Coated microfluidic devices for improved chiral separations in microchip electrophoresis

Chiral separations of fluorescein isothiocyanate-labeled amines have been performed in poly(vinyl alcohol) (PVA)-coated microfluidic glass chips. Baseline separation of enantiomers could be realized in coated devices while they could not be resolved in uncoated chips. The electroosmotic flow (EOF) in PVA-coated channels is suppressed over a wide pH range which leads to a considerable improved reproducibility of migration times in repetitive analysis. Due to the high resolution obtained in such devices, it was possible to reliable determine the enantiomeric purity with high accuracy. One percent of the minor enantiomer could be determined in the presence of large excess of the other enantiomer. As the EOF was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Applying PVA-coated channels considerable improved precision of migration times was found. The relative standard deviation of migration times was below 1% in PVA-coated devices. Accordingly, excessive rinsing or etching steps in order to stabilize the EOF could be omitted while this was necessary for a reliable operation of uncoated devices.     

Coating of powder-blasted channels for high-performance microchip electrophoresis

Channels in microfluidic glass chips manufactured with the alternative powder blasting technology were permanently coated with poly(vinyl alcohol) (PVA) in order to improve the performance in microchip electrophoresis. The performance of coated and uncoated powder-blasted (pb) devices as well as coated and uncoated wet chemical etched (wc) chips was compared in electrophoretic separations of fluorescently labeled test compounds. The limited electrophoretic resolution obtained in pb-chips could significantly be improved by coating the channels with PVA. The resolution of test compounds in such coated pb-devices was even higher than in uncoated wc-chips. PVA-coated pb-chips could also successfully be applied in chiral separations. While in an uncoated pb-chip using a cyclodextrins buffer only one broad signal was obtained, two well-resolved signals were obtained in a coated device.     

Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.     

Poly(ethylene glycol)-coated microfluidic devices for chip electrophoresis

Herein, we report on a strategy for durable modification of the channel surface in microfluidic glass chips with the neutral hydrophilic-coating material poly(ethylene glycol) PEG-1M-100. Applied in microchip electrophoresis such PEG-coated devices exhibit a suppressed electroosmotic flow and reduced analyte adsorption. The PEG-coated chips were successfully applied in chip electrophoresis of FITC-labelled amines and amino acids and native proteins as well as in chiral separations. The performance of the coated chips was found to be superior compared with uncoated microchips. The coated chips exhibited high stability and the relative standard deviation of migration times in PEG-coated devices was less than 2%.     

Separation of fluorescein isothiocyanate-labeled amines by microchip electrophoresis in uncoated and polyvinyl alcohol-coated glass chips using water and dimethyl sulfoxide as solvents of background electrolyte

On-chip capillary electrophoresis with uncoated and polyvinyl alcohol-coated glass channels in aqueous and nonaqueous dimethyl sulfoxide (DMSO) background electrolyte (BGE) solutions was applied in the separation of five amines derivatized with fluorescein-5-isothiocyanate. In aqueous BGE at pH 9.2, baseline separation of the analytes was not achieved on uncoated glass chips, but the separation was clearly improved when the chip channels were coated with polyvinyl alcohol (PVA). Separation was successful in nonaqueous DMSO electrolyte solution containing ammonium acetate and sodium methoxide, on both uncoated and PVA-coated glass microchips. The differences in the pK(a) values of analytes were probably amplified in DMSO, and all five analytes were at least partly dissociated and were separated. Because the viscosity of DMSO is higher than that of water, the migration times were longer in DMSO.     

