, however, the analyte zones for an electrokinetic injection of 200 μM solution of 2,3‐dihydroxybenzoic acid were observed with the 75 μm inner‐diameter capillary. The improvement in the ability to discriminate the analytical signal from the background found experimentally is consistent with modeling studies.     

On-column conductivity detection in capillary-chip electrophoresis

On-column conductivity detection in capillary-chip electrophoresis was achieved by actively coupling the high electric field with two sensing electrodes connected to the main capillary channel through two side detection channels. The principle of this concept was demonstrated by using a glass chip with a separation channel incorporating two double-Ts. One double-T was used for sample introduction, and the other for detection. The two electrophoresis electrodes apply the high voltage and provide the current, and the two sensing electrodes connected to the separation channel through the second double-T and probe a potential difference. This potential difference is directly related to the local resistance or the conductivity of the solution defined by the two side channels on the main separation channel. A detection limit of 15 microM (600 ppb or 900 fg) was achieved for potassium ion in a 2 mM Tris-HCl buffer (pH 8.7) with a linear range of 2 orders of magnitude without any stacking. The proposed detection method avoids integrating the sensing electrodes directly within the separation channel and prevents any direct contact of the electrodes with the sample. The baseline signal can also be used for online monitoring of the electric field strength and electroosmosis mobility characterization in the separation channel.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

Electroosmotic pump‐supported molecularly imprinted monolithic column for capillary chromatographic separation of nitrophenol isomers

In this work, a novel molecularly imprinted polymer (MIP) monolithic column with integrated in‐column electroosmotic pump (EOP) was designed and successfully prepared to facilitate the capillary chromatography with MIP column. A silica‐based EOP was synthesized at the detection end of the MIP monolithic capillary column by so‐gel to provide the hydrodynamic driven force for the capillary chromatography. Because of large surface area and low fluidic resistance of the silica monolith,a strong and steady EOF was generated by silica‐based EOP, indicating that the EOP was quite compatible with MIP capillary column. With the sufficient EOF provided by EOP, the electro‐driven based capillary chromatographic separation of nitrophenol isomers was achieved in 4‐vinylpyridine‐based MIP monolithic capillary, which was originally proved infeasible because of the EOF shortage. No significant influence upon the specific recognition of the MIP was found due to the setting of EOP after the detection window of the column. The influence of experimental parameters on the EOF such as voltage and pH value of running buffer was investigated. The column was also evaluated by capillary liquid chromatographic mode to compare with EOP‐driven capillary chromatography. Higher column efficiency was obtained by EOP‐driven separation with improved peak shape. The results suggested that EOP‐supported technique would be a good way to solve the problem of weak EOF generation in electro‐driven capillary chromatography.     

A Capillary Electrophoresis End‐Column Amperometric Detection System Incorporating Disposable Copper‐Plated Screen‐Printed Carbon Electrodes

We report here the development of copper‐plated screen‐printed carbon electrodes (designated as Cu‐SPE) to employ as electrochemical detectors for the determination of sugars by capillary electrophoresis (CE). A simple end‐column amperometric detection system with easily exchangeable (or even disposable) electrode and capillary in CE is described in this study. A complex alignment procedure was not required in this system based on the end‐column electrode arrangement using an 85 cm length and 20 μm (i.d.) capillary. The optimized separation voltage and applied potential were 9 KV and 0.4 V (vs. Ag/AgCl), respectively, for the detection of sugars using the Cu‐SPE. Good resolution was obtained by this proposed system with migration times of 28.8, 29.5, 29.9, 30.7, 31.2, and 32.0 min for galactose, glucose, arabinose, fructose, xylose, and ribose, respectively.     

Indirect Measurement of Yoctomole Alkaline Phosphatase by Capillary Electrophoresis with Electrochemical Detection

A method for indirectly detecting yoctomole (ymol) alkaline phosphatase was developed by capillary electrophoresis with electrochemical detection. In this method, disodium phenyl phosphate was used as the enzyme substrate and the product (phenol) of its hydrolysis reaction catalyzed by alkaline phosphatase was detected at the carbon fiber electrode. The optimum conditions of detection are 1.0×10^?2 mol/L Na₂B₄O₇ (pH 9.8) for the running buffer; 1.00×10^?3 mol/L disodium phenyl phosphate for the enzyme substrate; 20.0 kV for the separation voltage; 5 kV and 10 s for the injection voltage and injection time; 1.05 V (vs. saturated calomel electrode) for the detection potential and 10 min for the incubation time, respectively. In order to enhance the ratio of signal to noise, the shape and size of the working electrode, the shape of detection end of the capillary, and the capillary/electrode alignment method were studied in detail. When a single carbon fiber microcylinder electrode of 6 μm, a capillary of 10 μm ID with the etched detection end and the in‐capillary alignment were used, a ymol mass limit of detection for alkaline phosphatase was achieved.     

