Photothermal trace detection in capillary electrophoresis for biomedical diagnostics and toxic materials (invited)

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

Analysis and Detection by Capillary Electrophoresis

A photothermal lens method is used in combination with capillary electrophoresis for detecting samples of different drugs by electrokinetic chromatography. It is for the first time that drugs (Chloramphenicol, Dichlofenac, Pentoxifyllin, Oxprenolol) are determined with thermal lens spectroscopy. Direct and indirect separation techniques are applied to various classes of substances with different characteristic absorbance spectra. The combination of capillary electrophoresis and the high sensitivity of thermal lens spectroscopy allows the analysis of nanoliter volume samples occurring in biomedical diagnostics.

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.

It is shown that organo-aqueous separation buffers show much promise when used in capillary electrophoresis separations with photothermal (thermal lens) detection systems. Acetonitrile-water and methanol-water mixtures were selected, as conventionally used in capillary electrophoresis. It is shown that, despite more sophisticated experimental conditions (significant heat outflow from the capillary body) and peak detection, the theoretical ratio of the thermal lens signal for a binary mixture to the thermal lens signal for an aqueous solution (or the corresponding ratio obtained experimentally under bulk batch conditions) can be used to predict the sensitivity of thermal lens detection in capillary electrophoresis. The limits of detection for 2-, 3-, and 4-nitrophenols selected as model compounds in 70% v/v acetonitrile separation buffers are 1 x 10(-6) M, 1 x 10(-6) M and 3 x 10(-7) M, respectively, and are therefore decreased by a factor of six compared to thermal lens detection in aqueous separation buffers. The overall increase in the thermal lens detection sensitivity in a 100% ACN buffer is a factor of 13.

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.

This study presents a timely, reliable, and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of a single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of microliter sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 μL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 1.8 × 102, 1.7 × 103, and 1.0 × 103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83% and it was independent of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry required at least 4 × 103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2%. The adhered bacteria, either individual or in a mixture, were online analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.

Natural iron concentrations in real water samples have been determined by thermal lensing as a high performance capillary electrophoresis detector. 1,10-Phenanthroline was used as a chromogenic reagent, which forms a stable complex with Fe(II) but not with Fe (III). The interferences by other cations can be neglected by a combination of this photometric technique with high performance capillary electrophoresis. Laser induced thermal lens spectroscopy is demonstrated as a new technique for the measurement of small absorbances in the picoliter detection volume of a capillary. A limit of detection of 36 nmol/L with a signal to noise ratio of 7 : 1 was achieved using a 75 μm fused silica capillary.

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.

Aim: The aim of this work is to fabricate pipette tip electrodes for the capillary electrophoretic determination of nicotine and phenolic compounds in tobacco. Background: The content of nicotine affects not only the quality of tobacco products but also the health of smokers. Phenolic compounds are important flavor precursors in tobacco. The quantity of phenolic compounds is one of the most important evaluation indicators of tobacco quality. It is of high importance to determine nicotine and phenolic compounds in tobacco for quality control and the health of smokers. Objective: A method based on capillary electrophoresis and amperometric detection was developed for the simultaneous determination of nicotine, rutin, chlorogenic acid, quercetin, ferulic acid, gallic acid, and protocatechuic acid in tobacco leaves. Pipette electrodes were designed and fabricated for their amperometric detection. Method: Nicotine, rutin, chlorogenic acid, quercetin, ferulic acid, gallic acid, and protocatechuic acid were determined by capillary electrophoresis in combination with the detection electrodes that were fabricated by packing the composite of carbon nanotube and epoxy in pipette tips. Results: Detection potentials, the acidity and concentrations of background electrolyte, separation voltages, and injection times were optimized. At a high voltage of 12 kV, separation of the seven analytes could be achieved in less than 11 min in a piece of 40 cm long fused silica capillary with a background electrolyte of 50 mM borate buffer (9.2). Linearity was observed between the peak currents and the concentrations, with the limits of detection ranging from 0.1 to 0.2 μM for the seven analytes at the pipette electrodes. The method was applied in the simultaneous determination of nicotine and phenolic compounds with satisfactory assay results. Conclusions: The pipette tip electrodes were successfully coupled with capillary electrophoresis for tobacco analysis. The CE-AD method provides not only a simple approach for the quality control of tobacco and its preparations but also an alternative technique for the constituent and fingerprint investigation of other plants.