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.     

Chiral separation of NBD-amino acids by ligand-exchange micro-channel electrophoresis.

The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.     

Fabrication and performance of poly(methyl methacrylate) microfluidic chips with fiber cores

In this work, a piece of glass fiber was inserted into the channel of a poly(methyl methacrylate) (PMMA) electrophoresis microchip to enhance the electroosmotic flow (EOF) and the separation efficiency. The EOF value of the glass fiber-containing microchannel at pH 8.2 was determined to be 4.17 x 10(-4)cm2 V(-1)s(-1). The performance of the new microchip was demonstrated by its ability to separate and detect three purines coupled with end-column amperometric detection. In addition, a piece of trypsin-immobilized glass fiber was inserted into the channel of a PMMA microchip to fabricate a core-changeable microfluidic bioreactor that can be regenerated by changing the fiber. The in-channel fiber bioreactor has been coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the digestion and peptide mapping of bovine serum albumin and myoglobin.     

Versatile method for electroosmotic flow measurements in microchip electrophoresis

A novel versatile method for the determination of low or high electroosmotic mobility values in microdevices of variable microchannel design is presented. The electroosmotic flow (EOF) calculation is based on the difference between the apparent and effective mobilities of a reference compound. The proposed method uses microchip frontal electrophoresis for the determination of these mobilities. This requires simple monochannel microchip design and demonstrates versatile and time-saving procedure when compared to conventional current monitoring method when measuring low EOF. It has been applied successfully to the characterization of different coating procedure in glass and poly(dimethylsiloxane) microchips.     

Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis

Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15 s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear calibration curves. We interpret this effect as a variation in the volume of the injected sample plug caused by changes of the electroosmotic flow (EOF) due to the strong interaction of bivalent cations with the glass surface. This explanation is supported by the observation of severe peak tailing. Conducting microchip CE analysis in a glass microchannel, optimized conditions are received for the cationic species K+, Na+, Ca2+, Mg2+ using a background electrolyte consisting of 30 mmol/L histidine and 2-(N-morpholino)ethanesulfonic acid, containing 0.5 mmol/L potassium chloride to reduce surface interaction and 4 mmol/L tartaric acid as a complexing agent resulting in a pH-value of 5.8. Applying reversed EOF co-migration for the anionic species Cl-, SO42- and HCO3- optimized separation occurs in a background electrolyte consisting of 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mmol/L HEPES sodium salt, containing 0.05 mmol/L CTAB (cetyltrimethylammonium bromide) resulting in a pH-value of 7.5. The detection limits are 20 micromol/L for the monovalent cationic and anionic species and 10 micromol/L for the divalent species. These values make the method very suitable for many applications including the analysis of abundant ions in tap water as demonstrated in this paper.     

Convenient enantioseparation by monolithic imprinted capillary clamped in a chip with electrochemical detection

A microchip integrated with a monolithic imprinted capillary has been manufactured for performing the chip-based capillary electrochromatographic enantioseparation. The microporous monolith anchored on the inner wall of the microchannel was prepared by in situ chemical copolymerization, and characterized with scanning electron microscopy, IR spectroscopy, and solid-state UV-vis spectroscopy. The monolithic network with high porosity gave a large surface area, good permeability, low mass-transfer resistance, and thus high separation efficiency. A portable microchip was conveniently constructed by integrating an imprinted capillary with 5-cm length as the separation channel and a carbon fiber microdisk working electrode for amperometric detection. Using L-tyrosine (L-Tyr) as the template molecule, Tyr enantiomers could be baseline separated within 55 s under the optimized preparation and separation conditions. The linear ranges for online amperometric detection of both Tyr enantiomers were from 20 to 2400 μM. The microporous monolithic chip strategy exhibited excellent separation efficiency and promising analytical application in enantioseparation. It opens an avenue for high-throughput screening of chiral compounds.     

High-speed chiral separations on a microchip with UV detection

Fast chiral separations of a variety of basic and acidic compounds could be realized on microfluidic quartz chips. A microchip electrophoresis instrument equipped with a linear imaging UV-detector was used. The usually applied but troublesome fluorescence tagging in order to enable fluorescence detection could be omitted. Using sulfated cyclodextrins as chiral selectors baseline separation of 19 compounds could be achieved in less than 1 min with high reproducibility. The relative standard deviation of migration time was below 7%. The fastest separation could be performed in 2.5 s which is to date the fastest separation of enantiomers reported. It was possible to apply microchip electrophoresis (MCE) for the determination of high enantiomeric excess (ee) values, as exemplarily shown for pseudoephedrin where 2% of the minor enantiomer could reliably be determined beside high amount of the other isomer. Successful separation of a mixture of 3 chiral drugs could be performed in a single run in less than 11 s utilizing a separation length of only 12 mm. These results show that MCE has great potential for fast chiral analysis and high-throughput screening.     

