Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Collection and expansion of single cells and colonies released from a micropallet array

The ability to selectively grow out individual cells possessing unique characteristics from within a mixed population is of widespread importance for biomedical investigations. Generation of genetically engineered cell lines, transformation studies, cell-based assays, and stem cell studies are examples where single-cell cloning is of immense value. The vast majority of mammalian cells grow adherent to a surface; therefore, positive selection followed by cloning of cells while the cells remain adherent to their growth surface is an important goal. We recently demonstrated a microfabricated cell array combined with laser-based release of individual array elements for positive selection of single cells. In the current work, a strategy to collect single cells for clonal expansion is described. The system enabled cloning of individual cells with 80-90% efficiency. Single cells were selected and cloned from small populations of fewer than 10,000 cells. Strategies used by cells to migrate from the pallets to form colonies on the surface of the collection device were examined. Implementation of encoded array elements made it possible to follow specific cells throughout the selection, collection, and cloning procedure. Thus, a particular cell can be identified by any number of imaging techniques, isolated, and clonally expanded to generate a homogeneous cell line or a pure sample for genetic or biochemical analysis.     

Capillary electrophoretic method for the detection of bacterial contamination

There has been growing interest in separations-based techniques for the identification and characterization of microorganisms because of the versatility, selectivity, sensitivity, and short analysis times of these methods. A related area of analysis that is scientifically and commercially important is the determination of the presence or complete absence of microbes (in essence, a test for sample sterility). In such a test, it is not of immediate importance to identify a particular microorganism, but rather, to know with a high degree of certainty whether any organism(s) is (are) present. Current regulations require culture-based tests that can take up to 2 weeks to complete. As a rapid alternative, capillary electrophoresis-based methods are examined. Experimental formats are developed that promote the consolidation of all cell types into a single zone (peak) which is separated from the electroosmotic flow front and any other interfering molecular constituents. This process can be accomplished using a segment of dilute cetyltrimethylammonium bromide, which serves to temporarily reverse the migration direction of the cells, and another segment of solution containing a "blocking agent", which serves to stop the cell migration and focus them into a narrow zone. Relatively wide-bore capillaries can be used to increase sample size. This approach appears to be effective for a broad spectrum of bacteria, and analyses times are less than 10 min.     

Capillary electrophoretic separations of proteins using nonionic surfactant coatings

Capillary zone electrophoretic separations of proteins have been achieved by using nonionic surfactant coated capillaries. Capillaries were prepared by derivatization of the silica surface with octadecylsilane followed by the deposition of a layer of nonionic surfactant from an aqueous solution above the critical micelle concentration. This coating is of sufficient thickness and hydrophilicity to reduce both protein adsorption and electroosmotic pumping. This hydrophilic coating reduces electroosmotic pumping 5-8-fold while resolving proteins quickly and efficiently with good recovery. The coating provides a stable and reproducible means of deactivation, while the rate of electroosmotic pumping stays relatively constant throughout the pH range 4-11. This allows the pH to be varied to enhance selectivity without adversely affecting the flow rate.     

Capillary electrophoretic application of 1-alkyl-3-methylimidazolium-based ionic liquids

Ionic substances with melting points at or close to room temperature are referred to as ionic liquids. Interest in ionic liquids for their potential in different chemical processes is increasing, because they are environmentally benign and are good solvents for a wide range of both organic and inorganic materials. In this study, a capillary electrophoretic method for resolving phenolic compounds found in grape seed extracts is reported. The method, in which 1-alkyl-3-methylimidazolium-based ionic liquids are used as the running electrolytes, is simple and reproducible. The separation mechanism seems to involve association between the imidazolium cations and the polyphenols. The role of the alkyl substituents on the imidazolium cations was investigated and will be discussed.     

Micropallet arrays for the separation of single, adherent cells

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.     

Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.     

