Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: A review

Analysis and Detection by Capillary Electrophoresis

On-the-fly fluorescence lifetime detection (OFLD) in capillary electrophoresis (CE) was previously demonstrated using a commercial multiharmonic Fourier transform (MHF) spectrofluorometer interfaced to a commercial CE system. This paper discusses optimization of the interface design for minimization of background fluorescence and scattered light, thereby maximizing the signal-to-background ratio (S/B) of the dynamic measurement. Strategies included using various combinations of optical filters including a holographic filter and longpass or bandpass filters, tilting the capillary relative to the incident laser beam, employing a confocal design and adding an iris to remove out-of-focus light, using a microscope objective in the emission beam to increase the collection efficiency, and using square instead of ciruclar capillary columns. Significant improvements in S/B for on-column, on-the-fly detection of fluorescein in CE were achieved with most modifications.

Conductivity detection in capillary electrophoresis

The microwave hydrolysis and derivatization coupled with capillary electrophoresis detection were developed for the separation and determination of the amino acids in Panax notoginseng. The experimental conditions for the microwave hydrolysis and derivatization were examined and optimized. Several parameters of capillary electrophoresis, such as pH value of background electrolyte, borate concentration and applied voltage were optimized. Under the selected conditions, 11 amino acids were completely separated. The real sample was analyzed and the results were satisfactory. Compared with that of conventional heat hydrolysis and derivatization, the analytical time of this method was significantly shortened.

Macrocyclic antibiotics as effective chiral selectors for enantiomeric resolution by liquid chromatography and capillary electrophoresis

The influence of the separation voltage on end column electrochemical detection (EC) in capillary electrophoresis (CE) has been investigated using an electrochemical detector chip based on an array of microband electrodes. It is shown, both theoretically and experimentally, that the effect of the CE electric field on the detection can be practically eliminated, without using a decoupler, by positioning the reference electrode sufficiently close to the working electrode. In the present study, this was demonstrated by using an experimental setup in which neighboring microband electrodes on a chip, positioned 30 microns from the end of the CE capillary, were used as working and reference electrodes, respectively. The short distance (i.e., 10 microns) between the working and reference electrode ensured that both of the electrodes were very similarly affected by the presence of the CE electric field. With this experimental setup, no significant influence of the CE voltage on the peak potentials for gold oxide reduction could be seen for CE voltages up to +30 kV. The detector noise level was also found to be reduced.

Post-column reactor of coaxial-gap mode for laser-induced fluorescence detection in capillary electrophoresis

This article describes the application of capillary electrophoresis (CE) to the analysis of compounds relevant to the Chemical Weapons Convention (CWC). CE is an analytical technique that employs narrow‐bore, fused‐silica capillaries to perform high‐efficiency separations of analytes based on their mobilities in an electric field. In CE, analytes are dissolved in a buffer solution and placed in a capillary to which an electric field is applied. The analytes then migrate at a rate determined by their charge and size and are detected as they migrate past a detector. CE can analyze a broad range of compounds and is particularly applicable to the analysis of water‐soluble degradation products of scheduled compounds under the CWC. Compounds typically analyzed by CE include the hydrolysis products of nerve agents such as the alkylphosphonates and hydrolytic or oxidative products of the sulfur mustards such as thiodiglycol. The alkyphosphonates require the use of indirect ultraviolet (UV) detection or other detection methods as they do not possess a suitable UV chromophore. Degradation products of the sulfur mustards have been analyzed by micellar electrokinetic chromatography (MEKC) and direct UV detection. The ability of CE to analyze anionic, cationic, zwitterionic, and neutral CWC‐related compounds, whether they be chemical warfare (CW) degradation products or scheduled starting materials, without the requirement for elaborate sample processing or labeling procedures, is an important demonstration of the utility of CE for the analysis of CWC‐related compounds. CE can be an important screening method for rapid sample processing and further analysis for unambiguous identification. The relative ease of analysis should also increase its utility in field and on‐site analysis where minimizing the logistic burden is attractive.

