On-line enhancement and separation of nanoparticles using capillary electrophoresis

We describe a rapid, simple, and highly efficient capillary electrophoresis (CE)-based method for the analysis of nanoparticles (NPs). In this study, we used the reversed electrode polarity stacking mode (REPSM) of CE to assess the feasibility of enhancing the detection of Au NPs and Au/Ag NPs, optimizing parameters such as the length of time for which the REPSM was applied, the concentrations of the buffer and the sodium dodecylsulfate (SDS) surfactant, and the pH. Under the optimized on-line enhancement conditions [buffer: SDS (40 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 10.0; applied voltage: 20 kV; REPSM applied for 24s], the detection limits of the Au NPs and Au/Ag NPs increased by ca. 30- and 140-fold, respectively. In addition, when the NPs were subjected to on-line enhancement and separation by CE using diode array detection (DAD), this approach allowed chemical characterization of the NP species. Our results suggest that such CE analyses will be useful for accelerating the rates of fabrication and characterization of future nanomaterials.     

Capillary electrophoresis-electrospray mass spectra of the herbicides paraquat and diquat

The positive ion electrospray mass spectra of the quaternary ammonium salt herbicides paraquat and diquat are examined by on-line separation with capillary electrophoresis (CE) and by direct infusion of the analytes. The analytes are separated by CE in 7–10 min at pH 3.9 in 50% methanol-water by using several different separation buffer electrolytes. The capillary electrophoresis-electrospray ionization (CE-ES) mass spectra of paraquat and diquat consist primarily of doubly charged molecular ions, singly charged molecular ions, and singly charged deprotonated ions. The direct infusion spectra consist primarily of doubly charged molecular ions and singly charged deprotonated ions. The relative abundances of the doubly charged and deprotonated ions depend strongly on the presence or absence of ammonium ion in the CE separation buffer or the direct infusion solution. A deprotonation mechanism is proposed in which the free base ammonia is the deprotonating agent in the desolvating charged droplets or in the gas phase. The analytical potential of the CE-ES electrospray approach for environmental analyses is evaluated in terms of the precision of replicate injections, linear concentration range, and estimated detection limit.     

Ultrasensitive Capillary Electrophoresis of Oligoguluronates with Laser-Induced Fluorescence Detection

An ultrasensitive method, capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection, has been established for analysis of oligoguluronates. The oligoguluronates were derivatized with 9-aminopyrene-1,4,6-trisulfonic acid (APTS) by reductive amination at 60°C for 3.5 h. The effect on the separation of buffer pH and buffer concentration were examined. The APTS-oligoguluronates were successfully separated within 20 min by use of acidic (pH 2.5) phosphate buffer as electrolyte and a potential of 20 kV. The method was used to monitor the oligoguluronates produced by acidic hydrolysis of homopolymeric blocks of guluronic acids.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Determination of copper(I) and copper(II) ions after complexation with bicinchoninic acid by CE

A facile, sensitive, and selective method was developed for the simultaneous separation and determination of copper(I) [Cu(+)] and copper(II) [Cu(2+)] ions using CE with direct UV detection. The copper ions were complexed with a 1.5 mM bicinchoninic acid disodium salt solution at pH 8.7 prior to analysis. Acetate buffer (2 mM) was used as the CE running buffer. Parameters affecting CE separation such as sample pH, applied voltage, concentration of complexing agent, nature of the buffer solution, and interferences by other metal ions, were evaluated. The LODs for Cu(+) and Cu(2+) were 3.0 and 2.5 microg/mL (S/N = 3), respectively. The developed method allows the simultaneous determination of Cu(+) and Cu(2+) in less than 5 min with RSDs of between 5.3 and 9.5% for migration time and between 3.4 and 9.7% for peak areas, respectively. At optimum conditions, the percentage recoveries of Cu(+) and Cu(2+) were found to be 99.4 and 99.5%.     

Analysis of process impurities in the basic drug SB-253149 using capillary electrophoresis and on-line mass spectrometric detection

Capillary electrophoresis with on-line electrospray ionisation mass spectrometry (CE/ESI-MS) has been used to identify process impurities in a batch of the anti-atherosclerotic drug, SB-253149. The impurities were separated from the main drug compound by capillary electrophoresis (CE) using an ammonium formate buffer at low pH in an untreated fused silica capillary. The CE method was initially developed using UV as the detection mode and then later structural elucidation work was achieved using an ion trap mass spectrometer. To maintain peak resolution and peak shape when the CE system was coupled to the mass spectrometer, a modified capillary cassette linked to a coaxial sheath flow electrospray ionisation (ESI) interface was used. By performing MS/MS experiments in conjunction with chemical knowledge of the reactivities of SB-253149, it was possible to propose molecular structures for impurities detected in the batch of SB-253149. The results from this study revealed that most of the process impurities in SB-253149 were dimeric derivatives of the parent molecule as well as trace levels of the starting material. This type of information was vital in process control and optimisation for the synthetic route for this drug.     

