Miniaturized capillary electrophoresis system with ultraviolet photometric detection combined with flow injection sample introduction using a modified falling-drop interface

A miniaturized capillary electrophoresis (CE) system with UV-Vis detection was coupled to a flow injection (FI) system for achieving high throughput continuous sample introduction. The cassette of a commercial CE instrument was modified to hold a 6.5 cm long silica capillary and a flow-through waste reservoir. The cassette was inserted into the flow-cell chamber of a commercial UV detector, with the light beam focused on the capillary and collected by two ball lenses on the cassette. The capillary inlet, left outside the cassette and detector, was positioned on the top of a vertical 3.5 mm diameter glass rod, in close contact with an electrode. Samples injected through the FI system dropped freely on top of the pillar, covering the capillary inlet and electrode. Continuous sample introduction was achieved for CE separations under non-interrupted separation voltage, which was isolated from the FI system through the discontinuity of droplets. The newly developed interface and UV detection system was used for fast separation of sulphamethoxazole (SMZ) and trimethoprim (TMP) in sulphatrim tablets, achieving a high throughput of over 48 h⁻¹, and a low carryover of 2%. Separation efficiencies of 8 μm plate height and detection limits of 1.0 mg l⁻¹ for SMZ and 0.5 mg l⁻¹ (3σ) for TMP were obtained.     

Lamp-based native fluorescence detection of proteins in capillary electrophoresis

The potential of a recently developed lamp-based fluorescence detector for the analysis of underivatised proteins by capillary electrophoresis (CE) was investigated. Fluorescence detection (Flu) was achieved using optical light guides to deliver excitation light from a Xenon–Mercury lamp to the capillary detection window and to collect fluorescence emission and lead it to a photomultiplier. The performance of the detector was evaluated by monitoring the native fluorescence of the amino acid tryptophan and the proteins α-chymotrypsinogen A, carbonic anhydrase II, lysozyme and trypsinogen upon excitation at 280 nm. The test compounds were analysed using background electrolytes (BGEs) of sodium phosphate at pH 3.0 and 11.3. The results were compared to experiments of CE with UV absorbance detection. For tryptophan, a linear fluorescence response was obtained with a dynamic range of over 4 orders of magnitude, and a limit of detection (LOD) of 6.7 nM. This LOD was a factor of 200 more favourable than UV detection at 280 nm, and a factor of 20 better than detection at low-UV wavelengths. All tested proteins showed linear fluorescence responses up to 250 μg/mL. LODs were typically in the 10–20 nM range. These LODs were a factor of 25 lower than for UV detection at 280 nm, and comparable to UV detection at low-UV wavelengths. Overall, Flu yields much more stable baselines, especially with a BGE of high pH. The applicability of CE–Flu is demonstrated by the analysis of a degraded protein mixture, and of an expired formulation of the protein drug human growth hormone, indicating that protein degradation products can be selectively detected.     

Determination of mefenacet by capillary electrophoresis with electrochemiluminescence detection

Electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) separation system was used to the rapid analysis of mefenacet within 7 min. The linear response range of mefenacet was from 1.07 × 10⁻⁸ to 5.0 × 10⁻⁷ M with a detection limit of 4.0 × 10⁻⁹ M. This technique was also applied to analyze residues of mefenacet in seedling and soil.     

An on-column fracture/end-column reaction interface for chemiluminescence detection in capillary electrophoresis

Chemiluminescence (CL) offers a sensitive detection method for capillary electrophoresis (CE), but the implementation of CE–CL is usually under compromised operating conditions for CE, such as the prerequisite of extreme pH buffer for optimal CL reaction at the capillary outlet. This has sometimes significantly deteriorated the separation of CE. In this study, the development of a new interface makes it possible to optimize the operating conditions for CE separation and CL detection independently. The interface consists of an on-column fracture being installed in a reservoir near the capillary end to create an electrical connection and also serve as reagent addition entrance. The capillary terminal is inserted into an end-column reservoir for CL reaction and detection. In this arrangement, the applied electric field has been decoupled from the CL detection, which is proved to effectively improve CE's performance by allowing the use of optimal CE buffers. At the same time, it enables the optimization of CL detection independently. The applicability of this interface was evaluated by using acridinium ester (AE) and luminol systems. For AE system, the interfering products of CL reagent (⁻OH, HO₂⁻) have been prevented, and the pH range of CE buffer can be independent to the optimal pH value of AE CL reaction, which is usually below 3. The AE was detected using running buffer at pH 8.7, giving a detection limit of 0.1 nM (S/N = 3), and the theoretical plate numbers is as high as 56 000. The on-column fracture based configuration is simple, sensitive and easy to implement.     

