Heart-cutting two-dimensional capillary electrophoresis for the on-line purification and separation of derivatized amino acids

Heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was used for analysis of derivatized amino acids. A mixture of 12 amino acids derivatized with UV-active benzyl 4-(3-(2-chloroethyl)-3-nitrosoureido)butylcarbamate label served as a model of a moderately complex sample due to the presence of numerous derivatization byproducts. The first step of the heart-cutting 2D approach was sample cleanup by capillary zone electrophoresis (CZE) in borate electrolyte. Then, only a selected portion of the first-dimension separation was transferred into the second dimension of the separation by a specific voltage and pressure program. Finally, the zone of derivatized amino acids was separated by micellar electrokinetic chromatography in a borate-sodium dodecyl sulfate system. The whole 2D process can be performed in a conventional CE analyzer without any interface for connection of the two separation modes. Intraday repeatability of the total migration time was 2%. In general, the heart-cutting 2D-CE methodology in a single capillary can be adapted for any CE mode regardless of the direction and velocity of electroosmotic flow and position of the fraction of interest in the first dimension (i.e., first, last, or intermediate fraction).     

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190?000 theoretical plates are obtained for fluorescently labeled ovalbumin.     

Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

New avenue for mid-UV-range detection of underivatized carbohydrates and amino acids in capillary electrophoresis

Capillary electrophoresis (CE) appeared as an interesting alternative to chromatographic methods for carbohydrate analysis, but it can be difficult to implement, because of the lack of easily ionizable functions and chromophore groups. Recently, a promising method was proposed by Rovio et al. for the CE separation under extremely high alkaline conditions of neutral carbohydrates under their alcoholate form and their direct UV detection [Rovio et al. Electrophoresis 2007, 28, 3129-3135; and Rovio et al. J. Chromatogr. A 2008, 1185, 139-144], which is claimed to be due to the absorption of enediolate at 270 nm. Even so, most of the detected compounds in Rovio's paper (for example, sucrose) cannot give such enediolate, lacking a carbonyl group. In this work, a deeper insight was paid to the understanding of detection mechanism. In effect, unusual detection phenomena were observed in comparing reducing and nonreducing carbohydrate behaviors, which pointed to the existence of photochemical reactions in the detection window. A more systematic study of the influence of many parameters (carbohydrate nature, electrolyte pH, residence time in the detection window, and capillary diameter) was undertaken. In addition to this, most of this work was performed under cathodic (reversed) electro-osmotic flow conditions (using Polybrene-modified capillaries), to obtain much faster separations than under Rovio's conditions. This study also opens up new avenues for the detection in mid-UV range of non-UV-absorbing compounds bearing reducing moieties, such as amino acids.     

Batch-type chemiluminescence detection cell for sensitization and simplification of capillary electrophoresis

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10(-10)-6.5 x 10(-7) M (correlation coefficient, 0.999), with a detection limit (S/N = 3) of 2.5 x 10(-10) M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylgycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to postcolumn detection in capillary electrophoresis. When acetonitrile stacking was used under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about 1 order of magnitude higher than those obtained without stacking. The detection limit for luminol was 1.5 x 10(-11) M (S/N = 3), representing the highest sensitivity of luminol yet reported. Finally, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was approximately 2 orders of magnitude higher than that in the unenhanced reaction. A preliminary immunoassay using horseradish peroxidase-labeled anti-mouse IgG was also developed.     

Sialic Acid speciation using capillary electrophoresis: optimization of analyte derivatization and separation

Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was employed for rapid sialic acid speciation, facilitating the quantitative determination of N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) on glycoproteins. Derivatization of the sialic acids with 2-aminoacridone (2-AMAC), using classical reductive amination in a nonaqueous solvent, led to the spontaneous decarboxylation of the sialic acid residues as determined by CE-LIF and offline mass spectrometric analysis. Modification of both the labeling conditions, to drive the decarboxylation reaction to completion and the CE-LIF parameters to separate the neutral species by complexation with a neutral coated capillary and borate reversed polarity, led to a robust platform for the rapid, sensitive, and quantitative speciation of sialic acids. The method can readily be used for quality control of recombinant biopharmaceuticals.     

On-column coaxial flow chemiluminescence detection for underivatized amino acids by pressurized capillary electrochromatography using a monolithic column

A new method for pressurized capillary electrochromatography (pCEC) coupling with chemiluminescence (CL) detection using a modified on-column coaxial flow detection interface was developed. To evaluate the feasibility and reliability of the experimental setup, the typical CL compounds luminol and isoluminol were separated and detected by using this pCEC-CL system. A detailed investigation of CL detection interface and postcolumn CL reagent flow rate parameters was described. The excellent resolution and detection sensitivity was achieved by using 3-microm ODS-C18 packed column with 30% ACN (v/v), 5 mmol/L phosphate buffer (pH 8.0). Moreover, with the presence of Co(II) (1.0 x 10(-4) mol/L) in the mobile phase, the linear range of the concentration for luminol was 2.0 x 10(-9)-2.0 x 10(-6) mol/L with a detection limit (S/N = 3) of 2.0 x 10(-10) mol/L, and 2.5 x 10(4) theoretical plates was achieved. In addition, separation and detection of the underivatized amino acids (l-threonine and l-tyrosine) were accomplished by using a polymerized monolithic column based on the principle of the luminol-H2O2-Cu(II)-amino acid CL system. Under the optimum conditions, the mixture of amino acids was efficiently separated with satisfactory results.     

