Sensitivity improvement in capillary electrophoresis using organo-aqueous separation buffers and thermal lens detection

Analysis and Detection by Capillary Electrophoresis

Two applications of a near-field thermal lens capillary electrophoresis detector in the deep ultraviolet region (pump beam 257 nm wavelength) will be presented: (1) Capillary electrophoretic determination of the pharmaceuticals Tramadol, Verapamil, and Papaverin. Direct separation techniques were used for the different classes of substances with characteristic absorbance spectra. The combination of capillary electrophoresis and the highly sensitive detection with thermal lens spectroscopy permits the analysis of nanoliter volume samples common in biomedical diagnostics without any preconcentration step. (2) The determination of (nonfluorescent) nitro aromatic explosives in contaminated soil. These compounds are detected with the laboratory built thermal lens detector after their separation by micellar electrokinetic chromatography. Its shown that this type of detection makes it possible to obtain limits of detection 1–2 orders of magnitude lower than those obtained with classical absorption spectrometric detection.

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

Conductivity detection in 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.

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.

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

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.

We report the first successful implementation of a multiplexing method on a standard capillary electrophoresis system with UV detection that is independent of additional hardware. This was achieved using the Hadamard transform approach and employing vial exchange and voltage suspensions for translation of pseudorandom binary sequence elements into sample and background electrolyte injections of a capillary zone electrophoresis separation. Sequences exceeding peak capacity of the capillary were subdivided into shorter subsequences measured successively and realigned afterward based on EOF marker or analyte peaks. This way, we realized and deconvoluted modulation sequences as long as 8-bit (255 injections) for two systems containing either AMP or a mixture of the nucleotides (A,C,G,U)MP resulting in electropherograms of considerably improved signal-to-noise ratio. We achieved factors of intensity enhancement of around 6.9 and 5.2, respectively (theoretical maximum 8.0). This contribution, further, presents experimental and simulation studies on the effects on zones during injection and separation when experiencing voltage suspensions. Besides analysis of EOF behavior and influence of diffusion dispersion, we also provide data on the significance of specific electrophoretic errors such as peak position shift, inconsistent sample injection, and peak broadening on the quality of the inverse Hadamard transform. Moreover, the application of our approach to the practical analysis of a milk sample is described. The results demonstrate the applicability of multiplexing on unmodified standard CE instrumentation and establish a new suitable methodology to enhance the low sensitivity of on-column UV detection 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.

On-the-fly fluorescence lifetime detection in capillary electrophoresis (CE) is demonstrated. Virtually continuous detection is achieved by interfacing a commercial CE instrument with a commercial, multiharmonic Fourier transform lifetime fluorometer (MHF). The CE capillary cartridge was modified to allow the capillary to pass through a specially constructed capillary column mount capable of micropositioning in the MHF sample chamber. Optimization of the CE/MHF interface was achieved through the incorporation of a focusing lens, appropriate alignment of the laser beam on the capillary, and use of appropriate optical filters in the emission beam. Both fluorescence intensity and lifetime are recovered from the dynamic MHF data, which is analyzed using either conventional nonlinear least-squares or the maximum entropy method, which allows for lifetime recovery without a priori knowledge of the system. Continuous, on-the-fly fluorescence lifetime detection using the MHF technology adds the dimension of fluorescence lifetime without sacrificing the resolution and speed of CE. Its application to the CE separation of a mixture of the fluorescent dyes NBD-hexanoic acid and fluorescein is demonstrated.

A new approach for dual electrode electrochemical detection in capillary electrophoresis (CEEC) is described. In this approach, two identical capillaries, each containing an on-capillary electrode incorporated permanently onto its tip, were paired together for simultaneous sample injection and detection. This procedure permitted dual-parallel detection to be performed without the need for painstaking alignment of the electrodes with respect to one another and to the capillary outlet as is required for the off-capillary microelectrode systems usually employed in CEEC. As a result, independent detection at two electrodes held at different potentials or at two electrodes of different composition or structure could be performed simply and with wide flexibility. Fabrication of on-capillary electrodes was carried out by sputter-coating the exit end of the capillaries with a thin layer of Au or Pt. Dual electrode system performance was demonstrated by separation and analysis of phenol and catechol samples. In addition, the detection system was coupled with glucose oxidase for the selective detection of glucose.

A model which takes into account both stray light and polychromatic light was used to predict and evaluate linearity in on-capillary detection in capillary electrophoresis (CE). According to the model the stray light is the major factor which determines linearity under typical CE operating conditions. By calculating theoretical absorbance versus concentration plots, the influence of different levels of stray light and polychromatic light on linearity is demonstrated. Experimentally, six light-emitting diodes (LEDs) in the range from 563 to 654 nm were examined as light sources for on-capillary detection in CE. Fitting theoretical curves to measured linearity plots enabled determination of the values of both effective path length and stray light for a particular detection system. The detector linearity for the four LEDs was compared to mercury and tungsten lamps used with interference filters. For potassium permanganate as the test compound, the linear range for a 563 nm LED was two times greater than that for a mercury lamp operated at 546 nm. The relatively poor linearity of the mercury lamp detector is explained by its high level of stray light. The noise of the LED563-based detector was the same as for the mercury lamp, whereas the other LEDs of higher light intensity gave approximately half the noise of the mercury lamp. The lowest noise level of 3 x 10(-5) AU was obtained for the LED at 554 nm (determined at a detector time constant of 0.1 s).

Simultaneous laser-induced fluorescence, coaxial thermal lens spectroscopy and retro-reflected beam interference detection for capillary electrophoresis