Sample Plug Research Articles

Micropumps actuated negative pressure injection for microchip electrophoresis

A simple negative pressure pinched sample injection method was presented. This method combined diaphragm micropumps and a single voltage supply to generate controllable well-defined sample plug, and led to effective electrophoresis separation. The pinched plug was found to be favorable for obtaining representative and reproducible results that the RSD of the migration time and peak height of sodium fluorescein were 0.5 and 2.1%, respectively (n=25). The established method had been applied in separation of amino acid samples. This method has the advantages of well-defined plug, free sample bias effect, high reproducibility and convenience of controlling the negative pressure by the integrated pumps on the microchip. In addition, the single high voltage supply and the world-to-chip interface simplified the instrumentation, which is of benefit to the minimization and automation. These advantages demonstrate the potential of this method for a wide range of applications.

Pressure and electrokinetic injections for on‐line sample stacking neutral analytes in microemulsion electrokinetic chromatography with salt‐containing matrixes

An on-line technique for pressure and electrokinetic injections of long sample plugs with simultaneous stacking of neutral analytes (notoginsenoside R(1), ginsenoside Rg(1), ginsenoside Rf, ginsenoside Rh(1), ginsenoside Rd, ginsenoside Rg3) in microemulsion electrokinetic chromatography is presented. The effects of salt concentration, sample plug length, organic modification of the sample matrix, oil phase and SDS concentration on stacking efficiency were examined in order to optimize the two injection methods. In microemulsion electrokinetic chromatography, the effect of the type of oil and SDS content on stacking mechanism is often sophisticated. This study had demonstrated that the oil type and SDS content in microemulsion indeed markedly altered the affinity of microemulsion with analytes. Finally, in comparison with the electrokinetic injection method, the most apparent disadvantages of the pressure injection method were the relatively high LOD and poor reproducibility.

Separation and on-line preconcentration by stacking and sweeping of charged analytes in the plant by microemulsion electrokinetic chromatography with nonionic surfactants

A novel on-line technique for stacking and sweeping of long sample plugs with simultaneous determination of charged analytes in the plant (protocatechuic aldehyde, rosmarinic acid, danshensu, salvianolic acid B, and protocatechuic acid) by the nonionic microemulsion electrokinetic chromatography (MEEKC) is presented. The preconcentration efficiency provided about 9–28-fold for stacking and 7–14-fold for sweeping in the enhancements of LOD. The effects of oil phase, Brij-35 and buffer concentrations on stacking and sweeping efficiency were examined in order to optimize the two methods. In nonionic MEEKC, the effect of the type of oil and buffer contents on preconcentration mechanism is often sophisticated. This study had demonstrated that the oil type and buffer content in nonionic microemulsion indeed markedly altered the affinity of microemulsion with analytes. Finally, in comparison to the stacking method, the most apparent disadvantages of the sweeping method were the relatively high limits of detection and poor peak shapes.

Sampling and Electrophoretic Analysis of Segmented Flow Streams Using Virtual Walls in a Microfluidic Device

A method for sampling and electrophoretic analysis of aqueous plugs segmented in a stream of immiscible oil is described. In the method, an aqueous buffer and oil stream flow parallel to each other to form a stable virtual wall in a microfabricated K-shaped fluidic element. As aqueous sample plugs in the oil stream make contact with the virtual wall, coalescence occurs and sample is electrokinetically transferred to the aqueous stream. Using this virtual wall, two methods of injection for channel electrophoresis were developed. In the first, discrete sample zones flow past the inlet of an electrophoresis channel and a portion is injected by electroosmotic flow, termed the "discrete injector". With this approach at least 800 plugs could be injected without interruption from a continuous segmented stream with 5.1% RSD in peak area. This method generated up to 1,050 theoretical plates, although analysis of the injector suggested that improvements may be possible. In a second method, aqueous plugs are sampled in a way that allows them to form a continuous stream that is directed to a microfluidic cross-style injector, termed the "desegmenting injector". This method does not analyze each individual plug but instead allows periodic sampling of a high-frequency stream of plugs. Using this system at least 1000 injections could be performed sequentially with 5.8% RSD in peak area and 53,500 theoretical plates. This method was demonstrated to be useful for monitoring concentration changes from a sampling device with 10 s temporal resolution. Aqueous plugs in segmented flows have been applied to many different chemical manipulations including synthesis, assays, sampling processing and sampling. Nearly all such studies have used optical methods to analyze plug contents. This method offers a new way to analyze such samples and should enable new applications of segmented flow systems.