Fast electrophoretic separation optimization using gradient micro free-flow electrophoresis

The continuous nature of micro free-flow electrophoresis (mu-FFE) was used to monitor the effect of a gradient of buffer conditions on the separation. This unique application has great potential for fast optimization of separation conditions and estimation of equilibrium constants. COMSOL was used to model pressure profiles in the development of a new mu-FFE design that allowed even application of a buffer gradient across the separation channel. The new design was fabricated in an all glass device using our previously published multiple-depth etch method (Fonslow, B. R.; Barocas, V. H.; Bowser, M. T. Anal. Chem. 2006, 78, 5369-5374, ref 1). Fluorescein solutions were used to characterize the applied gradients in the separation channel. Linear gradients were observed when buffer conditions were varied over a period of 5-10 min. The effect of a gradient of 0-50 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the separation of a group of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) labeled primary amines was monitored as a proof of concept experiment. Direct comparisons to capillary electrophoresis (CE) separations performed under the same conditions were made. Gradient mu-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-beta-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-beta-CD concentrations using CE. Qualitatively, mu-FFE separations were predictive of the migration order and spacing of peaks in CE electropherograms measured under the same conditions. Data were fit to equations describing 1:1 analyte-additive binding to allow a more quantitative comparison between gradient mu-FFE and CE.     

Capillary zone electrophoretic detection of biological thiols and their S-nitrosated derivatives.

Reduced thiols (RSH) react with certain oxides of nitrogen to yield S-nitroso thiols (RSNO) possessing smooth muscle relaxant and platelet inhibitory properties. Nitrosated derivatives of the biological thiols--glutathione, cysteine, and homocysteine--have therefore been considered as bioactive intermediates in the metabolism of organic nitrates and the endothelium-derived relaxing factor with properties of nitric oxide. As yet, however, there is no established chemical method for identifying the biological S-nitroso thiols and, consequently, little is known of their distinguishing chemical characteristics or biochemistry. In this study, we demonstrate for the first time a simple, rapid, and reproducible method for separating these thiols from their S-nitrosated derivatives using capillary zone electrophoresis. Cysteine, homocysteine, and glutathione were separated from one another and from their corresponding disulfides in 0.01 M phosphate buffer, pH 2.5, by capillary zone electrophoresis and absorbance detection at 200 nm with measured elution times of 5.92-16.15 min; corresponding S-nitroso thiols were selectively detected at 320 nm and eluted at 2.50-18.20 min. These data support the specificity and reproducibility of this technique for separation and identification of thiols, disulfides, and S-nitroso thiol derivatives.     

Coupled isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis

We present a novel technique for coupling isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis (ITP). Bidirectional ITP simultaneously sets up sharp ITP interfaces between relatively high- and low-mobility cations and high- and low-mobility anions. These two interfaces can migrate toward each other and be described as ion concentration shock waves. We here demonstrate a bidirectional ITP process in which we use the interaction of these anionic and cationic ITP shock waves to trigger a transformation from ITP preconcentration to electrophoretic separation. We use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. The method provides faster and much less dispersive transition from ITP preconcentration to electrophoretic separation compared with traditional (unidirectional) transient ITP. It eliminates the need for intermediate steps between focusing and separation, such as manual buffer exchanges. We illustrate the technique with numerical simulations of species transport equations. We have validated our simulations with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high-resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP.     

Accelerated particle electrophoretic motion and separation in converging-diverging microchannels

Accelerated particle electrophoretic motions were visualized in converging-diverging microchannels on poly(dimethylsiloxane) chips. The accelerated particle electrophoretic separation is highly desirable in on-chip flow cytometry and high-speed electrophoresis. The effects of electric field, particle size, particle trajectory, and channel structure on the particle electrophoretic motion are examined. We find that the ratio of the particle velocity in the throat to that in the straight channel is significantly lower than their cross-sectional area ratio. This discrepancy may be attributed to the locally higher electric field around the two poles of a particle, as compared to other regions inside the microchannel. We also find that the particle velocity ratio is increased for smaller particles moving through symmetric converging-diverging channels under lower electric fields. These variations may be attributed to the negative dielectrophoretic force that is generated by the nonuniform electric field in the converging-diverging section. In addition, we find that particle trajectory has insignificant influences on the maximum velocity ratio obtained in the throat.     

Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips

The work presented herein describes highly efficient, two-dimensional (2D) electrophoretic separations of proteins in a PMMA-based microchip. Sodium dodecyl sulfate microcapillary gel electrophoresis (SDS micro-CGE) and micellar electrokinetic chromatography (MEKC) were used as the separation modes for the first and second dimension of the electrophoresis, respectively. The microchip was prepared by hot embossing into PMMA from a brass mold master fabricated via high-precision micromilling. The microchip incorporated a 30-mm SDS micro-CGE and a 10-mm MEKC dimension length. Electrokinetic injection and separation were used with field strengths of up to 400 V/cm. Alexa Fluor 633 conjugated proteins, ranging in size from 38 to 110 kDa, were detected using laser-induced fluorescence with excitation/emission at 633/652 nm. Average plate numbers (N) of 4.8 x 10(4) and 1.2 x 10(4) were obtained in the SDS micro-CGE and MEKC separation dimensions, respectively, for the investigated proteins corresponding to plate heights (H) of 0.62 and 0.87 microm. Effluents from the first dimension (SDS micro-CGE) were repetitively transferred into the second dimension every 0.5 s of run time in the first dimension with the electrophoresis run time in the MEKC dimension being 10 s. The 2D separation was performed on the investigated proteins in approximately 12 min and provided a peak capacity of approximately 1000.     

Capillary isoelectric focusing of yeast cells

In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (approximately 4-microm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-microm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/microL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.     

Capillary electrophoresis with electrochemical detection for chiral separation of optical isomers

The enantiomers of two amine derivatives were directly separated by capillary electrophoresis (CE), employing β-cyclodxtrin (β-CD) as a chiral additive in strongly alkaline solutions. The analytes were detected by electrochemistry, using a copper disk electrode at +675 mV vs Ag/AgCl reference electrode. Both the free enantiomers and the enantiomer-cyclodxtrin inclusion complexes could be detected using this approach, although the complexed forms gave lower oxidation currents than the free forms. Factors affecting the chiral CE separation of the analytes, such as working potential, concentration of running buffer and β-CD, and applied voltage, were extensively investigated. Under the optimum conditions, baseline separation of the enantiomers could be accomplished in less than 18 min. In addition, a successful application of the method to the enantiomeric purity determination confirmed its validity and practicability.     

Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents

A miniaturized analytical system for separating and detecting toxic organophosphate nerve agent compounds, based on the coupling of a micromachined capillary electrophoresis chip with a thick-film amperometric detector, is described. Factors influencing the on-chip separation and detection processes have been optimized. Using a MES buffer (20 mM, pH 5.0) running buffer, a 72-mm-long separation channel, and a separation voltage of 2000 V, baseline resolution is observed for paraoxon, methyl parathion, fenitrothion, and ethyl parathion in 140 s. Such miniaturization and speed advantages are coupled to submicromolar detection limits and good precision. Applicability to spiked river water samples is demonstrated, and the implications for on-site environmental monitoring and rapid security screening/warning are discussed.     

Capillary electrophoretic determination of different classes of organic ions by potentiometric detection with coated-wire ion-selective electrodes

Singly charged amines and sulfonic acids as cationic and anionic aliphatic model substances, respectively, were detected in capillary electrophoresis with all-solid-state ion-selective electrodes. The sensitivity for these compounds is a function of their lipophilicity. The signal detected is generally greater for molecules with a larger organic part. The utility of the method is further demonstrated by the detection of a group of aromatic compounds in the form of the anionic analgesics (S)-(+)-2-(4-isobutylphenyl)propionic acid, 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid, and o-acetylsalicylic acid. Further determined were the artificial sweeteners cyclamate, acesulfame-K, and saccharin. Detection limits for the different substances were between 2.5 × 10(-)(6) and 1 × 10(-)(5) M.     

In-situ sampling and separation of RNA from individual mammalian cells

In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromide-labeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed online with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of approximately equals 10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorporated with RNA molecules were eliminated by SDS treatment and thus did not influence the migration of RNA. Experiments were also performed with this technique to detect nucleic acid damage. Changes in the peak pattern were detected in the cells treated with hydrogen peroxide, which meant that strand breaks occurred in DNA and RNA. It was found that 60 mM caused the most severe damage to the nucleic acids.