Ultraviolet absorbance detection of colchicine and related alkaloids on a capillary electrophoresis microchip

The separation and UV absorbance detection of four toxic alkaloids, colchicine, thiocolchicine, colchicoside, and thiocolchicoside, on a microchip-based capillary electrophoresis device are reported. To increase the sensitivity of UV absorbance detection, optical cells with extended path lengths were integrated into the separation channel during the microfabrication process. The absorbance values realized on the microchip using these optical cells were proportional to the increase in average depths according to the Beer-Lambert Law, resulting in sensitivity enhancements by as much as five times. Linearity of response was observed from 5.0 to 500 mg L⁻¹ of colchicine, with detection limits ranging from 2 to 6 mg L⁻¹ depending upon the specific alkaloid and the dimension of the optical cell. The extraction of colchicine from spiked milk samples was performed and an average recovery rate of 83% with a relative standard deviation of 3.8% was determined using the optimized conditions on the microchip.     

Separation and determination of aloperine, sophoridine, matrine and oxymatrine by combination of flow injection with microfluidic capillary electrophoresis

A novel, rapid and accurate method for the separation and determination of aloperine (ALP), sophoridine (SRI), matrine (MT) and oxymatrine (OMT) has been developed by combination of flow injection (FI) with microfluidic capillary electrophoresis (CE) for the first time. In the present paper, a continuous sample introduction interface was described. The interface with an H-channel structure was produced using a non-lithographic approach. The H-channel structure was fixed on a planar plastic base utilizing a horizontal 6.5 cm-long separation capillary with two vertical sidearm tubes on each end that served as inlet and outlet flow-through electrode reservoirs. The inlet reservoir also functioned as interface for coupling to the FI system. The buffer solution used was a 50 mmol l⁻¹ borate solution with the pH adjusted to 8.80 with 2 mol l⁻¹ HCl. The performance of the system was demonstrated in the separation and determination of ALP, SRI, MT and OMT with UV detection at 215 nm, achieving baseline separation within 2 min. A series of samples was injected repeatedly without current interruption and subsequent rinsing, and the contents of these four bio-alkaloids in two marketed drugs were determined with satisfactory recovery by this proposed method.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: Application to labetalol hydrochloride

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C⁴D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol⁺) and its anionic counter-ion (Cl⁻) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software.This novel CE-C⁴D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time < 10 min), precise (repeatability of migration times < 0.7% and of corrected-peak areas < 3.3%; n = 6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.     

Highly sensitive fluorescence detection with Hg-lamp and photon counter in microchip capillary electrophoresis

A microchip capillary electrophoresis system with highly sensitive fluorescence detection is reported. The system was successfully constructed using an inverted fluorescence microscope, a highly sensitive photon counter, a photomultiplier tube (PMT) and a capillary electrophoresis microchip. This system can be applied to the fluorescence detection with various wavelengths (300-600 nm). Different fluorescence reagents require different excitation wavelengths. The wavelengths of UV light (300-385 nm), blue light (450-480 nm) and green light (530-550 nm) are employed to excite Titan yellow, fluorescence-5-isothiocyanate (FITC) and Rhodamine 6G, respectively. The detection limit (S/N = 3) of FITC is 7 × 10⁻¹⁰ M, which is 2-3 orders of magnitude lower than that obtained with the lamp-based fluorescence and PMT detection system and approaches the data gained by the laser-induced fluorescence detection. The linear relationship is excellent within the range of concentration 1.3 × 10⁻⁹ to 6.5 × 10⁻⁸ M FITC. It offers a new method to widen the application of the lamp-based fluorescence detection.     

Determination of purines and pyrimidines in beer samples by capillary zone electrophoresis

Ten purine and pyrimidine bases were separated using capillary zone electrophoresis (CZE) with direct UV detection at 254 nm as well as mass spectrometric (MS) detection using an electrospray ionization (ESI) interface. For this purpose a carrier electrolyte composition compatible with both methods of detection containing 300 mM diethylamine (DEA) was selected. Limits of detection were in the range between 0.1 and 0.3 mg l⁻¹ and calibration plots were found to be linear over at least two orders of magnitude. The applicability of the developed method for the analysis of real samples was demonstrated for some beer samples. A series of “Lager” beer samples from different breweries in Europe as well as a number of completely different types of beers were investigated with respect to their content in the selected purine and pyrimidine bases using the developed CE method with UV detection at 254 nm.     