Rapid and sensitive determination of anthraquinones in Chinese herb using 1-butyl-3-methylimidazolium-based ionic liquid with beta-cyclodextrin as modifier in capillary zone electrophoresis

The present study reported the ionic liquid (IL) used as running electrolyte in capillary zone electrophoresis (CZE) with beta-cyclodextrin (beta-CD) as modifier for the separation of anthraquinones extract of Chinese herb Paedicalyx attopevensis Pierre ex Pitard. The optimum running electrolyte was 60 mM 1-butyl-3-methylimizolium tetrafluoroborate (1B-3MI-TFB) solution with 4.0 mM beta-CD. The pH was 10.00 and the applied voltage was 20 kV with detection at 254 nm. The present method was compared with others and the effect of Joule heating was discussed as well. More significantly, this method is the development of the ionic liquids application to the capillary electrophoresis.     

Chiral separation of gemifloxacin in sodium-containing media using chiral crown ether as a chiral selector by capillary and microchip electrophoresis

Chiral crown ether, (+)-(18-crown-6)-tetracarboxylic acid (18C6H(4)), is an effective chiral selector for resolving enantiomeric primary amines owing to the difference in affinities between 18C6H(4) and each of the amine enantiomers. In addition to the destacking effect of sodium ion in the sample solution, the strong affinity of sodium ion to the polyether ring of crown ether is unfavorable to chiral capillary electrophoresis using 18C6H(4) as a chiral selector. In this report, the chiral separation of gemifloxacin dissolved in a saline sample matrix using 18C6H(4) was investigated. Adding a chelating agent, ethylenediaminetetraacetic acid (EDTA), to the run buffer greatly improved the separation efficiencies and peak shapes. The successful chiral separation of gemifloxacin in a urinary solution was demonstrated for both capillary and microchip electrophoresis.     

Fabrication and evaluation of single- and dual-channel (Pi-design) microchip electrophoresis with electrochemical detection

A new dual-channel microchip capillary electrophoresis (MCE) has been developed on glass substrates for the first time with electrochemical detection. Dual-channel (called Pi-design) as well as single-channel microchips have been fabricated on soda-lime glass using photolithography, wet etching and thermal bonding. Moreover, a laser writing system has been applied for the fabrication of photomasks with the different microchip designs (single- and dual-channel configurations). The microfabricated channels have been characterized by optical, confocal and scanning electron microscopy. The resulting single- and dual-channel microchips have been evaluated using an end-channel amperometric detector based on one (single-channel) or two (dual-channel) 100-mum gold wires aligned at the outlet of the separation channel. Parameters affecting the separation of several phenolic compounds (dopamine, p-aminophenol and hydroquinone) have been studied in the glass microchips. Thus, the influence of separation voltage, detection potential and background electrolyte has been examined in the single-channel microchip. Different total length microchannel has been compared. Furthermore, the possibility of carrying out two simultaneous measurements has been demonstrated in the new dual-channel microchip electrophoresis. The injection format has been checked and resulted to be critical, in such a way that a special and new form is employed for obtaining simultaneous signals at both channels. Analytical characteristics, such as sensitivity and reproducibility have been evaluated and resulted very adequate.     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

肝糖选择剂-毛细管电泳手性拆分盐酸度洛西汀对映体及分离机制

以肝糖为毛细管电泳手性选择剂,对盐酸度洛西汀对映体的分离进行研究,建立了拆分方法.考察了手性选择剂浓度、运行缓冲液的离子强度、pH值及分离电压对手性分离的影响.在3.0% (m/V)肝糖、50 mmol/L Tris-H3PO4(pH 3.0)的运行缓冲液中,分离电压25 kV时,盐酸度洛西汀两对映体分离度达1.84.对分离机制进行了初步探讨.     

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.     

Simultaneous multiple enantioseparation with a one-pot imprinted microfluidic channel by microchip capillary electrochromatography

A multi-template imprinted microchannel was prepared by a one-pot in situ imprinting process. The imprinted microchannel led to a novel chip-based strategy for simultaneous multiple enantioseparation. The one-pot imprinting process formed a multi-template imprinted porous thin layer (about 2 μm) on the inner wall of the capillary, which was characterized by scanning electron microscopy, infrared spectroscopy, and solid-state UV-vis spectroscopy. By fixing the imprinted capillary to a support substrate composed of poly(dimethylsiloxane) on a glass slide, a multi-analyte microchip was thus conveniently constructed. Using L-tyrosine (L-Tyr) and L-tryptophan (L-Trp) as the template molecules, two pairs of enantiomers were simultaneously baseline separated in a 6 cm separation channel within 120 s under the optimized preparation and electrochromatographic conditions. The separation showed excellent efficiency. The linear ranges for amperometric detection of four analytes using a carbon fiber microdisk electrode at +1.2 V (vs. Ag/AgCl) were from 20 to 500 μM for racemic Tyr and Trp. This multi-template imprinting strategy could be expanded for simultaneous separation and detection of additional pairs of enantiomers within a short analytical time. It could open up a promising avenue for high-throughput screening of chiral compounds.     