This article describes the application of capillary electrophoresis (CE) to the analysis of compounds relevant to the Chemical Weapons Convention (CWC). CE is an analytical technique that employs narrow‐bore, fused silica capillaries to perform high‐efficiency separations of analytes based on their mobilities in an electric field. In CE, analytes are dissolved in a buffer solution and placed in a capillary to which an electric field is applied. The analytes then migrate at a rate determined by their charge and size and are detected as they migrate past a detector. CE can analyze a broad range of compounds and is particularly applicable to the analysis of water‐soluble degradation products of scheduled compounds under the CWC. Compounds typically analyzed by CE include the hydrolysis products of nerve agents such as the alkylphosphonates and hydrolytic or oxidative products of the sulfur mustards such as thiodiglycol (TDG). The alkylphosphonates require the use of indirect ultraviolet (UV) detection or other detection methods as they do not possess a suitable UV chromophore. Degradation products of the sulfur mustards have been analyzed by micellar electrokinetic chromatography (MEKC) and direct UV detection. The ability of CE to analyze anionic, cationic, zwitterionic, and neutral CWC‐related compounds, whether they be chemical warfare (CW) degradation products or scheduled starting materials, without the requirement for elaborate sample processing or labeling procedures, is an important demonstration of the utility of CE for the analysis of CWC‐related compounds. CE can be an important screening method for rapid sample processing, and capillary electrophoresis coupled with mass spectrometry (CE‐MS) can provide further analysis for unambiguous identification.

Construction details and performance characteristics of an open-gap flow cell for fluorescence detection in capillary electrophoresis are described. The flow cell is created by separating two pieces of capillary by a small (90 μm) gap. The gap is surrounded with buffer and grounded, and the application of electric fields to both inlet and outlet capillaries causes the material in the inlet capillary to flow across the gap. The use of a simple confocal optical arrangement for laser-induced fluorescence detection allows straightforward application of the gap flow cell to detection in capillary electrophoresis. The signal-to-noise ratio is measured to be about a factor of 2 better than that for on-column confocal detection at nanomolar concentrations over a wide range of pinhole diameters. The detection limit for fluorescein isothiocyanate is in the low picomolar range. Detection of a simple mixture of amino acids that have been derivatized with fluorescein isothiocyan ate demonstrates the stability and utility of the gap. Increased tailing is observed with the gap cell, with average asymmetry of about 1.4 near the center of the gap. Dispersion characteristics as a function of position in the gap are interpreted as dilution of the analyte as it flows across the gap. Fortunately, resolution and theoretical plates, measured by using least-squares fitting, are not significantly different from on-column separations, in spite of the tailing.

Optimization of indirect ultraviolet detection in high-performance liquid chromatography and capillary electrophoresis

The construction, performance, and practical implementation of a rugged device for laser induced fluorescence (LIF) detection in capillary electrophoresis (CE) and liquid chromatography (LC) techniques is described. This device is based on a sheath flow cell (SFC) arrangement with attributes that include ruggedness, simplicity, ease of use, low cost, intra-day reproducibility, and small size. With this design, a pre-imaging mode of spatial filtering is employed to effectively reduce the contributions of both wall scatter and wall fluorescence to the background signal levels. A comparison of the pre-imaging spatial filtering and traditional methods of far-field spatial filtering is performed. This SFC system has been designed specifically for use in the UV spectral region, which can be very advantageous since it broadens the scope of the possible applications of LIF; however, fluorescence of the flow cell walls is particularly problematic in the UV region. The effect of flow cell geometry, slit widths, and detector position on fluorescent background levels are shown. The device has been applied to the fluorescence detection of three dansyl amino acids for both CE and capillary LC techniques. Efficiencies on the order of >105 plates/meter and 104 plates/meter are shown for CE and capillary LC techniques respectively. A comparison is made with CE between on-column and post-column detection demonstrating the latter preserves the integrity of a chromatographic separation.

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.

Chapter 4 Optical detection strategies in capillary electrophoresis

A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A approximately 50-microm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-microm inner diameter CE capillary window or in a flow cell consisting of a 250-microm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20x objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25-25 ppm concentrations, by using 1.4-3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.