Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated tapered fused-silica capillary with a beveled edge.

A tapered capillary tip containing a beveled edge was developed for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The optimal flow rate of a 75-microm-i.d., 90-microm-o.d. beveled tapered capillary tip was similar to a conventional flat tapered tip with a 25-microm orifice. Using a mixture of coptisine, berberine, and palmatine chloride, the sheathless CE/ ESI-MS sensitivity of a beveled 75 microm tapered tip capillary was found to be similar to a 25 microm flat tip. Although both tips offer similar CE/ESI-MS sensitivity, the beveled tapered capillary tip is more rugged and durable than a conventional 25-microm tapered capillary because of the larger outside diameter and inside diameter. To make electrical contact, the capillary tip was smeared with paint marker followed by the application of a carbon coating using a graphite pencil. Using this refined carbon-coating procedure, the capillary tip can be operated with aprotic solvents.     

On-line fourier transform infrared detection in capillary electrophoresis

The coupling of Fourier transform infrared (FT-IR) spectroscopy as a new on-line detection principle in capillary electrophoresis (CE) is presented. To overcome the problem of total IR absorption by the fused-silica capillaries that are normally employed in CE separations, a micromachined IR-transparent flow cell was constructed. The cell consists of two IR-transparent CaF2 plates separated by a polymer coating and a titanium layer producing an IR detection window, 150 microm wide and 2 mm long, with a path length of 15 microm. The IR beam was focused on the detection window using an off-axis parabolic mirror in an optical device (made in-house) attached to an external optical port of the spectrometer. The connections between the fused-silica capillaries and the flow cell were made by a small O-ring of UV-curing epoxy adhesive on the sharply cut ends of the capillaries, allowing the capillaries to be easily replaced. Aqueous solutions comprising mixtures of adenosine, guanosine, and adenosine monophosphate were used to test the system's performance. Conventional on-line UV detection was employed to obtain reference measurements of analytes after the IR detection flow cell. The limit of FT-IR detection for all analytes (in absolute amounts) was in the nano- to picogram range corresponding to concentrations in the low-millimolar range.     

Mass transport in a micro flow-through vial of a junction-at-the-tip capillary electrophoresis-mass spectrometry interface

When coupling capillary electrophoresis with postcolumn detection methods, such as mass spectrometry, the presence of postcolumn band broadening must be considered. The band broadening effects introduced by junction-at-the-tip CE-MS interfaces using a postcolumn micro flow-through vial are investigated by studying the hydrodynamic flow patterns and mass transport process inside the micro vial at the end of the CE separation capillary. Simulation results obtained by solving the Navier-Stokes and mass balance equations provide insights into the velocity field and concentration distribution of the analytes in the micro vial and demonstrate that, with a low flow rate of chemical modifier solution, the laminar flow streams confine the analyte molecules to the central part of the micro vial and thus maintain major features of the peak shapes. Peaks detected by UV and MS under similar experimental conditions were compared to verify the numerical prediction that the main features of the UV peak can be retained in the MS peak. Experiments also show that band broadening can be minimized when an appropriate chemical modifier flow rate is selected.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.     

Dual-channel method for interference-free in-channel amperometric detection in microchip capillary electrophoresis

A novel in-channel amperometric detection method for microchip capillary electrophoresis (CE) has been developed to avoid the interference from applied potential used in the CE separation. Instead of a single separation channel as in conventional CE microchips, we use a dual-channel configuration consisting of two different parallel separation and reference channels. A working electrode (WE) and a reference electrode (RE) are placed equally at a distance 200 microm from its outlet on each channel. Running buffer flows through the reference channel. Our dual-channel CE microchips consist of a poly(dimethylsiloxane) (PDMS) upper plate and a glass lower plate to form a PDMS/glass hybrid chip. Amperometric signals are measured without any potential shift and interference from the applied CE potential, and CE separation maintains its high resolution because this in-channel configuration does not allow additional band broadening that is notorious in end-channel and off-channel configurations. The high performance of this new in-channel electrochemical detection methodology for CE has been demonstrated by analyzing a mixture of electrochemically active biomolecules: dopamine (DA), norepinephrine, and catechol. We have achieved a 0.1 pA detectability from the analysis of DA, which corresponds to a 1.8 nM concentration.     

A colloidal graphite-coated emitter for sheathless capillary electrophoresis/nanoelectrospray ionization mass spectrometry

A colloidal graphite-coated emitter is introduced for sheathless capillary electrophoresis/nanoelectrospray ionization time-of-flight mass spectrometry (CE/ESI-TOFMS). The conductive coating can be produced by brushing the capillary tip to construct a fine layer of 2-propanol-based colloidal graphite. The fabrication involves a single step and requires less than 2 min. Full cure properties develop in approximately 2 h at room temperature and then the tip is ready for use. The coated capillary tip is applied as a sheathless electrospray emitter. The emitter has proven to bear stable electrospray and excellent performance for 50 microm i.d. x 360 microm o.d. and 20 microm i.d. x 360 microm o.d. capillaries within the flow rate of 80-500 nL/min; continuous electrospray can last for over 200 h in positive mode. Baseline separation and structure elucidation of two clinically interesting basic drugs, risperidone and 9-hydroxyrisperidone, are achieved by coupling pressure-assisted CE to ESI-TOFMS using the described sheathless electrospray emitter with a bare fused-silica capillary at pH 6.7. It is found that the signal intensity of m/z in sheathless CE/ESI-TOFMS at pH 6.7 is approximately 50 times higher than that at pH 9.0 for the two analytes, although the electroosmotic flow (EOF) at pH 9.0 provides sufficient flow rate (approximately 150 nL/min) to maintain electrospray.     