Cold vapor generation interface for mercury speciation coupling capillary electrophoresis with electrothermal quartz tube furnace atomic absorption spectrometry: Determination of mercury and methylmercury

A novel on-line coupled capillary electrophoresis (CE) cold vapor generation (CVG) with electrothermal quartz tube furnace atomic absorption spectrometry (EQTF-AAS) system for mercury speciation has been developed. The mercury species (inorganic mercury and methylmercury) were completely separated by CE in a 80 cm length × 100 μm i.d. fused-silica capillary at 20 kV and using a buffer of 100 mM boric acid and 10% (v/v) methanol (pH 8.30). The effects of the inner diameter of quartz tube, the acidity of HCl, the NaBH₄ concentration and N₂ flow rate on Hg signal intensity were investigated. Speciation of mercury was highlighted using CE-CVG-EQTF-AAS. The detection limits of methylmercury and mercury were 0.035 and 0.027 μg mL⁻¹, respectively. The precisions (RSDs) of peak height for six replicate injections of a mixture of 10 μg mL⁻¹ (as Hg) were better than 4%. The interface was used for speciation analysis of mercury in dry goldfish muscle.     

Voltammetric determination of boron by using Alizarin Red S

Cordycepin is the main active metabolite of Cordyceps militaris extracts; according to recent studies it has interesting therapeutic activities. A new capillary electrophoresis (CE) procedure with UV detection at 254 nm for determination of cordycepin was developed and optimized. Optimal conditions found were 20 mM sodium borate buffer with 28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 s and temperature 25 °C. Linearity was found over the 20-100 μg/mL concentration ranges of cordycepin. The developed method has been applied for determination of cordycepin in various pharmaceutical products. A comparison was made between CE and a high performance liquid chromatography (HPLC) method. Both of these methods gave comparable results. The shorter analysis time and low running cost are the main advantages of CE method.     

Enhancement of signal-to-noise level by synchronized dual wavelength modulation for light emitting diode fluorimetry in a liquid-core-waveguide microfluidic capillary electrophoresis system

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180°-phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N = 3) of 10 nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

Determination of terbinafine in pharmaceuticals and dialyzates by capillary electrophoresis

A capillary electrophoresis method has been developed for the separation and determination of terbinafine (TER) in various pharmaceutically relevant matrices. Capillary zone electrophoresis (CZE) separation and UV absorbance photometric detection were carried out in a 160 mm capillary tube with a 300 μm i.d., hydrodynamically (membrane) closed. The influences of pH, carrier cation and counterion on migration parameters of TER were studied and the following conditions were selected: a 20 mmol l⁻¹ glycine running buffer adjusted to pH 2.7 with acetic acid, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC) as an electro-osmotic flow (EOF) suppressor, a 250 μA driving current, and 20 °C. The optimized separation conditions were convenient for the determination of TER in commercial tablets and spray and in dialyzates. Here, the dialysis was used to investigate in vitro permeation of TER through the skin from the gel. The samples of dialyzates were examined with and without simple extraction procedure and the results were compared. A permeation profile of the drug present in the gel of given composition was obtained analyzing pretreated samples. The proposed electrophoretic method was successfully validated. It was suitable for the simple, sensitive, rapid and highly reproducible assay of TER. CZE analysis was completed within 5.5 min. The detection limit of TER was 1.73 μmol l⁻¹ at a 224 nm detection wavelength. The intra- and inter-laboratory precisions over the concentration range 6.0-60.0 μmol l⁻¹ were between 0.32-0.69% and 1.04-1.44% including R.S.D. of migration times and peak areas, respectively. The mean absolute recoveries of drugs from samples were found to be 98.34 (tablets) and 99.47% (spray). It is suggested that there are potentialities to determine TER present in unpretreated complex samples, as CZE in a hydrodynamically closed separation system may be easily on-line combinable with purification and preconcentration CE modes (e.g., isotachophoresis, ITP).     