A microfluidic device for performing pressure-driven separations

Microchannels in microfluidic devices are frequently chemically modified to introduce specific functional elements or operational modalities. In this work, we describe a miniaturized hydraulic pump created by coating selective channels in a glass microfluidic manifold with a polyelectrolyte multilayer (PEM) that alters the surface charge of the substrate. Pressure-driven flow is generated due to a mismatch in the electroosmotic flow (EOF) rates induced upon the application of an electric field to a tee channel junction that has one arm coated with a positively charged PEM and the other arm left uncoated in its native state. In this design, the channels that generate the hydraulic pressure are interconnected via the third arm of the tee to a field-free analysis channel for performing pressure-driven separations. We have also shown that modifications in the cross-sectional area of the channels in the pumping unit can enhance the hydrodynamic flow through the separation section of the manifold. The integrated device has been demonstrated by separating Coumarin dyes in the field-free analysis channel using open-channel liquid chromatography under pressure-driven flow conditions.     

p-tert-Butylcalix[4]arene-1,3-bis(allyloxyethoxy)ether coated capillaries for open-tubular electrochromatography.

The preparation and characterization of p-tert-butylcalix[4]arene-1,3-bis(allyloxyethoxy)ether (CA[4]-BAE) chemically coated capillaries via a free-radical reaction with vinyltriethoxysilane (VTES) that was attached onto the inner wall previously were carried out. IR spectra and decreased electroosmotic flow (EOF) suggested that the capillary was successfully coated with CA[4]-BAE. A slight slope of EOF versus pH at 5 < pH < 9 would help to make the separation reproducible. The CA[4]-BAE-coated capillary showed improved separations of toluidine isomers, naphthol isomers and polycyclic aromatic hydrocarbons (PAHs) compared with an uncoated capillary. The special selectivity indicates that there is a certain extent of host-guest interactions between the solutes and the CA[4]-BAE coating.     

Influencing electroosmotic flow and selectivity in open tubular electrochromatography by tetrakis(pentafluorophenyl)porphyrin as capillary wall modifier

A physically adsorbed and covalently bonded porphyrin derivative, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H₂TPFPP, has been used as a fused-silica capillary wall modifier in open tubular capillary electrochromatography (OT-CEC), and its influence on the electroosmotic flow (EOF) velocity and on the selectivity of OT-CEC separations of a set of model aromatic carboxylic acids has been tested. Whereas most of the coatings of this category bring about an increase in selectivity with a concomitant slow down of the EOF, H₂TPFPP coating, depending on pH of the background electrolyte used, resulted both in decreasing of EOF at pH 8.5 by 5% and in increasing of EOF by 10–43% at pH 6 and 5, respectively. The separation efficiency and the resolution of aromatic carboxylic acids separation in coated capillaries, namely in that one with covalent coating, were better than in the bare fused-silica capillary. The perspectives of H₂TPFPP as capillary wall modifier are visualized in introducing well defined electroosmotic properties of materials used for miniaturized separation channels preparation in chip-based electromigration devices.     

Electrophoretic separations on chips with hydrodynamically closed separation systems

This review focuses on capillary electrophoretic separations performed on capillary electrophoresis chips (CE chips) with hydrodynamically closed separation systems in a context with transport processes (electroosmotic flow (EOF)) and hydrodynamic flow (HDF)) that may accompany the separations in these devices. It also reflects some relevant works dealing with conventional CE operating under such hydrodynamic conditions. The use of zone electrophoresis (ZE), isotachophoresis (ITP) and their on-line combination (ITP-ZE) on the single-column and column-coupling CE chips with the closed separation systems and related problems are key topics of the review. Some attention is paid to sample pretreatment in the separations performed on the CE chips. Here, mainly potentialities of the ITP-ZE combination in trace analysis applications of the miniaturized systems are discussed in a broader extent. Links between the ZE separation and detection provide a frame for the discussion of current status of the detection on the CE chips. Analytical applications illustrate potentialities of the CE chips operating with the closed separation systems (suppressed HDF and EOF) to the determination of small ions present in various matrices by ZE, ITP and ITP-ZE.     