Anomalous radial migration of single DNA molecules in capillary electrophoresis

We report the unexpected radial migration of DNA molecules in capillary electrophoresis (CE) with applied Poiseuille flow. Such movement can contribute to anomalous migration times, peak dispersion, and size and shape selectivity in CE. When Poiseuille flow is applied from the cathode to the anode, DNA molecules move toward the center of the capillary, forming a narrow, highly concentrated zone. Conversely, when the flow is applied from the anode to the cathode, DNA molecules move toward the walls, leaving a DNA-depleted zone around the axis. We showed that the deformation and orientation of DNA molecules under Poiseuille flow was responsible for the radial migration. By analyzing the forces acting on the deformed and oriented DNA molecules, we derived an expression for the radial lift force, which explained our results very well under different conditions with Poiseuille flow only, electrophoresis only, and the combination of Poiseuille flow and electrophoresis. Factors governing the direction and velocity of radial migration were elucidated. Potential applications of this phenomenon include an alternative to sheath flow in flow cytometry, improving precision and reliability of single-molecule detection, reduction of wall adsorption, and size separation with a mechanism akin to field-flow fractionation. On the negative side, nonuniform electroosmotic flow along the capillary or microfluidic channel is common in CE, and radial migration of certain analytes cannot be neglected.     

Application of diamond microelectrodes for end-column electrochemical detection in capillary electrophoresis

Highly boron-doped diamond microelectrodes were employed in an end-column electrochemical detector for capillary electrophoresis (CE). The diamond microline electrodes were fabricated from conducting diamond thin films (exposed surface area, 300 x 50 microm), and their analytical performance as CE detectors was evaluated in a laboratory-made CE installation. The CE-ED system exhibited high separation efficiency for the detection of several catecholamines, including dopamine (DA), norepinephrine (NE), and epinephrine (E), with excellent analytical performance, for example, 155,000 theoretical plates for DA. The diamond-based electrochemical detection system also displayed low detection limits (approximately 20 nM for E at S/N = 3) and a highly reproducible current response with 10 repetitive injections of mixed analytes containing DA, NE, and E (each 50 microM), with relative standard deviations (RSD) of approximately 5%. The performance of the diamond detector in CE was also evaluated in the detection of chlorinated phenols (CP). When compared to the carbon fiber microelectrode, the diamond electrode exhibited lower detection limits in an end-column CE detection resulting from very low noise levels and highly reproducible analyses without electrode polishing due to analyte fouling, which makes it possible to perform easier and more stable CE analysis.     

Attenuated total internal reflectance infrared microspectroscopy as a detection technique for capillary electrophoresis

A novel detector for capillary electrophoresis (CE) using single-bounce attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the CE capillary is placed approximately 1 microm from the internal reflectance crystal at the focus of an ATR infrared microscope. Using pressure driven flow injection, concentration and volume detection limits have been determined for 25- and 10-microm-i.d. silica capillaries. Upon injection of 820 pL of succinylcholine chloride in a 10-microm capillary, a concentration detection limit of approximately 0.5 parts per thousand (ppt), or 410 pg, is found. The injection volume detection limit using a 108 ppt solution is 2.0 pL (216 pg). Sample separations using a programmed series of pressure, voltage, and again pressure on 25-, 50-, and 75-microm-i.d. capillaries are shown. CE separations of citrate and nitrate, as well as succinylcholine chloride with sodium salicylate using acetone as a neutral marker, are demonstrated. Several advantages of this CE-FT-IR technique include: (1) minimization of postcolumn broadening as a result of a small detector volume; (2) the ability to signal average spectra of the same aliquot, thereby improving the signal-to-noise in a stopped-flow environment; and (3) simplicity of design.     

Fourier transform capillary electrophoresis with laminar-flow gated pressure injection

Fourier transform capillary electrophoresis (FTCE) was developed as a method to improve signal-to-noise ratio (S/N) and resolution in capillary electrophoresis (CE) separation. In FTCE, multiple simultaneous CE separations were performed in the same channel system and interrogated using a single-point detector. To illustrate experimentally the improvement offered by FTCE in S/N ratio and resolution, we carried out a modest number (five) of multiple injections and separations. We show even with this small number of separations, S/N increased by a factor of 2.9, and theoretical plate height improved by a factor of more than 30. We demonstrated this technique with laser-induced fluorescence detection, but a wide variety of detection methods are compatible with FTCE.     

