Non-aqueous capillary electrophoresis of p-quinone anion radicals.

The electrophoretic detection of two kinds of p-quinone anion radicals arising from the electrolysis of benzoquinone and chloranil was achieved by employing an acetonitrile medium. Sufficient dehydration of a running solution was necessary for the detection of the benzoquinone anion radical. Oxygen in the running solution also caused a serious decrease in the amount of the benzoquinone anion radical during electrophoresis. The addition of methanol as a hydrogen-donor decreased the electrophoretic mobility of the benzoquinone anion radical significantly, while that of the chloranil anion radical was little changed. This result is interpreted in terms of hydrogen-bonding interaction between the p-quinone anion radicals and methanol, reflecting the magnitude of their proton-accepting ability (benzoquinone anion radical > chloranil anion radical).     

Non-aqueous capillary electrophoresis of tamoxifen and its acid hydrolysis products

Tamoxifen and its acid hydrolysis products were separated and tentatively identified by non-aqueous capillary electrophoresis with thermooptical absorbance and electrospray ionization mass spectrometry. Acid hydrolysis is a convenient method of generating tamoxifen degradation products. The parent compound and seven hydrolysis products were separated in 9 min.     

Non-aqueous capillary electrophoresis of biological samples after at-line solid-phase extraction

Summary Solid-phase extraction (SPE) was coupled at-line to capillary electrophoresis (CE) for the determination of a series of basic test compounds (i. e. tricyclic antidepressants). The analysis was performed using a non-aqueous CE buffer, which resulted in baseline separation of all test compounds. This is in marked contrast with CE using aqueous buffers where hardly any separation was obtained either with or without micelles. The SPE procedure was used to remove simultaneously most of the water from the sample, because no direct analysis of aqueous samples is possible when a non-aqueous CE buffer is used. With the present method the antidepressants can be determined in both urine and serum. Analyte detectability is increased up to 10-fold due to trace enrichment during the extraction process; the limits of detection (LODs; UV 214 nm) are 30–300 ng mL⁻¹ in urine and 300–1000 ng mL⁻¹ in serum. TheRSD values (n=5) of the within-day and between-day precision are below 9% and 11% respectively. Therefore, the present procedure can be used for drug monitoring.     

Non-aqueous capillary electrophoresis of the positional isomers of a sulfated monosaccharide

A non-aqueous capillary electrophoresis (NACE) method coupled to indirect absorbance detection has been developed for the separation of the three positional isomers of monosulfated fucose. The optimized electrolyte was composed of 12 mM ethanolamine, 2 mM trimesic acid buffer in a methanol-ethanol (1:1, v/v) mixture. As the retained electrolyte entails no separating agent other than the pH buffer, the NACE separation of the positional isomers has been ascribed mainly to selective ion-pairing with the electrolyte counter-ion and the possibility of a selective solvation effect in the alcohol mixture. In the absence of pure isomeric standards, peak identification was completed by MS and NMR spectroscopy and selective enzymatic desulfation. This method should be of interest for the structure elucidation of monosulfated fucose-based polysaccharides and for the screening of sulfoesterase of unknown activity.     

Non-aqueous capillary electrophoresis separation of fullerenes and C60 fullerene derivatives

As the interest in the use of fullerene compounds in biomedical and cosmetic applications increases, so too does the need to develop methods for their determination and quantitation in such complex matrices. In this work, we studied the behavior of C60 and C70 fullerenes in non-aqueous capillary electrophoresis, as well as two C60 fullerene derivatives not previously reported by any electrophoretic method, N-methyl-fulleropyrrolidine and (1,2-methanofullerene C60)-61-carboxylic acid. The separation was performed using fused-silica capillaries with an I.D. of 50?μm and tetraalkylammonium salts, namely tetra-n-decylammonium bromide (200?mM) and tetraethylammonium bromide (40?mM), in a solvent mixture containing 6?% methanol and 10?% acetic acid in acetonitrile/chlorobenzene (1:1?v/v) as the background electrolyte. Detection limits, based on a signal-to-noise ratio of 3:1, were calculated, and values between 1 and 3.7?mg/L were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with relative standard deviation lower than 15?%. For the first time, an electrophoretic technique has been applied for the analysis of C60 fullerene in a commercial cosmetic cream. A standard addition method was used for quantitation, and the result was compared with that obtained by analyzing the same cream by liquid chromatography coupled to mass spectrometry.     