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

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

A high-efficiency sample introduction system for capillary electrophoresis analysis of amino acids from dynamic samples and static dialyzed human vitreous samples

A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-mum inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others.

A low‐leakage sample plug injection scheme for crossform microfluidic capillary electrophoresis devices incorporating a restricted cross‐channel intersection

This study develops a crossform CE microfluidic device in which a single-circular barrier or a double-circular barrier is introduced at the cross-channel intersection. Utilizing a conventional crossform injection scheme, it is shown that these barriers reduce sample leakage and deliver a compact sample band into the separation channel, thereby ensuring an enhanced detection performance. A series of numerical and experimental investigations are performed to investigate the effects of the barrier type and the barrier ratio on the flow streamlines within the microchannel and to clarify their respective effects on the sample leakage ratio and sample plug variance during the injection process. The results indicate that a single-circular barrier injector with a barrier ratio greater than 20% and a double-circular barrier injector with a barrier ratio greater than 40% minimize the sample leakage ratio and produce a compact sample plug. As a result, both injectors have an excellent potential for use in high-quality, high-throughput chemical analysis procedures and in many other applications throughout the micro-total analysis systems field.

In-capillary derivatization and capillary electrophoresis separation of amino acid neurotransmitters from brain microdialysis samples

A new in-capillary derivatization method with naphtalene-2,3-dicarboxyaldehyde (NDA)/CN − has been developed for capillary electrophoresis with laser-induced fluorescence detection of brain microdialysate amino acids. Samples are sandwiched between two plugs of reagent mixture at the capillary inlet and subsequently separated. Highest derivatization yields are obtained by using a reagent to sample plug length ratio equal to 4, performing a first electrophoretic mixing followed by a zero potential amplification step before applying the separation voltage and using a NaCN to NDA concentration ratio equal to 1. This new single-step methodology allows the analysis of amino acid neurotransmitters in rat brain microdialysis samples.

Improved temporal resolution for in vivo microdialysis by using segmented flow.

Microdialysis sampling probes were interfaced to a segmented flow system to improve temporal resolution for monitoring concentration dynamics. Aqueous dialysate was segmented into nanoliter plugs by pumping sample stream into the base of a tee channel structure microfabricated on a PDMS chip that had an immiscible carrier phase (perfluorodecalin) pumped into the cross arm of the tee. Varying the oil flow rate from 0.22 to 6.3 microL/min and sample flow rate from 42 to 328 nL/min allowed control of plug volume, interval between plugs, and frequency of plug generation between 6 and 28 nL, 0.6 and 10 s, and 0.1 and 1.7 Hz, respectively. Temporal resolution of the system, determined by measuring fluorescence in individual sample plugs following step changes of fluorescein concentration at the sampling probe surface, was as good as 15 s. Temporal resolution was independent of both sampling flow rate and distance that samples were pumped from the sampling probe. This effect is due to the prevention of Taylor dispersion of the sample as it was transported by segmented flow. In contrast, without flow segmentation, temporal resolution was worsened from 25 to 160 s as the detection point was moved from the sampling probe to 40 cm downstream. Glucose was detected by modifying the chip to allow enzyme assay reagents to be mixed with dialysate as sample plugs formed. The resulting assay had a detection limit of 50 microM and a linear range of 0.2-2 mM. This system was used to measure glucose in the brain of anesthetized rats. Basal concentration was 1.5 +/- 0.1 mM (n = 3) and was decreased 60% by infusion of high-K(+) solution through the probe. These results demonstrate the potential of microdialysis with segmented flow to be used for in vivo monitoring experiments with high temporal resolution.