Optimisation of a Headspace Solid-Phase Micro- Extraction Procedure for the Determination of 2,4,6-Trichloroanisole and Various Related Compounds in Cork Washing Waste Water by Use of Gas Chromatography-Mass Spectrometry

This paper reports the use of an anionic cyclodextrin, heptakis(2,3-di-O-methyl-6-O-sulfato)-β-cyclodextrin (HDMS-β-CD), for chiral separations of pharmaceutical enantiomers by nonaqueous capillary electrophoresis (NACE). Enantiomer resolution was affected mainly by HDMS-β-CD concentration and the acidity of the background electrolyte (BGE). The effects of capillary length and applied voltage on enantiomer resolution were also investigated. Results showed that in a methanol solution of 20 mM phosphoric acid, 10 mM sodium hydroxide, and 10 mM HDMS-β-CD, seven anticholinergic drugs were separated to baseline but no chiral separation was obtained for three other similar drugs. NACE is suitable for routine, rapid separation of the enantiomers of pharmaceutical compounds.     

On-chip chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in a microchannel

A novel method of chiral separation based on protein-stationary phase immobilized in a poly(methyl methacrylate) microfluidic chip was developed. BSA conjugated with the shortened carboxylic single-walled carbon nanotubes (SWNTs) was employed as the chiral selector. Successful separation of tryptophan enantiomers was achieved in less than 70 s with a resolution factor of 1.35 utilizing a separation length of 32 mm. This is the first example of chiral separation based on SWNTs-BSA conjugates as stationary phase immobilized in microchip channel. The stability of the stationary phase in the channel was examined by microchip electrophoresis with laser-induced fluorescence detection. Factors that influenced the chiral separation resolution were examined. Under the optimized conditions, the proposed modified chip revealed adequate repeatability concerning run-to-run. These results show that the use of SWNTs-BSA conjugates within microfluidic channels hold great promise for a variety of analytical schemes.     

Enantiodifferentiation of N-benzyloxycarbonylaminophosphonic and phosphinic acids and their esters using cyclodextrins by means of capillary electrophoresis

Capillary electrophoresis was successfully applied for separation of the enantiomers of N-benzyloxycarbonyl-alpha-aminophosphonic and alpha-aminophosphinic acids as well as their ethyl and phenyl monoesters with the use of a range of commercially available cyclodextrins (alpha, beta and hydroxypropyl-gamma-cyclodextrins) as chiral selectors. The dependence of effectiveness of separation on type and concentration of these chiral selectors as well as on pH of background electrolyte was examined in some detail.     

Poly-N-hydroxyethylacrylamide (polyDuramide): a novel,hydrophilic, self-coating polymer matrix for DNA sequencing by capillary electrophoresis

A replaceable polymer matrix, based on the novel monomer N-hydroxyethylacrylamide (HEA), has been synthesized for application in DNA separation by microchannel electrophoresis. The monomer was found by micellar electrokinetic chromatography analysis of monomer partitioning between water and 1-octanol to be more hydrophilic than acrylamide and N,N-dimethylacrylamide. Polymers were synthesized by free radical polymerization in aqueous solution. The weight-average molar mass of purified polymer was characterized by tandem gel permeation chromatography-multiangle laser light scattering. The steady-shear rheological behavior of the novel DNA sequencing matrix was also characterized, and it was found that the viscosity of the novel matrix decreases by more than 2 orders of magnitude as the shear rate is increased from 0.1 to 1000 s(-1). Moreover, in the shear-thinning region, the rate of change of matrix viscosity with shear rate increases with increasing polymer concentration. Poly-N-hydroxyethylacrylamide (PHEA) exhibits good capillary-coating ability, via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly-N,N-dimethylacrylamide. Under DNA sequencing conditions, adsorptive PHEA coatings proved to be stable and to maintain negligible EOF for over 600 h of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in PHEA matrices generally improves with increasing polymer concentration and decreasing electric field strength. When PHEA is used both as a separation matrix and as a dynamic coating in bare silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within 3 h. These results demonstrate the good potential of PHEA matrices for high-throughput DNA analysis by microchannel electrophoresis.     

Separation of aminoalkanephosphonic acid enantiomers by indirect UV detection capillary electrophoresis with application of cyclodextrins

Indirect UV detection capillary electrophoresis (CE) was used for the separation of aminoalkanephosphonic acid (AP) enantiomers by applying commercially available cyclodextrins as chiral discriminators. The results show that the separation of the enantiomers depends on pH of the background electrolyte, the molar ratio of cyclodextrin to aminophosphonic acid, and on the type of the applied chiral selector. Optimization of process conditions allowed enantiomeric baseline separation or partial separation of 12 out of 14 alpha-aminophosphonic acids studied. This type of CE might therefore be successfully used for routine determination of enantiomeric purity of aminophosphonic acids.     

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).