Improved solid-phase microextraction device for use in on-line immunoaffinity capillary electrophoresis

A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.     

Immunoassay by capillary electrophoresis with quantum dots

The application of quantum dots in capillary electrophoresis immunoassay was studied for the first time. Quantum dots were conjugated with antibody and subsequently tested by electrophoretic separation of free antibody and antibody-antigen complex. Antibody was fluorescently labeled by quantum dots via conjugation procedures and its electrophoretic characteristics were effectively modified due to the attachment of quantum dots. The determination of human IgM by direct CE based immunoassay could be easily achieved by simply changing the pH value of separation buffer. Polymer additive influenced the separation too but the effect was not as significant as buffer pH adjustment. Satisfactory separation of complex from free antibody could be achieved with 20mM sodium tetraborate as separation buffer, at pH 9.8. The immunoassay application of quantum dots in CE offers considerable advantages and can be readily applied to other large bio-molecules.     

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.     

Direct Determination of Glyphosate in Environmental Waters Using Capillary Electrophoresis with Electrospray Condensation Nucleation Light Scattering Detection

An electrospray condensation nucleation light scattering detector (ESI-CNLSD) was coupled with capillary electrophoresis (CE) for analysis of glyphosate, a chemical of agricultural interest, which is otherwise difficult to detect owing to its lack of chromophores or fluorophores. To reduce the absorption of glyphosate on the CE capillary and to reduce the separation time, an N-cetyltrimethylammonium bromide (CTAB) pre-rinsing CE method was developed here. The protocol consisted of 15 min pre-rinsing of the capillary before analysis with CTAB solution and 5 min with ammonium acetate buffer at pH 2.8. The capillary inner wall coating established by this treatment lasted up to 10 h without bleeding to interfere with CNLSD signal. Calibration data were linear over two orders of magnitude, the instrument detection limit was 0.06 µg/mL and the method detection limit was 0.2 µg/mL. The method was applied to the analysis of local (rural area) lake water and commercial herbicide samples.     

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).     

Extremely highly efficient on-line concentration and separation of gold nanoparticles using the reversed electrode polarity stacking mode and surfactant-modified capillary electrophoresis

In this study, gold nanoparticles (Au NPs) were separated using the reversed electrode polarity stacking mode (REPSM) of a capillary electrophoresis (CE) system for on-line enhancement prior to performing surfactant-modified CE separation. Under optimized conditions [running electrolyte buffer, sodium dodecyl sulfate (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (10 mM) at pH 10.0; applied voltage, 20 kV; operating temperature, 25°C; REPSM strategy for sample on-line concentration; REPSM applied prior to initializing separation], two parameters were varied to further enhance the concentration and separation of the Au NPs: (i) the rate of polarity switching (from -20 to +20 kV) between the REPSM and surfactant-modified CE separation modes and (ii) the length of the capillary column. At a polarity switching rate of 1333 kV min(-1) and a column length of ca. 83.5 cm, the resolution of the separation of a mixture of 5.3- and 40.1-nm Au NPs was greater than 19; in addition, the numbers of theoretical plates for the 5.3- and 40.1-nm-diameter Au NPs were greater than 15,000 and up to 1.15×10(7), respectively-the latter being extremely high. Thus, this CE-based method for separating Au NPs provided high performance in terms of separation resolution and the number of theoretical plates, both of which were improved by greater than fivefold relative to those published previously. Notably, the sensitivity enhancement factors for the 5.3- and 40.1-nm-diameter Au NPs were improved (by ca. 20- and 500-fold, respectively) relative to those obtained using conventional surfactant-modified CE separation.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection,using a chemometrical optimization approach

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris‐H₃PO₄ buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris‐H₃PO₄ buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.     

Determination of chlorophenols in human urine based on the integration of on-line automated clean-up and preconcentration unit with micellar electrokinetic chromatography.

A new method was developed and validated for the determination of chlorophenols in human urine by using micellar electrokinetic chromatography (MEKC) coupled via a mechanic arm to an on-line automatic clean-up and preconcentration unit for urine samples. Separation is accomplished by using a selective buffer consisting of 15 mM borate, 25 mM phosphate and 100 mM sodium dodecyl sulfate (SDS) at pH 9.1 in addition to a positive power supply of 25 kV at 18 degrees C. The proposed capillary electrophoresis (CE) method allows the separation of 11 chlorophenols within 7 min with a reproducibility as relative standard deviation (RSD) between 2.6% and 7.2%, and limits of detection (LODs) between 0.08 and 0.46 microg/mL for all chlorophenols. Urine samples were previously hydrolyzed with 37% HCl at 80 degrees C for 60 min and then cleaned on a C-18 mini-column. Recoveries ranged from 58% to 103%. The preconcentration treatment affords limits of determination between 4 and 12 ng/mL for all chlorophenols except pentachlorophenol and 4-chlorophenol, which could not be determined. The overall analysis time, including on-line clean-up, preconcentration and electrophoretic separation is 20 min per sample.     