The use of cationic surfactants and ionic liquids in the detection of microbial contamination by capillary electrophoresis

A rapid test of whether a laboratory sample contains any microorganisms is important and necessary for many areas of science and technology. Currently, most of the standard procedures for the detection of aerobic bacteria, anaerobic bacteria and fungi, require the preparation of microbial cultures in respective growth media, which are dramatically slow. Different approaches providing fast analysis such as CE are becoming more desired. To compensate for the natural electrophoretic heterogeneity of microbes, various buffer additives were examined to stack all bacteria and fungi in a sample plug into a single peak. This peak was removed from the molecular contaminants in the sample to prevent false positives. Both cationic surfactants and ionic liquids (IL) were investigated as run buffer additives and they are both widely applicable to different species of bacteria and fungi. Given that high concentrations of surfactants can potentially lyse cells, dicationic IL offer attractive auxiliary buffer additives for use in CE-based sterility tests. The analysis can be completed in 10 min, thus providing a great advantage over traditional direct inoculation methods that require several weeks to complete.

Adhesion-Based Cell Sorter With Antibody-Coated Amino-Functionalized-Parylene Surface

An adhesion-based cell-separation device is developed for the extraction of rare cells from a cell mixture. The cell-separation principle mimics leukocyte recruitment from blood vessels in our body, where leukocytes are decelerated by antigen-antibody interaction at the sites of inflammation or injury. Separation of cell mixture can be accomplished by simply introducing the sample plug through an antibody-immobilized microchannel without any pre- or postprocessing. A new class of amino-functionalized parylene (diX AM) is employed in order to provide amino group on the channel-wall surface. The amount of immobilized biomolecules on diX AM surface is characterized through quartz-crystal-microbalance measurements. The number density of immobilized biotin is as large as , which indicates an amount of amino group enough to immobilize biotin and other biomolecules in a closely packed state. It is shown by the measurement of cell velocity in the CD31-coated diX AM microchannel that the flowing velocity of human endothelial cells are reduced by up to 70% due to specific adhesion of CD31 antigens and antibodies. The results are further analyzed by using a 2-D membrane-peeling model, with which the cell velocity can be estimated under different conditions of antibody number density and bulk mean velocity. Based on the experimental results, a cell-separation device for treating a 1- cell-mixture plug is designed and microfabricated. A mixture of human endothelial cells and leukocytes is successfully separated into plugs of each cell type within a shorter period of time as compared to conventional cell-separation methods which require sample preprocessing.

Flow-Injection Coulometric Detection Based on Ion Transfer and Its Application to the Determination of Chlorpromazine.

A flow-injection coulometric method for the determination of chlorpromazine based on ion transfer into a plasticized poly(vinyl chloride) (PVC) membrane, was developed. The detector used consists of a flow-through cell that incorporates a plasticized poly(vinyl chloride) (PVC) membrane which contains tetrabutylammonium tetraphenylborate as electrolyte. The membrane is located between the flowing solution and an inner aqueous electrolyte solution. Two pairs of electrodes, each pair formed by a reference electrode and a working electrode, are used, one pair in each solution. The potential between the reference electrodes was controlled by a four-electrode potentiostat with ohmic drop compensation. A potential step capable of producing the transfer of the chlorpromazine ion into the membrane was applied during the passage of a wide portion of sample plug through the cell and the corresponding quantity of the electricity was measured. In the selected conditions, a linear relationship was observed between the quantity of electricity and chlorpromazine concentrations over a range of 1×10-6 −1×10-4 M. The detection limit was 2 × 10-7 M. Good repeatability and between-day reproducibility was obtained. No interference was observed on the part of some common ions and pharmaceutical excipients. The method proposed was applied satisfactorily to the determination of chlorpromazine in pharmaceuticals and human urine.

Multiphase flow predictions from carbonate pore space images using extracted network models

A methodology to extract networks from pore space images is used to make predictions of multiphase transport properties for subsurface carbonate samples. The extraction of the network model is based on the computation of the location and sizes of pores and throats to create a topological representation of the void space of three‐dimensional (3‐D) rock images, using the concept of maximal balls. In this work, we follow a multistaged workflow. We start with a 2‐D thin‐section image; convert it statistically into a 3‐D representation of the pore space; extract a network model from this image; and finally, simulate primary drainage, waterflooding, and secondary drainage flow processes using a pore‐scale simulator. We test this workflow for a reservoir carbonate rock. The network‐predicted absolute permeability is similar to the core plug measured value and the value computed on the 3‐D void space image using the lattice Boltzmann method. The predicted capillary pressure during primary drainage agrees well with a mercury‐air experiment on a core sample, indicating that we have an adequate representation of the rock's pore structure. We adjust the contact angles in the network to match the measured waterflood and secondary drainage capillary pressures. We infer a significant degree of contact angle hysteresis. We then predict relative permeabilities for primary drainage, waterflooding, and secondary drainage that agree well with laboratory measured values. This approach can be used to predict multiphase transport properties when wettability and pore structure vary in a reservoir, where experimental data is scant or missing. There are shortfalls to this approach, however. We compare results from three networks, one of which was derived from a section of the rock containing vugs. Our method fails to predict properties reliably when an unrepresentative image is processed to construct the 3‐D network model. This occurs when the image volume is not sufficient to represent the geological variations observed in a core plug sample.