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.     

Non-aqueous capillary electrophoresis with red light emitting diode absorbance detection for the analysis of basic dyes

Non-aqueous capillary electrophoresis was evaluated for the separation of five hydrophobic basic blue dyes for application in forensic dye analysis. The use of a red light emitting diode as a high intensity, low-noise light source provided sensitive detection of the blue dyes while also allowing the evaluation of solvents that absorb strongly in the UV region. Excellent peak shapes and separation selectivity were obtained in methanol, ethanol, acetonitrile and dimethylsulfoxide, however water, tetrahydrofuran, dimethylformamide and acetone were unsuitable as solvents due to poor peak shapes and a lack of sensitivity, most likely due to adsorption onto the capillary wall. Due to the known compatibility of methanol with capillary electrophoresis–mass spectrometry, this solvent was examined further with the relative acidity/basicity of the electrolyte being optimised with an artificial neural network. The optimised method was examined for the separation of ink samples from 6 fibre tip and 2 ball point blue or black pens and showed that a unique migration time for the main dye component in seven of the eight pens could be obtained.     

Non-aqueous capillary electrophoresis for simultaneous separation and determination of three major active components in traditional medicinal preparations

A simple and sensitive non-aqueous capillary electrophoresis method has been developed for simultaneous assay of three bioactive components (puerarin, daidzein and wogonin) in three traditional medicinal preparations for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. capillary using a non-aqueous buffer system of 20% acetonitrile, 25 mm ammonium acetate and apparent pH 9.00, with applied voltage and capillary temperature of 20 kV and 16 degrees C, respectively. The relative standard deviations (RSDs) of the migration times and the peak areas of the three analytes were in the ranges 2.5--4.0% and 3.2--3.9%, respectively. Detection limits of puerarin, daidzein and wogonin were 0.090, 0.145 and 0.090 microg mL(-1), respectively. In the tested concentration range, good linear relationships (correlation coef fi cients: 0.9998 for puerarin, 0.9998 for daidzein and 0.9978 for wogonin) between peak areas and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with recoveries from 91.0 to 114.0%.     

Non-aqueous capillary zone electrophoresis method for the analysis of paroxetine, tamoxifen, and their main metabolites in urine

The potential of non-aqueous capillary electrophoresis (NACE) was investigated for the simultaneous separation of paroxetine, tamoxifen, and their main metabolites. Baseline separation of the studied solutes was obtained on a  μm capillary using a non-aqueous buffer composed of 18 mM ammonium acetate and 1.1% acetic acid in 80:20 (v/v) methanol-acetonitrile, with a temperature and voltage of 22 °C and 15 kV, respectively. Clomipramine was used as internal standard. Aspects such as stability of the solutions, linearity, accuracy, precision and ruggedness were examined in order to validate the proposed method. Detection limits obtained for all the studied compounds ranged between 3.0 and 7.1 μg l⁻¹. The developed method is sensitive and robust and was used to determine paroxetine, tamoxifen, and their metabolites at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of an extraction-pre-concentration step with a pre-conditioned C₁₈ cartridge. Determination of these analytes in the urine of four females urines was demonstrated.     

Cyclic capillary electrophoresis

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages. An enhancement-stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement-stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero-integrated. The ratchet-generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion-induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement-stages are critically dependent on the electrical field-dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer-controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet-generating pulses. These other pulses can be either the previously used, shorter field-inversion pulses or high-frequency periodic oscillations previously found to sharpen peaks.     