A fluorescence detection scheme for capillary electrophoresis of N-methylcarbamates with on-column thermal decomposition and derivatization

This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50,000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats.     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.     

Sensitive chemiluminescence immunoassay by capillary electrophoresis with gold nanoparticles

This technical note describes a new chemiluminescence immunoassay hyphenated to capillary electrophoresis (CE-based CL-IA) with gold nanoparticles (AuNPs) technique for biological molecules determination. AuNPs were used as a protein label reagent in the light of its excellent catalytic effect to the CL reaction of luminol and hydrogen peroxide. AuNPs conjugate with antibody (Ab) to form tagged antibody (Ab*), and then Ab* link to antigen (Ag) to produce an Ab*-Ag complex by a noncompetitive immunoreaction. The mixture of the excess Ab* and the Ab*-Ag complex was baseline separated and detected within 5 min under the optimized conditions. This new protocol was evaluated with human immunoglobulin G (IgG) as the target molecule. The calibration curve of IgG was in the range of 0.008-5 μg/mL with a correlation coefficient of 0.995. The detection limit (S/N = 3) of IgG was 1.14 × 10(-3) μg/mL (7.1 pmol/L, 0.39 amol). The proposed AuNPs enhanced CE-based CL-IA method was successfully applied for the quantification of IgG in human sera from patients. It proves that the present method could be developed into a new and sensitive biochemical analysis technique.     

High-voltage capacitively coupled contactless conductivity detection for microchip capillary electrophoresis

Contactless conductivity detection was carried out on a planar electrophoresis device by capacitive coupling using an ac excitation voltage of 500 V(p-p) and a frequency of 100 kHz. It was possible to carry out detection in this way through a cover plate of 1 mm thickness. Better sensitivity is obtained, however, by placing the electrodes into troughs that allow tighter coupling to the separation channel. The 3 x S/N detection limits are 0.49, 0.41, and 0.35 microM for the small inorganic ions K+, Na+, and Mg2+. The detection of heavy metals is demonstrated with the example of Mn2+, Zn2+, and Cr3+ with detection limits of 2.1, 2.8, and 6.8 microM, respectively. The universal nature of the method is further illustrated by the detection of citric and lactic acids, which are of interest in food and beverage analysis, and detection of three antiinflammatory nonsteroid drugs, 4-acetamidophenol, ibuprofen, and salicylic acid, as examples of species of pharmaceutical interest.     

Electrogenerated chemiluminescence of Au nanoclusters for the detection of dopamine

Electrogenerated chemiluminescence (ECL) emission was observed from the water-soluble, bovine serum albumin (BSA)-stabilized Au nanoclusters for the first time. The possible ECL mechanism was discussed according to the presented results and ascribed to the effective electron transfer from the conduction-band of excited indium tin oxide (ITO) to Au nanoclusters (NCs). A simple label-free method for the detection of dopamine has been developed based on the Au NCs ECL in aqueous media. The Au NCs could be an effective candidate for new types of ECL biosensors in the future due to their fascinating features, such as good water solubility, low toxicity, ease of labeling, and excellent stability.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

Positively charged sol-gel coatings for on-line preconcentration of amino acids in capillary electrophoresis

A novel on-line method is presented for the extraction and preconcentration of amino acids using a sol-gel-coated column coupled to a conventional UV/visible detector. The presented approach does not require any additional modification of the commercially available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. Sol-gel coatings were created by using N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride (C18-TMS) in the coating sol solutions. Due to the presence of a positively charged quaternary ammonium moiety in C18-TMS, the resulting sol-gel coating carried a positive charge. For extraction, the pH of the samples was properly adjusted to impart a net negative charge to amino acids. A long plug of the sample was then passed through the sol-gel-coated capillary to facilitate extraction via electrostatic interaction between the positively charged sol-gel coating and the negatively charged amino acid molecules. Focusing of the extracted amino acids was accomplished through desorption of the extracted amino acid molecules carried out by local pH change. Two different methods are described. Both methods showed excellent extraction and preconcentration effects. Preconcentration results obtained on sol-gel-coated columns were compared with the CZE analysis performed on bare fused-silica columns with traditional sample injections. The described procedure provided a 150,000-fold enrichment effect for alanine. The two methods provided acceptable repeatability in terms of both peak height and migration time.