Isoelectric Focusing in a Microfluidically Defined Electrophoresis Channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.

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.

Electrophoresis of Membrane-Associated Molecules in Packed Beds of Bilayer-Coated Particles

We describe a new method for large-scale separation of membrane-associated biomolecules using a packed bed of bilayer-coated particles as an electrophoretic medium. The basic strategy is as follows. First, solid particles coated with fluid lipid bilayers and those coated with fluid lipid bilayers containing the sample to be separated (lipids or membrane proteins) are packed in a column so as to form a “sample plug.” Next, electrophoretic separation of the sample components is carried out by applying a dc electric field along the column. Finally, the separated fractions are collected by taking the particles out from the column. The above strategy is based on the continuity of the bilayer throughout the columnthe bilayers on adjacent particles are fused together under a densely packed condition. We demonstrate proof-of-concept experiments using fluorescently labeled lipids as the sample. This method will provide an easy way for production of functional particles coated with membrane-associated biomolecules.

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.

“Drop-and-Sip” Fluid Handling Technique for the Reagent-Release Capillary (RRC)-based Capillary-Assembled Microchip (CAs-CHIP): Sample Delivery Optimization and Reagent Release Behavior in RRC

The experimental conditions of the sample delivery inside the reagent-release capillary-based capillary-assembled microchip (RRC-based CAs-CHIP) were optimized and the reagent release procedure in the RRC is discussed. Recently, our group introduced the basic concept of the "drop-and-sip" fluid handling technique (Anal. Chem., 2007, 79, 908). A microliter volume of sample solution is dropped on the inlet hole and is sipped into another hole, producing a sample plug flow in the main poly(dimethyl siloxane) (PDMS) channel, concurrently filling each sensing capillary that faces the main PDMS channel. However, the detailed evaluation of the successful sample delivery condition and the reagent release behavior in the RRC has not been fully discussed. Under our experimental conditions, ca. 0.6 - 2.4 s of sample plug-RRC contact time allowed the successful sample introduction into the RRC by capillary force without any reagent leakage or disturbance of the sample plug flow. On the other hand, reagent release behavior inside the RRC is governed by both convective and diffusive mass transport, which leads to a faster mixing time of the sample with reagents immobilized inside the RRC compared to that expected from the simple diffusion alone.

Optimization of sample transfer in two-dimensional microfluidic separation systems

Multidimensional microfluidic separation systems combining a first dimension microchannel with an array of parallel second dimension microchannels can suffer from non-uniform sample transfer between the dimensions, sample leakage, and injection plug tailing within the second dimension array. These factors can significantly reduce overall two-dimensional separation performance. In this paper, numerical and analytical models reveal an optimized chip design which combines multidimensional backbiasing and an angled channel geometry to ensure leakage-free and uniform interdimensional sample transfer, while also minimizing injected sample plug lengths. The optimized design is validated experimentally using a multidimensional chip containing five second dimension channels.

New advances in on-line sample preconcentration by capillary electrophoresis using dynamic pH junction

The small injection volumes and narrow dimensions characteristic of microseparation techniques place constraints on concentration sensitivity that is required for trace chemical analyses. On-line sample preconcentration techniques using dynamic pH junction and its variants have emerged as simple yet effective strategies for enhancing concentration sensitivity of weakly ionic species by capillary electrophoresis (CE). Dynamic pH junction offers a convenient format for electrokinetic focusing of dilute sample plugs directly in-capillary for improved detection without off-line sample pretreatment. In this report, we highlight new advances in dynamic pH junction which have been reported to enhance method performance while discussing challenges for future research.