Chiral separation of six diastereomeric flavanone-7-O-glycosides by capillary electrophoresis and analysis of lemon juice

Inexpensive and disposable polyester microchips were fabricated through photolithographic and wet-chemical etching procedure, followed by replication using an imprinting method at room temperature. Laboratory-scale laser-induced fluorescence equipment was employed as a detection system. The generation of electroosmotic flow (EOF) on the polyester channels was discussed in this paper. Surfactants in the running buffer had a significant effect on the EOF depending on their types. The epsilon potential of the electric double layer formed by adsorbing sodium lauryl sulfate molecules on the wall of polyester channels seemed to be constant within the buffer pH investigated. EOF could also be suppressed to zero by adding polyoxyethylene 23 lauryl ether into the running buffer. The separation of two laser dyes was obtained using polyester chips through both micellar electrokinetic chromatography and capillary zone electrophoresis. The polyester channels modified with 10-undecen-1-ol exhibited a dramatically high-separation efficiency compared with the conventional fused-silica capillary tubes.     

Conductivity detection and quantitation of isotachophoretic analytes on a planar chip with on-line coupled separation channels

A poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors and intended, mainly, to isotachophoresis (ITP) and ITP-capillary zone electrophoresis (CZE) separations was developed recently. The present work was aimed at assessing its performance relevant to the detection and quantitation of the ITP analytes. Hydrodynamic (HDF) and electroosmotic (EOF) flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the ITP separations with model analytes carried out in this context. When the surfaces of the detection electrodes of the conductivity sensors on the chip were appropriately cleaned qualitative indices of the test analytes [relative step heights (RSHs)], provided by a particular detection sensor, agreed within 1% (expressed via RSDs of the RSH values). Their long-term reproducibilities for one sensor, as estimated from 70 ITP runs repeated in 5 days, were 2% or less. Sensor-to-sensor and chip-to-chip fluctuations of the RSH values for the test analytes were 2.5% or less. In addition, experimentally obtained RSH values agreed well with those predicted by the calculations based on the ITP steady-state model. Reproducibilities of the migration velocities attainable on the CC chips with suppressed EOF and HDF, assessed from the migration time measurements of the ITP boundary between well-defined positions on the separation channels of the chips (140 repeated runs on three chips), ranged from 1.4 to 3.3% for the migration times in the range of 100-200 s. Within-day repeatabilities of the time-based zone lengths for the test analytes characterized 2% RSDs, while their day-to-day repeatabilities were less than 5%. Chip-to-chip reproducibilities of the zone lengths, assessed from the data obtained on three chips for 100 ITP runs, were 5-8%.     

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis.

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.     

A split microchannel design and analytical model to compensate for electroosmotic instabilities in micro-separations

Organic polymers offer many advantages as materials for the construction of microfluidic devices but suffer frequently from the limitation that the electrodynamic flow they support can exhibit considerable instability. This article describes a split-channel microfluidic device that can be used to compensate for changes in electroosmotic flow. The design of the separation system divides an analyte plug after injection between two separation channels of differing length. The two channels are later recombined for single point detection, eliminating the need for a scanning optical detection system. The utility of this simple design lies in the fact that the migration time of any analyte can be referenced to its twin in the parallel separation channel. This eliminates the need for a separate electroosmotic marker and allows mobilities measured in multiple devices to be compared quantitatively. Using a model adopted from the literature, the data from the split channel system can be used to precisely account for the drift that characterizes electrophoretic separations made in a polymer chip. The relative standard deviations of the analyte mobilities measured for replicate runs on multiple devices were reduced from values as high as 20% to ca. 1% RSD. This internal standardization procedure also appears to address other sources of drift in the electroosmotic flow (EOF) supported by the polymer microchannel, eliminating the need for careful monitoring of either the temperature or reservoir pH between separation runs.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecysilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecylsilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Semi-permanent surfactant coatings for inorganic anion analysis in capillary electrophoresis

Capillary electrophoretic separations of inorganic anions are performed using a capillary coated with a mixture of the cationic surfactant didodecyldimethylammonium bromide (DDAB) and the zwitterionic surfactant 1,2-dilauroyl-sn-phosphatidylcholine (DLPC). These double-chained surfactants form semi-permanent coatings on the capillary wall, which allows the excess surfactant to be removed from the buffer prior to separation. Interactions between surfactant aggregates in the buffer and analyte anions are thus eliminated. The electroosmotic flow (EOF) can be altered from fully reversed (100% DDAB) to near zero (100% DLPC) using different ratios of DDAB and DLPC. Controlling the EOF allows for improved resolution of the anions while maintaining a rapid, co-EOF separation, free from analyte-surfactant additive interactions.     

Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3-11. More than 1,000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, beta-lactoglobulin) and fragments of dsDNA phiX174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs.     

Dynamic coating of a capillary with room-temperature ionic liquids for the separation of amino acids and acid drugs by capillary electrophoresis

A room-temperature ionic liquid (IL), 1-ethyl-3-methyl-imidazolium tetrafluoroborate (1E-3MI-TFB), used for the coating of a silica capillary enables one to reduce or invert the electroosmotic flow (EOF) in capillary zone electrophoresis. Excellent separations of amino acids and ary lalkanoic acids were obtained. Such separations could not be obtained in a naked capillary in the presence of the cationic surfactants cetyltrimethylammonium bromide (CTAB) or polycationic polymer hexadimethrine bromide (HDB). The results indicate that 1E-3MI-TFB not only modulates the EOF but also acts as a discriminator. Further experiments indicate that the interaction between hydrogen at C-2 carbon of IL and acid drugs plays an important role in the separation. The text was submitted by the authors in English.     

Influence of pH*-value of methanolic electrolytes on electroosmotic flow in hydrophilic coated capillaries

The dependency of EOF on the H+-concentration and the related so called pH* value of methanolic electrolytes has been examined with poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and uncoated capillaries. These results were compared with the pH dependency of EOF of these capillaries using aqueous buffers. In uncoated capillaries the dependency of EOF on the pH(*)-value is very similar for aqueous and methanolic electrolytes. The EOF increases with increasing H+-concentration and pH-hysteresis is observed. In PVA coated capillaries the EOF is strongly reduced over wide pH* or pH ranges for both methanolic electrolytes and aqueous buffers. The EOF in PEG coated capillaries is surprisingly directed to the anode with methanolic electrolytes whereas a reduced cathodic EOF is observed in aqueous electrolytes. The anodic EOF of PEG-coated capillaries in methanolic electrolytes is independent of the pH*-value. The usefulness of PEG- and PVA-coated capillaries for adjusting the EOF in non-aqueous electrolytes for the analysis of isomeric organic acids was demonstrated.     

Isoelectric focusing in cyclic olefin copolymer microfluidic channels coated by polyacrylamide using a UV photografting method

As an alternative material to glass or silicon, microfluidic devices made from a cyclic olefin copolymer (COC) were fabricated. This material is of interest because of the relative ease of fabrication, low costs, and solvent resistance. However, as a result of the strong hydrophobic interactions normally present, COC surfaces are not suitable for protein separations. To reduce the protein adsorption and make COC suitable for protein separations, UV-initiated grafting of polyacrylamide was used to coat the surface of COC devices. The change in surface properties caused by different graft times was studied. The surface hydrophilicity and electroosmotic mobility were characterized by contact angle and electroosmosis measurements. Isoelectric focusing was performed to test protein separations in polyacrylamide-coated COC microchannels. A single protein, carbonic anhydrase, was used to analyze the focusing effects and peak capacities in uncoated and polyacrylamide-coated COC devices. Peak capacities ranging from 75 to 190 were achieved with a polyacrylamide-coated surface. A mixture of two proteins, conalbumin labeled with Alexa Fluor 488 and beta-lactoglobulin A labeled with Alexa Fluor 546, was used to test protein separations. Linear and rapid separation of proteins was achieved in the polyacrylamide-coated COC microfluidic device.