Size-based characterization by the coupling of capillary electrophoresis to Taylor dispersion analysis

In this work, a new methodology is presented for performing capillary electrophoresis (CE) coupled to Taylor dispersion analysis (TDA). The CE step allows the separation of the different compounds of the injected mixture, while the diffusion coefficient related to each sample zone can be derived from the subsequent TDA step. TDA is an absolute and straightforward nonseparative method allowing the determination of the diffusion coefficient (or hydrodynamic radius) from the peak dispersion obtained in an open tube under Poiseuille laminar flow conditions. With a mass concentration sensitive detector, the hydrodynamic radius derived from TDA is a weight average value calculated upon all the molecules present in the sample zone. Since CE can be hardly coupled to light scattering detection for technical reasons (low volumes, short detection path length), TDA represents an interesting alternative for the size characterization, without calibration, of sample mixtures using CE-based separation techniques. The coupling of CE to TDA can be implemented on a commercial CE apparatus.     

Integrated refractive index optical ring resonator detector for capillary electrophoresis

We developed a novel miniaturized and multiplexed, on-capillary, refractive index (RI) detector using liquid core optical ring resonators (LCORRs) for future development of capillary electrophoresis (CE) devices. The LCORR employs a glass capillary with a diameter of approximately 100 mum and a wall thickness of a few micrometers. The circular cross section of the capillary forms a ring resonator along which the light circulates in the form of the whispering gallery modes (WGMs). The WGM has an evanescent field extending into the capillary core and responds to the RI change due to the analyte conducted in the capillary, thus permitting label-free measurement. The resonating nature of the WGM enables repetitive light-analyte interaction, significantly enhancing the LCORR sensitivity. This LCORR architecture achieves dual use of the capillary as a sensor head and a CE fluidic channel, allowing for integrated, multiplexed, and noninvasive on-capillary detection at any location along the capillary. In this work, we used electro-osmotic flow and glycerol as a model system to demonstrate the fluid transport capability of the LCORRs. In addition, we performed flow speed measurement on the LCORR to demonstrate its flow analysis capability. Finally, using the LCORR's label-free sensing mechanism, we accurately deduced the analyte concentration in real time at a given point on the capillary. A sensitivity of 20 nm/RIU (refractive index units) was observed, leading to an RI detection limit of 10-6 RIU. The LCORR marries photonic technology with microfluidics and enables rapid on-capillary sample analysis and flow profile monitoring. The investigation in this regard will open a door to novel high-throughput CE devices and lab-on-a-chip sensors in the future.     

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.     

Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly(dimethylsiloxane) device

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 microm was determined to be the optimum decoupler size, with effective voltage isolation lasting for approximately 6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 microm was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/microM, r(2) = 0.9996) and epinephrine (2.1 microM, 2.6 pA/microM, r(2) = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode.     

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.     

Experimental characterization of end-column electrochemical detection in conjunction with nonaqueous capillary electrophoresis

An end-column electrochemical detector arrangement for capillary electrophoresis (CE) based on a 75-microm-i.d. capillary and a 25-microm microdisk electrode is characterized. The investigations were carried out using a nonaqueous (acetonitrile-based) buffer and ferrocene model compounds which offer high reliability for voltammetric measurements. The positioning of the microdisk electrode relative to the capillary outlet is the most important parameter for optimization of detection performance as it determines the characteristics of mass transport toward the electrode and the effect of ohmic potential drop resulting from the electrophoretic current on the actual detection potential. On the basis of spatially resolved studies, it was concluded that for the detection system used the microdisk electrode should be placed in a central position relative to the capillary outlet at a distance within the range of 75-100 microm. The presence of a high-voltage electric field had no negative effect on baseline noise, which was demonstrated by comparison of capillary flow injection based on gravity flow and CE experiments. Even a faster stabilization of the baseline was observed by increasing the separation voltage.     

Separation and determination of polycyclic aromatic hydrocarbons by solid phase microextraction/cyclodextrin-modified capillary electrophoresis

A sensitive method for the determination of polycyclic aromatic hydrocarbons (PAHs) by solid phase microextraction coupled with cyclodextrin (CD)-modified capillary electrophoresis (CE) using UV detection has been developed. A glass fiber was prepared and used for absorbing 16 EPA priority PAHs from diluted samples until equilibrium was reached. After the glass fiber was connected to a separation capillary via an adapter, the absorbed analytes were directly released into the CE buffer stream, and electrophoretic separation was effected using a 50 mM borate, pH 9.2, buffer containing 35 mM sulfobutyloxy-β-CD, 10 mM methyl-β-CD, and 4 mM α-CD. Separation was effected since neutral PAHs differentially partitioned between the neutral and charged CD phases. Under 30 kV applied potential, separation was achieved in less than 15 min with high resolution and number of theoretical plates. Pyrene as low as 8 ppb was detected, while the highest limit of detection was 75 ppb for acenaphthene. Very satisfactory reproducibility with respect to migration time and peak area was obtained for repetitions using the same separation capillary and adapter, where only the extraction fiber was discarded after each analysis.