Formation of a resistive region at the anode end in DNA capillary electrophoresis.

We have studied the formation of a resistive region in the capillary during DNA separation. This effect is caused by an unequal change in the mobilities of cations and anions at the interface between the running buffer solution and the capillary. We studied the motion of the resistive region boundary by sequential removal of portions of the affected capillary end. We found that in the process of developing the resistive region the distribution of the electric fields in the capillary changes from uniform to extremely nonuniform, with a very high field (above 1 MV/cm) in the resistive region and a reduced field (80 V/cm) in the rest of the capillary. Though theoretically a resistive region may appear either at the anode (detection) or the cathode (injection) end of the capillary, all previous publications report the formation of the resistive region at the cathode side. In our experiments, however, the anomalous region is formed at the anode. Thus, the separated DNA peaks move towards the slowly progressing resistive region. Our results indicate that the DNA is stopped at the boundary and does not enter the region. When the resistive region is clipped off the peak motion resumes. This suggests that there exists a potential barrier at the resistive layer boundary that prevents the drift of the peaks towards the anode. The formation of the resistive region interferes with a normal separation process causing a gradual decrease of the capillary current and the deceleration and eventual quenching of the peak motion. For the ABI chemistry, we experimented with adding polymers to the electrode buffer to equate the transference numbers for anions and cations, and found the conditions at which this effect is completely eliminated.     

An investigation into the stability of microemulsions in electrophoresis

A typical oil‐in‐water microemulsion (ME) was applied for the capillary electrophoretic separation of nonsteroidal anti‐inflammatory drugs (NSAIDs). As the high‐pH ME was introduced into the capillary and a voltage was applied, we observed an unusual phenomenon during the preconditioning process: a sharp inflection point occurred at 56.2 min ( ± 4.8%) (N = 15). Both before (region A) and after (region B) that point, a steady state was observed. Highly reproducible results were obtained for this event. Two different absorbance patterns were observed for the hydrostatic injection of either freshly prepared ME or ME collected from the outlet of the capillary column during the steady state of region B. The latter had an inflection point at approximately 9 min, whereas the former had a constant absorbance over the entire range. To further characterize this property, regions A and B were used for the separation of NSAIDs. The results showed that both the detection limit and the reproducibility of the separation were superior in region B. For deep insight into the stability of the ME in an electric field, the effects of the oil, cosurfactant, pH, and voltage were systematically investigated in the neat ME. From our findings, it can be concluded that the inflection time might be the migration time of the ME in an electric field, and it might actually be the result of equilibration. Moreover, the existence of many unexpected phenomena seems to be the result of a change in the properties of ME droplet in an electric field.     

电场对Ni3（dpa）4Cl2金属串配合物结构影响的理论研究

应用密度泛函UBP86方法对具有分子导线潜在应用性的金属串配合物Ni3(dpa)4Cl2进行研究,分析了外电场对配合物的几何构型和电子结构的影响.结果表明,零电场条件下存在沿着Ni63+轴及轴向配体Cl的Ni—Ni及Ni—Cl离域作用.沿金属轴Cl4→Cl5方向施加外电场,可使高电势端的Ni2—Cl4键长增大而Ni1—Ni2键长减小,低电势端的Ni3—Cl5键长减小而Ni1—Ni3键长增大;分子能量降低,偶极矩线性增大;HOMO与LUMO能隙减小,前线占据轨道分布向低电势方向移动且轨道能升高,空轨道分布则向高电势方向移动且轨道能降低,其中沿着金属轴方向离域的前线轨道分布及其轨道能随电场的变化尤为显著.在电场作用下,电荷分布发生改变,低电势端Cl5的负电荷向高电势端Cl4转移,但金属和桥联配体的电荷变化很小;同样,在电场作用下,配合物存在明显的结构变化和电子转移现象,呈现出类似导电过程中电子定向转移的变化规律.     

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.     

Highly sensitive analysis of catecholamines by counter‐flow electrokinetic supercharging in the constant voltage mode

Electrokinetic supercharging is one of the most powerful sample‐stacking methods that combines field amplified sample injection and transient ITP. In counter‐flow electrokinetic supercharging, a constant counter pressure is applied during sample injection in order to counterbalance the movement of the injected sample zone. As a result, there will be a pronounced increase in the amount of sample injected and the portion of the capillary available for electrophoresis. In this report, counter‐flow electrokinetic supercharging optimization factors such as the electric field application in the constant voltage and constant current modes, the magnitude of counter pressure, and the terminating electrolyte concentrations were investigated. The enrichments obtained with a 30 min injection of 10 nM catecholamines in 5 mM terminating electrolyte solution in the constant voltage mode applying a counter pressure of 1.3 psi were 41000‐fold for dopamine, 50 000‐fold for norepinephrine, and 32 000‐fold for epinephrine, yielding detection limits of 1.3, 1.4, and 1.2 nM, respectively, with absorbance detection at 200 nm.     