Determination of salidroside and tyrosol in Rhodiola by capillary electrophoresis with graphene/poly(urea-formaldehyde) composite modified electrode

This report describes the fabrication and application of a novel graphene/poly(urea-formaldehyde) composite modified electrode as a sensitive amperometric detector of CE. The composite electrode was fabricated on the basis of the in situ polycondensation of a mixture of graphenes and urea-formaldehyde prepolymers on the surface of a platinum disc electrode. It was coupled with CE for the separation and detection of salidroside and tyrosol in Rhodiola, a traditional Chinese medicine, to demonstrate its feasibility and performance. Salidroside and tyrosol have been well separated within 6 min in a 40 cm long capillary at a separation voltage of 12 kV using a 50 mM borate buffer (pH 9.8). The prepared graphene-based CE detector offered significantly lower detection potential, yielded enhanced signal-to-noise characteristics, and exhibited high resistance to surface fouling and enhanced stability. It showed long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 15).     

采用隔离池的毛细管电泳-间接紫外吸收法测定茶叶中的氨基酸

改进的毛细管电泳-间接紫外吸收法采用了自制隔离池,以对氨基苯甲酸(PAB)为背景电解质,对茶叶中的氨基酸进行了测定。PAB能够提高分离效率,降低检出限。隔离池的使用避免了PAB的电极反应,降低了基线噪声,维持了两缓冲液池间的电流导通。研究了背景电解质的浓度、pH值以及电渗流改性剂的种类和浓度对氨基酸分离的影响。在优化的实验条件下,16种氨基酸在14 min内达到了基线分离,峰面积的相对标准偏差小于5%(n=5),检出限为1.7~4.5 μmol/L,回收率为83.0%~106%。该法快速、便捷和灵敏,已成功应用于茶叶中11种游离氨基酸的检测。     

DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions

The goal of this work was to reduce the capillary electrophoresis (CE) separation time of DNA sequencing fragments with linear polyacrylamide solutions while maintaining the previously achieved long read lengths of 1000 bases. Separation speed can be increased while maintaining long read lengths by reducing the separation matrix viscosity and/or raising the column temperature. As urea is a major contributor to the separation buffer viscosity, reducing its concentration is desirable both for increase in the separation speed and easier solution replacement from the capillary. However, at urea concentrations below 6 M, the denaturing capacity of the separation buffer is not sufficient for accurate base-calling. To restore the denaturing properties of the buffer, a small amount of an organic solvent was added to the formulation. We found that a mixture of 2 M urea with 5% v/w of dimethyl sulfoxide (DMSO) resulted in 975 bases being sequenced at 70 degrees C in 40 min with 98.5% accuracy. To achieve this result, the software was modified to perform base-calling at a peak resolution as low as 0.24. It is also demonstrated that the products of thermal decomposition of urea had a deleterious effect on the separation performance at temperatures above 70 degrees C. With total replacement of urea with DMSO, at a concentration of 5% v/w in the same linear polyacrylamide (LPA)-containing buffer, it was possible to increase the column temperature up to 90 degrees C. At this temperature, up to 951 bases with 98.5% accuracy could be read in only 32 min of separation. However, with DMSO alone, some groups of C-terminated peaks remained compressed, and column temperature at this level cannot at present be utilized with existing commercial instrumentation.     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl-L-arginine and structurally related amino acids: quantitation in human plasma

We report the development of efficient electrophoretic methods for the separation and quantification of L-arginine and six naturally occurring derivatives that are structurally and functionally related. Capillary electrophoresis (CE) employing a concentrated borate buffer at pH 9.4 achieves the separation of mixtures containing dimethyl-L-arginine, NG-monomethyl-L-arginine, L-arginine, L-homoarginine, L-ornithine, and L-citrulline as 4-fluoro-7-nitrobenzofurazan derivatives. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) is attained with baseline resolution by micellar electrokinetic chromatography (MEKC) when a high concentration of deoxycholic acid is added as a surfactant to the same running buffer. The influence of buffer type, concentration, and pH on the separation was studied to optimize separation conditions. The limit of quantitation (LOQ) for asymmetric dimethyl-L-arginine in aqueous solution was determined to be 20 microM using UV absorption in a CE separation and 0.1 microM using laser induced fluorescence (LIF) detection in an MEKC separation. This newly developed method was successfully applied for the quantitation of asymmetric dimethyl-L-arginine and L-arginine in human plasma samples at levels that might be used as a clinical diagnostic for cardiovascular disease (0.125 microM LOQ).