Ligand-exchange capillary electrophoresis

The fundamentals of ligand-exchange capillary electrophoresis are discussed, and the potential of the method in solving major problems, such as the separation of enantiomers and the determination of biologically active compounds poorly absorbing in the UV region (sugars, amines, and amino acids) is considered.     

Forensic capillary electrophoresis

Capillary electrophoresis (high-performance electrophoresis) has recently become established as a versatile and powerful separation technique. It has a broad applicability and can be used with a variety of detection systems. An introduction is given to this analytical technique, then some applications to forensic problems are discussed.     

Carbon nanotubes in capillary electrophoresis, capillary electrochromatography and microchip electrophoresis

Carbon nanotubes are among the plethora of novel nanostructures developed since the 1980s. Nanotubes have attracted considerable interest by the scientific community thanks to their extraordinary physical and chemical properties. Research areas have flourished in recent years and now include the nano-electronic, (bio)sensor and analytical field along with many others. This review covers applications of carbon nanotubes in capillary electrophoresis, capillary electrochromatography and microchip electrophoresis. First, carbon nanotubes and a range of electrophoretic techniques are briefly introduced and key references are mentioned. Next, a comprehensive survey of achievements in the field is presented and critically assessed. The merits and downsides of carbon nanotube addition to the various capillary electrophoretic modes are addressed. The different schemes for fabricating electrochromatographic stationary phases based on carbon nanotubes are discussed. Finally, some future perspectives are offered.      

Non-equilibrium capillary electrophoresis of equilibrium mixtures--appreciation of kinetics in capillary electrophoresis

We describe a new electrophoretic method (patent pending), Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and demonstrate its application to the study of protein-DNA interactions. A single NECEEM experiment allows for the determination of equilibrium and kinetic parameters of protein-DNA complex formation. The equilibrium mixture is prepared by mixing protein and DNA; it contains three components: free protein, free DNA, and the protein-DNA complex. A small plug of such a mixture is injected onto a capillary and the three components are separated under non-equilibrium conditions using a run buffer that does not contain the components of the equilibrium mixture. The protein-DNA complex decays during the NECEEM separation; the resulting electropherograms contain characteristic peaks and exponential curves. A simple analysis of a single electropherogram reveals two parameters: the equilibrium dissociation constant of the protein-DNA complex and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the ratio of the two experimentally-determined constants. NECEEM was applied to find the equilibrium and kinetic parameters of interaction between an E. coli single-stranded DNA binding protein and a fluorescently-labeled oligonucleotide. The constants determined by NECEEM are in good agreement with those obtained by other methods. The new method is simple, fast, and accurate. It can be equally applied to other non-covalent molecular complexes.     

Nonaqueous capillary electrophoresis using a titania-coated capillary

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.     

Non-aqueous capillary electrophoresis for separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its medicinal preparations

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five samples.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

Whole-column fluorescence-imaged capillary electrophoresis

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.     

Detection in capillary electrophoresis

A review of the four major, on-line, capillary electrophoresis (CE) detection modalities is presented. It is shown that each detection method, fluorescence, absorbance (conventional and nonconventional), electrochemical and refractive index, have distinct advantages and limitations when applied to analysis in a CE format. Various aspects of CE detection are considered and a perspective regarding the applicability of the technique is provided. It is shown that because of widely varying detection limits (ranging from single molecule to 10(-5) M) and detection scheme complexity, the particular application should dictate the selection of detection methodology in CE.     

Haplotyping by capillary electrophoresis

The investigation of the genetic background and phenotype structures of complex diseases, such as cardiovascular or psychiatric disorders and tumors, is one of the most scrutinized fields of the post genomic era. Besides the multiplex analysis of genetic markers and polymorphisms throughout the whole genome, more and more attention is focused on the interaction between the etiological factors of these traits. Haplotype determination, rather than multiplex genotyping seems to be one of the first building blocks of this endeavor. This review focuses on the importance and theoretical background of haplotyping, and summarizes the recent examples of novel and emerging haplotyping techniques by capillary gel electrophoresis based DNA fragment analysis, a powerful tool for the examination of the inheritance of complex traits.