径向电场调制毛细管电泳法用于蛋白质分离

利用自制的双向电场控制毛细管电泳新系统,考察了蛋白质的分离情况.结果发现,在低pH值下,通过施加径向电场,不仅可改变电渗流的大小和方向,而且能抑制蛋白质的吸附,进而实现对蛋白质分离效率和分离速度的调控.研究结果表明,可通过物理化学方法实现毛细管电泳的动态或随机调控,这对许多生物样品分离有实际意义.     

Electrohydrodynamic Velocity and Pumping Measurements in Water and Alcohols

Bubble and particle velocities in water and alcohols, under the influence of an electric field, were investigated in this work. Air bubbles were injected into the liquids through an electrified metal capillary insulated by glass with its tip left exposed. The end of the capillary from which the bubbles were released was conical in shape. Due to an electric field formed between the noninsulated capillary tip and a ground electrode immersed in the solvent, small bubbles were formed and used as tracers for the electrohydrodynamic (EHD) flow field. The pressure inside the capillary was measured for all liquids used in this study. For water, ethanol, and n-propanol, it was found that, at relatively low applied voltage, the pressure increases with voltage, reaches a maximum (pressure breakpoint), and then sharply decreases. This behavior is a result of the competition between the electric force appearing at the interface and the force due to the EHD flow near the capillary tip. The electric force tends to increase the pressure inside the capillary, while the EHD flow tends to decrease this pressure. For isopropanol and butanol, the pressure breakpoint was not observed in the range of voltage applied in the experiments. The EHD flow velocity was measured by using microbubbles and particles as flow tracers. An adaptive phase-Doppler velocimeter was employed to measure the velocity of bubbles, while the velocity of particles was measured by trajectory visualization of fluorescent particles. A discrepancy was observed between the two methods because of the location at which the measurements were made. It was found that average velocities of both bubbles and particles increase linearly with applied voltage. Experiments were also conducted to investigate pumping of water, which is a result of the EHD velocity near the capillary tip. The pumping flow rate was linearly related to the applied voltage and agreed well with EHD velocity measurements obtained from particle trajectories. Copyright 2000 Academic Press.     

Capillary electrophoresis coupled with in‐column fiber‐optic laser‐induced fluorescence detection for the rapid separation of neodymium

In this study, in‐column fiber‐optic (ICFO) laser‐induced fluorescence (LIF) detection technique is coupled with capillary electrophoresis (CE) for the rapid separation of neodymium for the first time. The effects of buffer concentration, buffer pH, and separation voltage on the CE behaviors, including electrophoretic efficiency and detection sensitivity, are investigated in detail. Under the optimal condition determined in this study (15 mM borate buffer, pH 10.50, separation voltage 24 kV), neodymium could be separated effectively from the neighboring lanthanides (praseodymium and samarium) within several minutes, and the limit of detection for neodymium is estimated to be at the ppt level. The ICFO‐LIF‐CE system assembled in this study exhibits unique performance characteristics such as low cost and flexibility. Meanwhile, the separation efficiency and detection sensitivity of the assembled CE system are comparable to or somewhat better than those obtained in the previous traditional CE systems, indicating the potential of the assembled CE system for practical applications in the fields of spent nuclear fuel analysis, nuclear waste disposal/treatment, and nuclear forensics.     

The transitional isoelectric focusing process

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.     

DNA aggregation and cleavage in CGE induced by high electric field in aqueous solution accompanying electrokinetic sample injection

The phenomenon of peak area decrease due to high injection voltage (V_inj, e.g. 10–30 kV, 200–600 V/cm in the 50 cm capillary) was found in the analysis of very dilute DNA fragments (<0.2 mg/L) by using high‐sensitive electrokinetic supercharging‐CGE. The possibility of DNA cleavage in aqueous solution was suggested, in addition to the aggregation phenomenon that is already known. The analysis of intentionally voltage‐affected fragments (at 200 V/cm) also showed decreased peak areas depending on the time of the voltage being applied. Computer simulation suggested that a high electric field (a few kV/cm or more) could be generated partly between the electrode and the capillary end during electrokinetic injection (EKI) process. After thorough experimental verification, it was found that the factors affecting the damage during EKI were the magnitude of electric field, the distance between tips of electrode and capillary (D_e/c), sample concentration and traveling time during EKI in sample vials. Furthermore, these factors are correlating with each other. A low conductivity of diluted sample would cause a high electric field (over a few hundred volts per centimeter), while the longer D_e/c results in a longer traveling time during EKI, which may cause a larger degree of damage (aggregation and cleavage) on the DNA fragments. As an important practical implication of this study, when the dilute DNA fragments (sub mg/L) are to be analyzed by CGE using EKI, injection voltage should be kept as low as possible.