Combination of flow injection with capillary electrophoresis: 8. Miniaturized capillary electrophoresis system with flow injection sample introduction and electrogenerated chemiluminescence detection

The combined flow injection (FI)-capillary electrophoresis (CE) system was further exploited by coupling to an electrogenerated chemiluminescence (ECL) detection system. A low-cost miniaturized CE system was developed on a chip platform to provide easy interface both with FI sample introduction and with ECL detection. A falling-drop interface was employed to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A plexiglas reservoir at the capillary outlet served as both the reaction and detection cell for the ECL reaction, with Ru(bpy)₃²⁺ reagent continuously flowing through the cell. An optical fiber was positioned within the reservoir close to the capillary outlet for transferring the ECL emission to the PMT. The relative positions of the capillary outlet, working electrode and optical fiber as well as reagent renewal flow-rate were optimized to achieve both good sensitivity and separation efficiency under non-interrupted sampling conditions, involving large numbers of samples. An on-column joint often used in other works for isolating the ECL detection system from the CE separation voltage was not found necessary. The performance of the system was illustrated by the baseline separation of proline, valine and phenylalanine with a high throughput of 50 h⁻¹ and plate height of 14 μm for proline under 147 V cm⁻¹ field strength. Detection limits (3σ) were 1.2, 50 and 25 μM and peak height precisions were 1.4, 5.4 and 4.3% R.S.D. (n=9) for proline, valine and phenylalanine, respectively.     

On-line capillary electrophoresis enrichment by combining chitosan trapping with surfactant assisted sample stacking for the ultratrace determination of organic acids in Plateau alfalfa roots

In this paper, four organic acids constituents of Plateau alfalfa roots have been identified and detected by a novel capillary electrophoresis (CE) strategy which combined chitosan (CS) trapping and cetyltrimethyl ammonium bromide (CTAB) assisted sample stacking. Under the optimized condition, organic acids, i.e., aconitic acid, gallic acid, citric acid and l-malic acid were concentrated and separated within 3 min. Validation parameters of this method (such as detection limits, linearity and precision) were also investigated and the limit of detection (LOD) was 2.41-53.9 ng mL⁻¹. Linearity was obtained over the magnitude range of 5-4000 ng mL⁻¹ approximately for different organic acids and 3 × 10²-1.5 × 10⁴ folds enrichment was achieved. The method has been applied to the determination of organic acids in roots of normal grown Plateau alfalfa and stressing affected Plateau alfalfa. Satisfactory results and recoveries were obtained in the analysis without costly and complicated sample pretreatment.     

Potential of porphyrins as chromogenic reagents for determining metals in capillary electrophoresis

Although capillary electrophoresis (CE) with photometric detection is a well-established technique for the determination of various inorganic ions, its limited sensitivity has hindered greater development in this area. In this work, we used a mixture of metals consisting of Co(II), Ni(II), Zn(II) and Mn(II) to demonstrate that the sensitivity of CE with ultraviolet–visible (UV–vis) detection can be improved by using chromogenic reagents such as porphyrins. To this end, the metals were reacted with 5,10,15,20-tetrakis(4-sulphophenyl)-porphine dodecahydrate (TPPS₄) to obtain their respective porphyrinato complexes, which were then separated by CE with a citrate buffer and detected at 410 nm. The ensuing electrophoretic method has a limit of detection (LOD) of 3 × 10⁻⁶ M (180 μg L⁻¹) for Co(II), 2 × 10⁻¹⁰ M (0.012 μg L⁻¹) for Ni(II), 4 × 10⁻⁶ M (260 μg L⁻¹) for Zn(II) and 4 × 10⁻⁹ M (0.219 μg L⁻¹) for Mn(II). The method is a highly promising choice for the ultratrace determination of Ni(II) and Mn(II).     

Ultrasensitive determination of cobalt in single hair by capillary electrophoresis using chemiluminescence detector

By use of the high separation capability of capillary electrophoresis (CE) and ultrasensitive chemiluminescence (CL) detection, a CE-CL method was proposed for the ultrasensitive determination of trace cobalt in a single hair, which is potentially useful in construction of the fingerprint of trace elements in the hair collected from crime scenes. In this work, the CE experimental conditions, CL experimental conditions and the digestion methods for the analysis of a single hair sample for cobalt were investigated in detail. The relative limit of detection (LOD, 3σ) was 0.01 ng/mL, and the absolute LOD was 2.4 × 10^− 16 g considering the sampling volume of 24 nL. Using a dry digestion method, the analytical results for certified reference hair samples by the proposed method were in good agreement with the certified values. Finally, this method was successfully used to detect trace cobalt in a single hair from three adults. It has potential applications in forensic analysis.     

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.